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University of Veterinary Medicine Hannover
Seroprevalence of Rift Valley fever virus specific
antibodies in livestock in Egypt and expression
studies of virus related proteins in mammalian and
arthropod cells
INAUGURAL - DISSERTATION
in partial fulfillment of the requirements of the degree of
Doctor of Veterinary Medicine
- Doctor medicinae veterinariae -
(Dr. med. vet.)
Submitted by
Claudia Mroz
Stadtlohn
Hannover 2017
Academic supervision: Prof. Dr. Martin H. Groschup
Institute for novel and emerging infectious diseases
Friedrich-Loeffler-Institute
Greifswald-Insel Riems
1. Referee: Prof. Dr. Martin H. Groschup
Institute for novel and emerging infectious diseases
Friedrich-Loeffler-Institute
Greifswald-Insel Riems
2. Referee: Prof. Dr. Ludwig Haas
Department of Virology
University of Veterinary Medicine Hannover
Day of oral examination: 9th of November 2017
Sponsorship:
This work was supported by the Federal Foreign Office (German Partnership 303
Program for Excellence in Biological and Health Security).
to my family
Table of contents
Chapter 1: Introduction ................................................................................................ 1
Chapter 2: Literature review ........................................................................................ 5
2.1. History of RVFV ......................................................................................................... 5
2.2. History of RVFV in Egypt .......................................................................................... 6
2.3. Virus-host-interaction ............................................................................................... 7
2.4. Vectors ........................................................................................................................ 8
2.5. Pathogenesis ............................................................................................................. 8
2.5.1. RVFV in humans ........................................................................................................................ 9
2.5.2. RVFV in ruminants ................................................................................................................... 10
2.5.3. RVFV in camels ........................................................................................................................ 11
2.5.4. RVFV in other mammalian animals ....................................................................................... 11
2.6. Virus characterization ............................................................................................. 12
2.6.1. Viral replication cycle ............................................................................................................... 12
2.6.2. Virulence factors ....................................................................................................................... 13
2.7. Vaccination ............................................................................................................... 14
2.7.1. Live-attenuated vaccines ........................................................................................................ 14
2.7.2. Inactivated vaccines................................................................................................................. 15
2.7.3. Recombinant vaccines ............................................................................................................ 15
2.7.4. Vaccination in Egypt ................................................................................................................ 16
2.8. Detection, treatment, protection and surveillance .............................................. 17
2.8.1. Detection ................................................................................................................................... 17
2.8.2. Treatment .................................................................................................................................. 17
2.8.3. Prevention and control............................................................................................................. 17
2.8.4. Surveillance in Egypt ............................................................................................................... 18
Chapter 3: Seroprevalence of Rift Valley fever virus in livestock during inter-
epidemic period in Egypt, 2014/15 ..................................................... 19
Chapter 4: Rift Valley fever virus infections in Egyptian cattle and their
prevention .............................................................................................. 21
Chapter 5: Comparative expression kinetics of five different Rift Valley fever
proteins in mammalian and insect cells ............................................ 22
5.1. Abstract .................................................................................................................... 23
5.2. Background .............................................................................................................. 24
5.3. Material ..................................................................................................................... 26
5.4. Results ...................................................................................................................... 30
5.5. Discussion ................................................................................................................ 32
5.6. Conclusions ............................................................................................................. 35
5.7. Tables ........................................................................................................................ 36
5.8. Figures ...................................................................................................................... 37
Chapter 6: Comparison of different serological test systems to detect anti
RVFV IgG antibodies in cattle and camel sera ................................. 45
6.1. Abstract .................................................................................................................... 46
6.2. Short communication .............................................................................................. 47
6.3. Tables ........................................................................................................................ 50
Chapter 7: General Discussion ......................................................................... 52
Chapter 8: Summary ........................................................................................... 59
Chapter 9: Zusammenfassung .......................................................................... 62
Chapter 10: Bibliography ..................................................................................... 65
Chapter 11: Acknowledgements ......................................................................... 76
Manuscripts extracted from the doctorate project:
1. C. Mroz, M. Gwida, M. El-Ashker, M. El-Diasty, M. El-Beskawy, U. Ziegler, M.
Eiden, M.H. Groschup; Seroprevalence of Rift Valley fever virus in livestock
during inter-epidemic period in Egypt, 2014/15; BMC Veterinary Research,
2017 Apr 5; 13(1):87.
(doi: 10.1186/s12917-017-0993-8)
2. C. Mroz, M. Gwida, M. El-Ashker, U. Ziegler, T. Homeier-Bachmann, M.
Eiden, M.H. Groschup; Rift Valley fever virus infections in Egyptian cattle and
their prevention; Transboundary and emerging diseases, 2017 Jan 24; 1–10.
(doi: 10.1111/tbed.12616)
3. C. Mroz, K.M. Schmidt, S. Reiche, M.H. Groschup, M. Eiden; Comparative
expression kinetics of five different Rift Valley fever proteins in mammalian
and insect cells; Virology Journal (under review)
4. C. Mroz, K.M. Schmidt, U. Ziegler, M. Rissmann, M. Gwida, B.O. EL Mamy, K.
Isselmou, B. Doumbia, M. Eiden, M.H. Groschup; Comparison of different
serological test systems to detect anti RVFV IgG antibodies in cattle and
camel sera (to be submitted)
Furthermore, parts of the results have already been
published in poster presentations:
Junior Scientist Symposium, Mariensee, Germany, 2014:
“Rift Valley fever – an emerging disease in Europe?”
Junior Scientist Zoonoses Meeting, Munich, Germany, 2015:
“The seroprevalence of Rift Valley fever virus infections in Egypt”
Junior Scientist Zoonoses Meeting, Göttingen, Germany, 2016:
“Seroprevalence of Rift Valley fever virus infections in Egyptian livestock”
List of abbreviations
cELISA Competition enzyme linked immunosorbent assay
CI Confidence interval
CO2 Carbon dioxide
Cy3 Cyanine 3
DIVA Differentiating Infected from Vaccinated Animals
ELISA Enzyme linked immunosorbent assay
FBS Fetal bovine serum
Gn Glycoprotein n (-terminal)
Gc Glycoprotein c (-terminal)
Hpi Hours post infection
IgG Immunoglobulin G
IgM Immunoglobulin M
IFA Indirect immunofluorescence assay
IFN-ß Interferon-beta
kDa Kilodalton
L segment Large segment
M segment Medium segment
mab Monoclonal antibody
MOI Multiplicity of infection
mRNA Messenger ribonucleic acid
ND50 Neutralizing dose of 50%
nm Nanometer
N protein Nucleocapsid protein
NP Nucleocapsid protein
NSs Nonstructural protein (S-segment)
NSm Nonstructural protein (M-segment)
OD Optical density
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PVDF membrane Polyvinylidene difluoride membrane
RNA Ribonucleic acid
RNP Ribonucleoprotein
RT PCR Reverse transcriptase polymerase chain reaction
RT qPCR Reverse transcriptase quantitative polymerase chain reaction
RVF Rift Valley fever
RVFV Rift Valley fever virus
SDS Sodium dodecyl sulfate
S segment Small segment
TCID50 50% tissue culture infective doses
VNT Virus neutralization test
List of figures
Figure 5.1. First appearance of RVFV MP12 derived proteins in Vero 76
cells and C6/36 cells 37
Figure 5.2.
Distribution of RVFV MP12 derived proteins in Vero 76 cells
and C6/36 cells at different time points of infection 38
Figure 5.3.
Expression of NSs protein in cells infected with RVFV MP12
at a MOI of 1 39
Figure 5.4.
Western blotting of infected cell lysates on sequential time points 40
Figure 5.5.
Protein secretion in supernatants of infected Vero 76 cells (a) and
C6/36 cells (b) detected by western blotting 41
Figure 5.S1.
SDS Page analysis and Coomassie blue staining of recombinant
NSs and NSm protein 42
Figure 5 S2.
Comparative protein expression in RVFV MP12 infected cells 43
List of tables
Table 5.1. Characteristics of produced monoclonal antibodies 36
Table 6.1.
Overview of positive, negative and inconclusive results in each
of the three test methods with calculation of the diagnostic
sensitivity and specificity of the ELISA and the IFA 50
Table 6.2.
Combination of the results from different methods 51
1
Chapter 1: Introduction
Rift Valley fever (RVF) is a vector-borne zoonotic disease, caused by the Rift Valley
fever virus (RVFV) of the order Bunyavirales, family Phenuiviridae, genus
Phlebovirus. The virus causes large and devastating epidemics in animals and
humans throughout Africa and causes hemorrhagic fever and severe illness to both,
humans and animals. Multiple mosquito species are able to transmit the virus to
susceptible mammalian hosts like humans, ruminants, camels and diverse wildlife
species. In particular, infected sheep suffer from high numbers of abortions and
mortality of newborns whereas the symptoms, such as nasal discharge, fever and
hepatitis are less severe in older animals. Goats, cattle, buffalos or camels are
generally believed to be more refractory to the infection. Humans can also become
infected by direct contact with infectious tissues or body fluids when handling with
infected animals or products thereof. Most infections are mild with flu-like, mild febrile
symptoms but in 1-2% of cases patients develop severe symptoms including ocular
disease, encephalitis up to a hemorrhagic fever syndrome.
RVFV consists of a three-parted single stranded RNA genome which is packaged in
the enveloped virus particle. The small (S) segment encodes the N protein, which
regulates genome packaging in the form of ribonucleoprotein complexes. Two
envelope glycoproteins Gn and Gc are encoded from the medium (M) segment. The
large (L) segment of the genome encodes for the RNA dependent RNA polymerase
which is responsible for processing mRNA after infection. Furthermore, the genome
encodes for nonstructural proteins, the M segment for the NSm as well as for the 78
kDa protein and the S segment encodes for the NSs protein. The NSs protein
displays the major virulence factor of RVFV which targets the innate host defense by
interacting with different key components of the interferon response. As an additional
virulence factor the NSm protein manipulates the apoptosis of infected cells.
The disease was first described in 1931 when massive abortions and neonatal
fatalities were reported in a sheep flock in the Kenyan Great Rift Valley. From then on
RVF outbreaks emerged at irregular intervals of 4-15 years in more than 30 African
countries. Serious epidemics were reported especially in Egypt, Kenya, South Africa,
Mauritania and Senegal. During inter-epidemic periods the Rift Valley fever virus
2
presumably circulates at low level between the mosquito population and wildlife
ruminants which generally display no clinical signs. Several mosquito species, of the
Aedes genus in particular, can transmit the virus transovarially and enable the
survival of the virus during drought periods in infected eggs. In years with exceptional
rainfalls an explosive increase of the mosquito population leads to transmission from
infected mosquitos to naïve animals including livestock. Rapid spread of the virus
may result in a switch form the endemic to an epidemic cycle noticeable by clinical
animals and, subsequently, infections of humans. Until 1977 the RVFV was limited to
sub-Saharan countries but in 1977/78 the most serious epidemic so far occurred in
Egypt in the Nile river delta, which demonstrated the long range spreading potential
of RVFV even to Mediterranean Africa. More than 200,000 people were affected,
nearly 600 patients died and a large number of sheep, goats and cattle were
affected, which led to massive economic losses. Egypt has an exceptional position
compared to other endemic countries in sub-Saharan Africa since it is not affected by
heavy rainfalls influenced by El Niño. Further outbreaks re-occurred in 1993, 1994,
1997 and, most recently, in 2003 in Egypt but the sources of these epidemics were
not fully understood. Beside re-introduction from other endemic countries, the
maintenance of the virus was considered to be a possible cause.
Previous seroepidemiological studies suggested a cryptic virus circulation in the
country. All the more the transmission and existence of RVFV in Egypt is still not fully
understood and the current epidemiological status of the Egyptian livestock not
completely clear to date. Therefore, this thesis intended to provide insights into the
serological RVFV status in different livestock species in Egypt (manuscript I and parts
of manuscript II).
After the first severe epidemic in 1977 the Egyptian government established a control
program, which included the vaccination of all susceptible animals. Interestingly,
further outbreaks occurred in Egypt after untypically short inter-epidemic periods,
which already indicates a failure of the vaccination program pursued.
Because little is known about the seroconversion in the livestock population after
vaccination and the effectiveness of the vaccination program a further objective of
3
this work was the comparison of the antibody levels of vaccinated and non-
vaccinated cattle (manuscript II).
Understanding how a virus overcomes the defense mechanisms of the host is
essential for developing effective therapies and to implement efficacious control and
prevention measures. The nonstructural proteins, NSs and NSm as the main
virulence factors, have been analyzed in several studies to clarify their role in the
infection of mammalian host cells and in the mosquito vector. So far, their exact
function is still not completely understood. Therefore, the final part of this thesis
aimed at providing tools to visualize NSs and NSm during RVFV infections. For this
purpose, monoclonal antibodies against both proteins were produced, characterized,
utilized and the expression kinetics and locations in mammalian and insect cells were
compared as well as the interplay with the other viral proteins NP, Gn and Gc
(manuscript III).
Seroloprevalence studies are important approaches to identify RVFV endemic areas
as characterized by constant low level virus circulation. For this purpose, various
ELISA (Enzyme linked immunosorbent assays) have been developed and used in
different laboratories. The virus neutralization test (VNT) is considered as the gold
standard for serological testing, as it allows the highly specific discrimination of
antibodies also to other Phenuiviridae family members. However, the VNT requires
handling live viruses under appropriate biosafety conditions, allows testing only small
numbers of sera and takes at least one week until a final result is obtained.
Therefore, another aim of this thesis was to establish a novel diagnostic
immunofluorescence assay in order to enable a high-throughput serological testing of
large numbers of ruminant and camel sera (manuscript IV).
In summary, the main objective of this work was to provide insights into the
seroepidemiological status of RVFV in Egyptian livestock, to evaluate the current
risks of new outbreaks in this country and to appraise the vaccination program of
Egyptian cattle. Moreover, a new serological assay should be developed and
compared with existing test methods to provide the highest possible test accuracy.
These studies therefore will contribute to a better understanding of the RVFV
circulation during inter-epidemic periods and raise attention concerning the
4
implemented control strategies in Egypt. Finally, the generation and application of
monoclonal antibodies directed against the NSs and the NSm protein were carried
out to elucidate their role in the mosquito vectors and mammalian hosts.
5
Chapter 2: Literature review
2.1. History of RVFV
Rift Valley fever virus was first described and characterized in 1931 when massive
losses in newborn lambs were detected in a single farm near the lake Naivasha in the
Great Rift Valley in Kenya [1]. From then on, the occurrence of RVF was reported
consistently in many sub-Saharan countries. After continuing outbreaks in Kenya
over decades, RVFV caused a large epidemic in South Africa in 1950-1951 with
more than 500,000 abortions and 100,000 deaths in sheep followed by diseased
humans after contact with infected animals [2-4]. In years with exceptionally high
rainfalls further epidemics occurred in eastern and southern Africa, especially in bush
and wooded savanna grasslands. In 1987, the first RVFV epidemic was reported in
West Africa with 220 human fatalities in southern Mauritania and northern Senegal
after closing of the Diama Dam on the Senegal River [4-6]. Unlikely to those in East
and South Africa, RVF epidemics in West Africa occur only at irregular intervals
frequently even without obvious associations with extensive rainfalls. Subsequent to
epidemics in Sudan in 1973 and 1976, RVFV was introduced to Egypt in 1977-1978,
which is the northernmost affected African country to date, causing a massive
epidemic (see 2.2.) [7-9]. Outside the mainland of Africa, RVFV was isolated from
mosquitos for the first time in Madagascar in 1979. No clinical signs in humans or
animals were reported then. In Madagascar, the first clinically noticeable epidemic
occurred in 1990-1991 when high rates of abortions in cattle were detected after two
consecutive rainy seasons. Nearly 20 years later, in 2008-2009 the next epidemic
occurred in many parts of Madagascar, affecting livestock and humans [10, 11]. The
emerging importance of RVFV was underlined in 2000 when a significant epidemic
occurred on the Arabian Peninsula. A large number of infected sheep and goats,
around 1,500 human infections and over 200 human deaths, were reported in Saudi
Arabia and Yemen during this massive outbreak [3, 12]. In 2006-2008 the RVFV
affected several countries including Kenya, Somalia, Tanzania and Sudan,
sometimes even causing severe outbreaks. Further epidemics occurred in South
Africa, Swaziland and on the Comoros and Mayotte islands in 2007-2009 [13-18].
RVFV has been found in over 30 African and Arab countries to date and is endemic
in many African countries - usually leading to visible epidemics in irregular intervals.
6
Most recently, outbreaks were reported in Uganda in March 2016 when the disease
was confirmed in goats and humans, in Niger in September 2016 affecting more than
1,000 sheep, goats and cattle as well as in Mali in October 2016 when abortions in
goats and sheep were detected. [19].
2.2. History of RVFV in Egypt
The virus was introduced into Egypt in 1977 through the occurrence of a large RVF
epidemic near the Nile Delta [3]. At the beginning of October 1977 increasing
numbers of people were affected with febrile illness in the villages of the Sharqia
governorate. During October further, human cases were reported near the town
Zagazig in the Sharqia governorate as well as in the governorates of Qalyubia and
Giza. Since the end of August an increase in mortality rates and in abortions was
observed in sheep, cattle, domestic buffalos and camels in the Sharqia and Aswan
governorates. Elevated numbers of human cases with encephalitis, ocular disease
and hemorrhagic syndromes were reported [20-22]. The number of cases decreased
over the winter but in July 1978 further cases of human and animal diseases recurred
in the Sharqia governorate as well as in upper Egypt in the governorates of Minya
and Asyut. RVFV was isolated till December of 1978. However, detailed
epidemiological data do not exist [8, 21, 23-25]. In the end the first RVF outbreak in
1977/1978 lead to the infection of more than 200,000 humans of which nearly 600
patients died and also to high death tolls among domestic livestock. The most
probable route of introduction of the RVFV to Egypt was through the entry of infected
animals e.g. camels or infected humans from endemic countries like the Sudan. In
Sudan recurrent outbreaks took place in the years 1973 and 1976 [22, 26].
Furthermore, Culex pipiens was the most prominent mosquito species during 1977
and transmissions from Culex to humans were considered to play an important role
during the epidemic [8, 24]. Recently, phylogenetic analyses demonstrated that the
RVFV strains of the 1977 outbreak are closely related to strains from Zimbabwe in
1974 which illustrates that Zimbabwe may be the origin of the Egyptian RVFV
lineages [27].
After a long inter-epidemic period RVF reoccurred in 1993 in the Aswan and
Damietta governorates when approximately 40 human cases were reported [28].
Patients experienced acute fever with severe headache, myalgia as well as back pain
7
but only one case of a diabetic woman with neurological complications ended fatal.
No severe hepatic or hemorrhagic symptoms were observed during the outbreak.
Veterinary services recognized a high incidence of abortions predominantly in cattle
and domestic buffalos. However, sheep and goats were also affected. Animals which
aborted their fetuses were tested positive to RVFV [28, 29]. Isolates of the RVFV in
this outbreak were relatively similar to those from the first outbreak in 1977 which led
to the suggestion that the virus remained in the country or was re-introduced from the
same source of infection [8].
A further outbreak occurred in 1994 in the Behera and Kafr el Sheikh governorates
where RVFV was isolated from infected cattle and sheep. This outbreak was
described as a vaccination failure as the live-attenuated Smithburn vaccine was used
in the same year in the country [8]. From April to August 1997 a high incidence of
abortions in sheep and cattle and high mortality rates in young lambs and calves
were recognized in upper Egypt in the governorates of Aswan and Assuit [8, 30]. The
abortion rate in ewes was 60-70% whereas cattle showed approximately 30-40%.
Also, young lambs harbored higher mortality rates (50-60%) compared to calves (25-
30%). This unusual behavior of RVFV indicated a failure of the applied RVF
vaccination program in Egypt [8]. Recently, RVF occurred in summer 2003 in various
Egyptian governorates when increased cases of acute febrile illness were recognized
by the hospital-based electronic disease surveillance system [31]. Confined to the
Nile Delta area, predominantly in the governorate of Kafr el Sheikh, in this epidemic
mainly humans were affected with no reported clinical cases in livestock. It was the
first time that Culex antennatus was found naturally infected with RVFV in Egypt [8,
31].
2.3. Virus-host-interaction
Rift Valley fever virus epidemics occur in irregular intervals. In eastern and southern
African countries the virus circulates within two overlapping cycles: in periods with
normal rainfall the virus shows only low-level endemic activity and circulates within
the mosquito population with sporadic transmissions to wildlife ruminants like buffalos
or susceptible livestock [3, 32]. These inter-epidemic periods vary from 4 to 15 years
in areas with grassland and up to 35 years in drought areas [32]. Periods with
extensive rainfall may induce a shift from the endemic to the epidemic cycle when the
8
number of infected mosquitos exceeds a critical value followed by transmission of the
virus to naïve ruminants and the development of high viremic titers as well as clinical
signs. Secondary infections of mosquitos arise after they feed from infected
ruminants leading to a rapid spread of the virus. Humans can also become infected,
mainly through the contact with infectious material when handling diseased animals
[3, 9, 32]. Usually epidemics are limited to a few months, but they may persist in
humid zones for years.
2.4. Vectors
The Rift Valley fever virus is transmitted by mosquitos and was isolated from more
than 53 species out of eight genera (Aedes, Culex, Anopheles, Eretmapodites,
Mansonia, Coquillettidia, Eumelanomyia, Ochlerotatus) [3, 9, 33]. Additional
arthropod species like ticks, biting midges and other flies are considered to be
mechanical vectors, with no proof of their role in the amplification cycle of RVFV [33].
Mosquitos from the genera Aedes and Culex show the highest vector competence for
RVFV. Moreover, the transovarian transmission of Aedes species ensures the
maintenance of RVFV during inter-epidemic periods. Thus, the virus is able to remain
in drought-resistant eggs of the floodwater-breeding Aedes species over long periods
of time [3, 9, 32, 33].
2.5. Pathogenesis
The susceptibility to RVFV infections depends on species and age. Animals can get
infected by bites of infected mosquitos or by direct contact with infectious materials
[34]. Direct human-to-ruminant contact represents the main transmission route for
humans. Humans as “dead-end” hosts are dispensable for the sustainment of the
virus-life cycle.
Clinical signs of infected animals and humans range from inapparent infections to
sudden deaths [4, 9, 32]. Main replication sites of RVFV are liver and spleen but also
the brain is frequently affected [32, 35]. Highly susceptible animals show viremia for
up to 10 days whereas more resistant breeds display a viremia only for one to three
days [32].
9
2.5.1. RVFV in humans
Veterinarians, health personnel, farmers and workers from the slaughterhouse who
are in direct contact with body fluids and tissues of possibly infected animals during
necropsy, slaughtering and butchering are at high risk of becoming infected. In
addition, these persons are more likely to also be exposed to infected mosquitos [33].
Infections in humans follow four different clinical progressions [4]. Most commonly,
infections in humans appear as a self-limiting, febrile illness, similar to a mild
influenza infection. Typically, clinical symptoms appear abruptly after four to six days
and may include malaise, severe chills, dizziness, weakness, severe headache,
nausea and a sensation of fullness over the liver region. Fever, decreased blood
pressure, body pain, vomiting and diarrhea may follow. The symptoms subside and
the body temperature reaches normal after the viremic phase (three to four days) [7,
36, 37]. Repeated high fever and severe headache may occur after the first recovery
[20, 36, 38]. In the phase of convalescence, however, symptoms including general
weakness, malaise, headache, pain during ocular motility and a sense of imbalance
may last for weeks [36]. Beside this flu-like course of the disease the Rift Valley fever
virus infection can be associated with more severe forms including neurological
disorders, vision loss or hemorrhagic fever syndrome. One to two percent of the
patients may develop one of these progresses [3, 4]. Often patients with more severe
RVF develop ocular manifestations with the loss of central vision or blurred vision at
various times after infection. Macular edema with retinal hemorrhage, exudates,
vasculitis and infarction or haze of the vitreous body may occur in one or both eyes.
Furthermore, retinal detachment, uveitis and arterial occlusion is possible. Often a
complete recovery of vision does not occur or takes several months [9, 36, 39-41].
Neurological disorders are related to encephalitis with symptoms like confusion,
disorientation, drowsiness, coma, neck stiffness, hemiparesis, paraparesis or
convulsions. The cerebrospinal fluid of those patients contains high amounts of anti-
RVFV antibodies and increased numbers of white blood cells correlating to viral
meningitis or meningoencephalitis [7, 36, 37, 42]. Frequently, fatal cases of RVF
involve the development of the hemorrhagic fever syndrome, the most severe course
of RVFV infection which includes hepatitis, thrombocytopenia, icterus and multiple
hemorrhages. Fatality rate is less than one per cent in humans but may increase up
10
to 20% during large epidemics [3, 9, 36]. Typically, patients with hemorrhagic fever
syndrome sicken abruptly with flu-like symptoms, followed by macular rash,
ecchymosis, bleeding from gastrointestinal tract, low blood pressure, hematemesis,
melena, diarrhea, throat pain, pneumonitis, jaundice or hepatosplenomegaly. The
majority of the patients die three to six days after the onset of the first symptoms.
Beside extensive necrosis of hepatocytes patients may develop fatal renal failure or
disseminated intravascular coagulation (DIC) [1, 9, 20, 22, 36].
2.5.2. RVFV in ruminants
The course of RVFV infections in livestock depends on animal species, breed as well
as the age. Generally, the susceptibility of young animals is higher than that of adult
animals which results in the development of more severe symptoms in young animals
[1, 3, 4, 9, 33]. Sheep represent the most susceptible species and newborn lambs,
younger than one week, show peracute infections with mortality rates of up to 100%.
After an incubation period of 12 to 24 hours they suffer under a rapid progression of
symptoms with elevated body temperature up to 42°C, loss of appetite, lethargy and
death within 24 to 72 hours. Hepatic necrosis is the most common lesion in newborn
lambs and aborted fetuses. Sheep older than one week up to three weeks may also
develop a fatal disease or a milder infection with fever, hemorrhagic diarrhea, muco-
purulent nasal discharge and decreased activity. Adult sheep, older than three
months, are relatively resistant to RVFV infections and display only mild infections
with no or mild clinical signs. In adult sheep the mortality rate is ranging from 10 to
30%. However, pregnant ewes may show characteristic abortion storms with abortion
rates ranging from 5 to 100%. Multiple organ infections and necrosis of the fetus as
well as infections of the placental cotyledons and caruncles result in fetal losses and
abortions. In addition, 20% of aborting ewes die as a consequence of the infection [1,
3, 4, 9, 36, 43].
Likewise, goats are also highly susceptible, albeit infections with RVF are less severe
than in sheep, with only mild symptoms and a faster recovery. The disease is less
fatal in adult goats (<10%), but abortions are also frequent (up to 80%) [3, 4, 9].
After RVFV infection calves may develop acute illnesses with fever for one to four
days, loss of appetite, diarrhea, lethargy and dyspnea with a mortality rate ranging
from 10 to 70%. Adult cattle often show mild infections with abortions being the only
11
clinical sign. More severe forms may include fever, inappetence, lethargy,
hematochezia, epistaxis and a drop in milk production. The mortality rate in adult
cattle is similar to that of adult goats (5-10%) [3, 4, 9, 32].
Other ruminants like African or Asian buffalos are also susceptible to RVFV
infections. In general buffalos seem to show no clinical signs of infections;
nevertheless, abortion may occur. As indicated by the revealed high seroprevalence
rates, wildlife buffalos might play an important role in the maintenance of RVFV
during inter-epidemic times [3, 4, 9, 44].
2.5.3. RVFV in camels
For a long time, the role of camels and their susceptibility to the RVFV was unclear.
Clinical signs in camels were rarely noted. However, in 2010 during a RVFV outbreak
in Mauritania infected camels displayed two clinical forms. Either camels suffered
from a hyper-acute form with sudden deaths in less than 24 hours. Or they developed
an acute form with fever, ataxia, edema in the neck region, dyspnea, blood-tinged
nasal discharge, icterus, severe conjunctivitis, foot lesions and hemorrhages.
Hemorrhagic symptoms often lead to the death of the animal within a few days. The
second form of the RVF infection was also a combination of abortions and nervous
symptoms. Also, the infection of adult camels may be inapparent with the only sign of
the disease being abortions. [3, 45, 46].
2.5.4. RVFV in other mammalian animals
Rodents like mice or hamster are highly susceptible to laboratory infections with
RVFV. Infections are mostly fatal and the pathologic findings reflect those in newborn
lambs. Surviving mice often develop fatal limb paralysis and encephalitis. At the late
phase of infection mice develop acute hepatitis but syndromes like hemorrhagic fever
or ocular disease do not occur. Laboratory experiments could further demonstrate
infections of dogs, cats and ferrets with flu-like symptoms. In contrast, no symptoms
or virus replications were observed in rabbits, guinea pigs, birds, horses and pigs
[36]. Seroconversion was also determined in various wild-life species such as the
warthog, black rhino, zebra, kudu, impala, waterbuck, bat and nonhuman primate [3,
44].
12
2.6. Virus characterization
RVFV is a member of the family Phenuiviridae, genus Phlebovirus. The enveloped
particles, 80-120 nm in diameter, contain the typical tri-segmented single stranded
RNA genome which is organized in viral ribonucleocapsids (RNPs). The RNPs
contain numerous copies of the nucleocapsid proteins and the RNA-dependent RNA
polymerase [7]. Complementary RNA sequences at the ends of each segment result
in a circular pan-handled secondary structure of the segments. The envelope is
composed of a liquid bilayer of glycoprotein-heterodimers forming an ordered
icosahedral shell of 122 capsomers [7, 47]. The M segment is synthesized in
negative orientation. One open reading frame encodes for a precursor polyprotein
which is co-translationally cleaved into four proteins: two glycoproteins Gn and Gc as
well as two nonstructural proteins, the NSm and the 78 kDa protein. However, five
initiation codons upstream to the Gn coding sequence result in the synthesis of
different precursor proteins. Beginning of the translation from the first initiation codon
result in the synthesis of the 78 kDa protein. The NSm protein is synthesized from the
second start codon and the Gn protein is synthesized from the second, third or fourth
codon as a poly-Gn-Gc protein [7, 48, 49]. The L segment contains the genetic
information for the RNA-dependent RNA polymerase (L protein) of RVFV which is
responsible for replication and transcription of the viral RNA genome. The S segment
encodes for the nucleoprotein and the nonstructural protein NSs in an ambi-sense
manner. The intergenic region of the S segment is the most variable region of the
genome of RVFV strains (11%) [7]. The nucleoprotein (NP) is transcribed from the
sense portion of the S segment whereas the NSs protein exhibits an ambi-sense
coding strategy.
2.6.1. Viral replication cycle
The virion of RVFV binds to a host cell receptor which is not yet completely
characterized. Possible receptor candidates are DC-SIGN [50] and heparan sulfate
[51]. After binding, the virion enters the cell in a pH dependent manner, presumably
through a clathrin-mediated endocytic pathway [36, 52]. Ribonucleocapsids and L
protein are released into the cytoplasm where all transcription and replication steps
take place, similarly to other negative stranded RNA viruses. After encapsidation, the
13
L protein begins the primary transcription of the mRNA of the N and NSs protein as
early as 40 minutes after infection [36]. Generally, each segment is transcribed into a
complementary RNA (cRNA), an exact copy of the genome from which the
messenger RNA (mRNA) is generated. Additionally, the virion contains some cRNA
of the S ambi-sense segment which leads to an early transcription and replication of
the nonstructural protein NSs immediately after infection [47, 53]. General replication
of viral proteins starts around one to two hours after the infection of the cells. Each
segment contains specific untranslated regions (UTRs) at the 5’ and 3’ ends which
serve as promoters for transcription and replication by the L protein. Transcription of
viral mRNA utilizes a cap-snatching strategy, by which mRNA of the host is cleaved
and the fragments are used as primers to synthesize viral mRNA [47]. Accumulation
of produced N and L proteins initiate further RNA replication. Assembly and budding
of viral particles take place in the Golgi apparatus of infected cells, supported by
interactions between Gn proteins and encapsidated genomic segments. Fusion of
the vesicles with the Golgi apparatus and then with the cell membrane results in the
release of the RVFV particles [52].
2.6.2. Virulence factors
NSs protein, the major virulence factor of the RVFV, forms filamentous structures in
the nucleus of infected cells. This feature is unique for RVFV and is not shared with
other Bunyaviruses [54-56]. NSs protein is a multifunctional protein which interferes
with the host innate immune response to viral infections. As a main strategy, the NSs
protein suppresses the general host mRNA synthesis by interacting with the p44
subunit of the basal transcription factor complex IIH (TFIIH) and the TFIIH p62 as
well as by promoting the degradation of the RNA-dependent protein kinase [57-59].
Furthermore, the NSs protein counteracts the antiviral interferon (IFN) system [59,
60]. The NSs protein binds specifically to Sin3A-associated protein (SAP30), a
subunit of the Sin3A complex which regulates the gene expression. The formation of
this complex results in the suppression of the transcriptional activation of IFN-ß
promotor [57, 61].
NSm is an additional virulence factor of the RVFV and the only phleboviral NSm
which acts as an anti-apoptotic regulator. It suppresses the apoptosis of infected host
14
cells by inhibiting the cleavage of caspase-8 and caspase-9, components of the
mammalian apoptotic pathway [62]. Moreover, there is evidence that NSm plays an
important role in the transmission of the virus to mosquitos by reason of a lower
infection rate of mosquitos when infecting with mutants which lack the NSm protein
[63].
2.7. Vaccination
RVFV shows a relatively low genetic diversity and infection induces humoral
immunity leading to protection against fatal courses of RVF [7, 36]. These
characteristics enable the development of vaccines to protect animal and human
health and to impede viral spread. Although many vaccines for animals were
developed in the past, no licensed vaccines for human use are yet available.
Vaccination of susceptible livestock is therefore the method of choice to protect
humans and livestock from the severe consequences of a RVFV infection [9, 64].
2.7.1. Live-attenuated vaccines
The first available animal vaccine, the Smithburn live attenuated vaccine, was
developed by serial passages of the Entebbe strain in mice in 1949. The Entebbe
strain was originally isolated from mosquitos in western Uganda in 1944. Since 1952
the Smithburn vaccine was used in South Africa, since 1960 in Kenya and also in
Egypt [4, 34, 35, 65-67]. Live-attenuated vaccines provide a long-lasting immunity
directly after the first immunization [68]. However, the Smithburn vaccine exhibits
residual pathogenic effects for gestating and newborn livestock, which is associated
with foetal abnormalities or abortions [4, 34, 69]. Moreover, despite the attenuation of
this strain, a reversion to the original pathogenic strain could not be excluded.
Therefore, the Smithburn vaccine should be used in endemic areas of RVF only [70,
71] as recommended by the FAO. Hence, it is the most widely used vaccine in Africa
[68].
In order to generate a highly attenuated and safe live vaccine, the RVFV MP12
vaccine was developed. The pathogenic strain ZH548, isolated from an Egyptian
human case in 1977, was attenuated by 12 passages in the presence of the mutagen
5-fluorouracil. Chemical mutagenesis resulted in mutations in all three segments of
15
the genome [34, 35, 67, 70, 72-75]. A single dose of this vaccine already induces a
safe and efficacious immunity in sheep and cattle. In addition, the RVFV MP12
vaccine strain is currently being examined to prove its efficacy and safety as a human
vaccine [34]. Divergently, some studies demonstrate apoptotic properties of the
RVFV MP12 strain [3, 67, 75].
An additional strain of RVFV, the Clone 13 is naturally attenuated by the lack of 70%
of the coding sequence for the NSs protein [76]. This strain is highly immunogenic in
mice, sheep and cattle without showing pathogenic effects. The related RVFV strain
74HB59 was isolated 1974 in the Central African Republic from a human case and
was further analyzed by Muller and colleagues who found out that an individual clone
was highly immunogenic in mice and hamster. So far, the Clone 13 is the only live
attenuated vaccine without inducing abortion and is therefore a promising vaccine
candidate [34, 35, 66, 67, 70].
2.7.2. Inactivated vaccines
Inactivated RVFV vaccines were developed in order to limit the possible
transformation of live attenuated vaccine strains into pathogenic mutants. The first
inactivated vaccine, based on the Entebbe strain was inactivated by formalin [34, 70].
Alternatively, other chemical substances or heat are used for inactivation of the virus.
These vaccines are safe but the application in the field is inconvenient. To ensure a
sufficient immunity level the vaccine needs to be primed twice and boosted annually.
Adjuvants like aluminum hydroxide are used to increase the immunogenicity of
inactivated vaccines [34, 35, 66, 67, 70, 71].
2.7.3. Recombinant vaccines
Recombinant vaccines comprise a group of promising vaccine candidates which
include viral constituents to induce the production of neutralizing and therewith
protective antibodies in immunized animals. Various methods for the expression of
viral proteins are available. Recombinant vaccines offer the possibility to distinguish
between wild-type infections and vaccine associated immune responses (DIVA).
Virus like particles (VLPs) are replication-deficient viral particles which consist of the
viral envelope and the nucleoprotein. VLPs express the immunogenic components of
16
the virus and induce protective antibody titers yet pose less of a risk than the use of
fully functional virus particles. They are promising candidates but thus far, difficulties
with the large-scale production of VLPs limit the application of these candidates [15,
34, 70, 71, 75].
Another approach is the production of RVFV derived proteins, especially
glycoproteins, in host cells via viral vectors like capripoxviruses, Newcastle disease
virus or modified vaccinia virus Ankara. Processing of the glycoproteins in host cells
induces an immune response and results in the production of neutralizing antibodies.
The Lumpy skin disease virus, combined with RVFV glycoproteins, may bring about a
protection against both viral diseases. These candidates are still being refined but
are already considered to be promising vaccine alternatives for the future [15, 34, 64,
70, 75, 77].
Similar to the vector based vaccination, the immunization with DNA vaccines induces
the expression of recombinant proteins followed by the formation of an immunity
against wild-type RVFV [15, 34, 64, 67, 70, 75].
2.7.4. Vaccination in Egypt
Shortly after the devastating epizootic in 1977, Egypt focused on the production of
effective vaccines. In 1980, an inactivated RVF vaccine based on the ZH501 isolate,
a RVFV strain which was isolated from a human patient during the 1977´ outbreak in
Zagazig in the Sharqia governorate, was generated. ZH501 was passaged twice
through the brain of suckling mice and combined with the adjuvant aluminum
hydroxide gel. This vaccine is safe and various test trials confirmed its efficacy.
Various studies, using different adjuvants, are carried out to optimize the immune
response to this vaccine. Interim vaccination of livestock with the Smithburn vaccine
was insufficient and lead to further RVF outbreaks in the country [8, 68, 78].
Therefore, the ZH501 formalin-inactivated vaccine is the preferred vaccine in Egypt
to vaccinate all susceptible livestock in well managed farms twice per year. The
nation produces a total of 14 million doses per year [8, 68, 79-81].
17
2.8. Detection, treatment, protection and surveillance
2.8.1. Detection
RVFV can be detected directly through the isolation of the virus from cell culture or
alternatively by the intracerebral inoculation of suckling mice [82]. RNA isolation
followed by quantitative real-time reverse transcriptase polymerase chain reaction
(RT qPCR) is the method of choice for rapid detection of viral genomes. RNA can be
isolated from tissues including liver, brain, spleen, aborted fetuses as well as from
lymph nodes or body fluids like serum, whole blood or plasma from infected animals
or patients [3, 34, 35, 82]. ELISA enable the identification of viral antigens in blood or
serum samples. Immunhistopathology and pathological examinations of tissues can
also be used for the detection of RVFV infections [35, 82].
Serological tests are used to detect specific antibodies generated against RVFV
proteins after infection or immunization. The virus neutralization test (VNT) is able to
identify neutralizing antibodies as early as three days after infection. VNT, as the gold
standard, is highly specific and cross reactions to other Phleboviruses are unlikely.
ELISA and hemagglutination inhibition tests can detect antibodies six or seven days
after infection. Further serological tests include the immunofluorescence assay, the
complement fixation and the immunodiffusion [9, 34, 35, 82].
2.8.2. Treatment
Currently, no licensed therapeutics for the treatment of humans and animals are
available. However, in recent animal experiments Favipiravir (T-705) was
demonstrated to protect against peracute Rift Valley fever virus infection and to
reduce delayed-onset of the neurologic disease [83]. In case of a RVF supported
care is highly recommended [3, 9].
2.8.3. Prevention and control
RVFV can only be successfully controlled through a close interaction of agricultural,
entomological, veterinary and medical efforts [3]. Individual efforts to prevent the
infection of humans and animals play an important role during high risk periods.
Humans can protect themselves by wearing long clothes or using repellents to limit
the exposure to mosquitos. Wearing protective equipment when handling blood or
18
other samples from animals can also reduce the risk of contamination. Animals can
likewise be protected from the exposure to mosquitos by using repellents or by indoor
housing of the animals. Moreover, in order to protect humans, preventive vaccination
of livestock is essential as these animals play a key role in the life circle of RVFV.
Restrictions of animal movements can also prevent further viral spread within and
outside affected areas. Actions to regulate the mosquito population should target the
larval stages at identified breeding sites. Treatments against adult mosquitos are
expensive and difficult to implement [3, 84, 85]. Early detection of the virus through
the monitoring of viral activities utilizing sentinel herds or through hospital based
surveillance of human infections may prevent large scale outbreaks. Moreover, the
monitoring and assessment of rainfall rates can help to predict high risk periods in
which specific precautions should be applied to prevent animal and human infection
[3, 84-89].
2.8.4. Surveillance in Egypt
Egypt implemented a continuous monitoring program for sheep, goats, cattle and
camels. Furthermore, governorates adjacent to other countries like Sudan
implemented the monthly testing of livestock. Animals which are intended for
importation are also tested for the presence of IgG and IgM antibodies. Control of the
movement of animals into and throughout the country is also part of the Egyptian
disease control program [68, 90]. As mentioned above, Egypt is the only African
country which performs a biannual vaccination of all susceptible sheep, cattle and
camels with an inactivated vaccine based on the ZH 501 [68, 81]. Hospital based
early warning systems which can detect an increase in the number of febrile illnesses
enable rapid diagnosis of human RVF cases [31].
19
Chapter 3: Seroprevalence of Rift Valley fever virus in
livestock during inter-epidemic period in
Egypt, 2014/15
Manuscript I
C. Mroz, M.Gwida, M. El-Ashker, M. El-Diasty, M. El-Beskawy, U. Ziegler, M. Eiden
and M.H. Groschup
Abstract
Background: Rift Valley fever virus (RVFV) caused several outbreaks throughout
the African continent and the Arabian Peninsula posing significant threat to human
and animal health. In Egypt the first and most important Rift Valley fever epidemic
occurred during 1977/78 with a multitude of infected humans and huge economic
losses in livestock. After this major outbreak, RVF epidemics re-occurred in irregular
intervals between 1993 and 2003. Seroprevalence of anti-RVFV antibodies in
livestock during inter-epidemic periods can be used for supporting the evaluation of
the present risk exposure for animal and public health. A serosurvey was conducted
during 2014/2015 in non-vaccinated livestock including camels, sheep, goats and
buffalos in different areas of the Nile River Delta as well as the furthermost southeast
of Egypt to investigate the presence of anti-RVFV antibodies for further evaluating of
the risk exposure for animal and human health. All animals integrated in this study
were born after the last Egyptian RVF epidemic in 2003 and sampled buffalos and
small ruminants were not imported from other endemic countries.
Results: A total of 873 serum samples from apparently healthy animals from different
host species (camels: n =221; sheep: n = 438; goats: n =26; buffalo: n = 188) were
tested serologically using RVFV competition ELISA, virus neutralization test and/or
an indirect immunofluorescence assay, depending on available serum volume. Sera
were assessed positive when virus neutralization test alone or least two assays
produced consistent positive results. The overall seroprevalence was 2.29% (95%CI:
1.51–3.07) ranging from 0% in goats, 0.46% in sheep (95%CI: 0.41–0.5), and 3.17%
in camels (95%CI: 0.86–5.48) up to 5.85% in buffalos (95%CI: 2.75–8.95).
20
Conclusion: Our findings assume currently low level of circulating virus in the
investigated areas and suggest minor indication for a new RVF epidemic. Further the
results may indicate that during long inter-epidemic periods, maintenance of the virus
occur in vectors and also most probably in buffaloes within cryptic cycle where
sporadic, small and local epidemics may occur. Therefore, comprehensive and well-
designed surveillance activities are urgently needed to detect first evidence for
transition from endemic to epidemic cycle.
BMC Veterinary Research, 2017 Apr 5; 13(1):87
doi: 10.1186/s12917-017-0993-8
21
Chapter 4: Rift Valley fever virus infections in Egyptian
cattle and their prevention
Manuscript II
C. Mroz, M. Gwida, M. El-Ashker, U. Ziegler, T. Homeier-Bachmann, M. Eiden and
M.H. Groschup
Abstract
Rift Valley fever virus (RVFV) causes consistently severe outbreaks with high public
health impacts and economic losses in livestock in many African countries and has
also been introduced to Saudi Arabia and Yemen. Egypt with its four large outbreaks
in the last 40 years represents the northernmost endemic area of RVFV. The purpose
of this study was to provide an insight into the current anti-RVFV antibody status in
immunized as well as non-immunized dairy cattle from the Nile Delta of Egypt. During
2013-2015, a total of 4,167 dairy cattle from four governorates including Dakahlia,
Damietta, Gharbia and Port Said were investigated. All cattle were born after 2007
and therewith after the last reported Egyptian RVFV outbreak in 2003. The samples
derived from vaccinated animals from 26 different dairy farms as well as non-
immunized cattle from 27 different smallholding flocks. All samples were examined
following a three-part analysis including a commercially available competition ELISA,
an in-house immunofluorescence assay and a virus neutralization test. Additionally, a
subset of samples was analyzed for acute infections using IgM ELISA and real-time
reverse transcriptase PCR. The results indicated that the RVFV is still circulating in
Egypt as about 10% of the non-immunized animals exhibited RVFV-specific
antibodies. Surprisingly, the antibody prevalence in immunized animals was not
significantly higher than that in non-vaccinated animals which points out the need for
further evaluation of the vaccination program. Due to the substantial role of livestock
in the amplification and transmission of RVFV, further recurrent monitoring of the
antibody prevalence in susceptible species is highly warranted.
Transboundary and emerging diseases, 2017 Jan 24; 1–10
doi: 10.1111/tbed.12616
22
Chapter 5: Comparative expression kinetics of five
different Rift Valley fever proteins in
mammalian and insect cells
Manuscript III
C. Mroz1, K.M. Schmidt1, S. Reiche2, M.H. Groschup1, M. Eiden1
1 Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Greifswald - Isle of
Riems, Germany
2 Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut,
Greifswald - Isle of Riems, Germany
This manuscript was submitted to Virology Journal (under review)
23
5.1. Abstract
Background: Rift Valley fever is still an emerging disease with a high impact in
public health and economic aspects. Climate changes and globalization increase the
risk of spreading the Rift Valley fever virus (RVFV) to countries outside of known
endemic areas. The virus circulates within an enzootic cycle between mosquitoes
and largely unknown wild life hosts before abruptly spreading via amplifying vectors
to domestic ruminants. The switch between enzootic and epizootic cycles is mainly
triggered by unusual and heavy rainfalls leading to an explosive increase of
competent mosquito species. The contribution of multiple host and vector species
during both cycles requires divergent amplification and maintenance strategies that
have not been fully explored and described on the molecular level.
Methods: We have investigated the expression kinetics of five different RVFV
proteins in vertebrate (Vero76) and mosquito-derived (C6/36) cells after infection with
RVFV MP12 vaccine strain. Expression dynamics were monitored by Western Blot
analysis and indirect immunofluorescence using monoclonal antibodies (mabs)
directed against the nucleocapsid protein (NP), the glycoproteins Gn and Gc, as well
as newly generated mabs against the nonstructural proteins NSs and NSm.
Results: Our results demonstrate significant differences of these detected viral
proteins in their expression levels, accumulation and distribution patterns in
vertebrate compared to mosquito-derived cells. Most intriguing differences were
observed for the nonstructural NSs protein, which formed filamentous structures
within the nucleus of infected mammalian cells. In mosquito cells, NSs appeared in
homogenous distribution throughout the cytoplasm. Small dots within the nucleus
only developed in late stage of infection.
Conclusions: Together these results reflect the discrepancies of virus replication
and interaction within the mammalian host compared to the mosquito vector.
Greatest differences in protein expression, distribution and accumulation are related
to proteins with major significance for the replication strategy of the virus as
demonstrated for NSs in Vero 76 cells and for NSm in C6/36 cells.
24
5.2. Background
Rift Valley fever (RVF) is an emerging zoonotic disease caused by Rift Valley fever
phlebovirus [3] out of the novel designated family Phenuiviridae, order Bunyavirales
[91]. Widely spread throughout many African countries as well as the Arabian
Peninsula the corresponding Rift Valley fever virus (RVFV) poses an important public
health threat. It occurs in irregular intervals and is characterized by fever, hepatitis,
neonatal mortality, and abortions in livestock mainly in sheep [3, 4]. In humans the
disease is often characterized by a self-limited, flu-like illness. Manifestations of the
disease, including hemorrhagic fever syndromes, ocular disease or encephalitis can
be observed in severe forms in 1-2% of cases [3, 36, 37]. RVFV can be transmitted
by more than 30 mosquito species out of six genera mainly to ruminants and camels,
which serve as amplifying vectors [9]. During inter-epidemic periods the virus
circulates at low levels in mosquito vectors and is transmitted transovarially by
infected Aedes floodwater mosquitos [32]. A transition to an epidemic cycle can be
initiated by heavy rainfalls accompanied by a subsequent increase of the infected
mosquito population leading to increased virus transmission to susceptible hosts as
well as a broad amplification by secondary arthropod vectors like Culex or Anopheles
species [3, 32].
Similarly to all members of the order Bunyavirales the RVFV is an enveloped RNA
virus with a three segmented genome in negative or ambisense polarity [47]. The L
segment encodes for the RNA-dependent RNA polymerase L. The M segment
contains the genetic information for the glycoproteins Gn and Gc and two accessory
proteins, a 14 kDa nonstructural protein (NSm) and a differentially expressed 78 kDa
protein [3, 4, 35, 47]. The glycoproteins are highly immunogenic and induce the
formation of neutralizing antibodies in infected hosts [75]. The nonstructural protein
NSm serves as a virulence factor by suppressing the virus-induced apoptosis of host
cells [62]. NSm might also play an important role in the mosquito vector, since
mutants lacking the NSm protein showed a lower infection rate in mosquitos [63]. The
role of the 78 kDa protein is still not fully understood but it might also play a role
during virus replication in mosquitos [92, 93]. The S segment utilizes an ambisense
strategy. In negative orientation the S segment encodes for the nucleoprotein, which
25
forms ribonucleoproteins associated with the RNA-dependent RNA polymerase L.
Another nonstructural protein, the NSs is encoded in antisense orientation [36]. As a
major virulence factor of RVFV the NSs interferes with the host innate immune
response. This includes blocking interferon-ß (IFN-ß) gene expression through
different pathways, downregulation of the protein kinase R and by interacting with the
host cell transcription factor TFIIH in vertebrates [58, 59, 94]. Together this results in
facilitating viral replication and proliferation of infected cells. During infection, NSs
forms filamentous structures in the nucleus of infected cells by interacting with
regulatory DNA regions leading to chromosome cohesion and segregation defects
[57, 95, 96]. However, in insect cells the function and distribution of these viral
proteins is not fully understood. Therefore, the question arose whether these proteins
show differences in their expression and localization indicating differences in their
function during infection in mammalian hosts and mosquito vectors. To investigate
this further, we undertook a comparative expression study of RVFV proteins in
mammalian Vero 76 and Aedes-derived C6/36 cells infected with the attenuated
RVFV vaccine strain MP12. These studies were performed with indirect
immunofluorescence assays and immunoblotting utilizing monoclonal antibodies
(mabs) against NP, Gn, Gc as well as newly generated monoclonal antibodies
against NSs and NSm.
26
5.3. Material
5.3.1. Cells and virus
The SP2/0 myeloma cells, the mammalian Vero 76 cells, and the insect C6/36 cells
were obtained from the Collection of Cell Lines in Veterinary Medicine, Friedrich-
Loeffler-Institut, Germany. The SP2/0 myeloma cells were grown in RPMI 1640
media (Gibco, life technologies, Thermo fisher, Germany) at 37°C in vented flasks.
The Vero and C6/36 cell lines were grown in minimal essential media with 10% fetal
bovine serum (FBS). Vero cells were incubated at 37°C and C6/36 cells at 28°C in
closed flasks. The RVFV MP12 vaccine strain was kindly supplied by Richard Elliot
(University of Glasgow, Center for virus research) and was handled under BSL-2
conditions. Virus titer was determined as 50% tissue culture infective doses (TCID50)
using Vero 76 which yielded a titer of 106,9 TCID50/ml. Calculation was carried out by
the Spearman-Kärber method [97, 98].
5.3.2. Expression of recombinant NSs and NSm protein
RNA was isolated from cell culture supernatant from RVFV MP12-infected cells using
a QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer ‘s
instructions. The genes encoding the nonstructural protein NSs and NSm of RVFV
MP12 were amplified by a one-step RT PCR. The amplification of the gene encoding
the NSs protein was performed from the S segment with NSS_3 (5´-
CCGGATCCGATTACTTTCCTGTGATATCTG-3´) as forward primer and NSs_4 (5´-
CCAAGCTTCTAATCAACCTCAACAAATC-3´) as reverse primer. These primers
contain a BamH1 and a HindIII restriction site, respectively. The gene encoding the
NSm protein was amplified from the M segment using NSm_4 (5´
CCGAATTCATTATTAGAGTGTCTCTAAGCTCC-3´) as forward primer and NSm_5
(5´-CCCTCGAGAGCAAAAACAACAGGTGCCAAAGC-3´) as reverse primer. These
primers contain an EcoRI and XhoI restriction site, respectively. All primers were
synthesized and HPLC purified by Eurofins MWG Operon (Ebersberg, Germany).
Amplification by one-step RT PCR was performed using a Super Script III One-Step
RT PCR with Platinum Taq Kit (Invitrogen, USA) according to the manufacturer ‘s
instructions. The amplified NSs and NSm genes were sub-cloned into pBluescriptK/S
27
vector (Agilent Technologies, Denmark) and amplified in XL1-blue Escherichia coli
cells (Invitrogen). The NSs gene was subsequently cloned into the bacterial
expression vector pQE40 (Qiagen) via the restriction sides BamH1 and HindIII (New
England Bio Labs, Germany). The restriction sides XhoI and EcoR1 (New England
Bio Labs) were used to clone the NSm gene into the bacterial expression vector
pET21a (Novagen, UK). The corresponding clones were designed as NSspQE40 and
NSmpET21a, respectively. Expression and purification of recombinant RVFV NSs
and NSm proteins were carried out under denaturing conditions as described
previously by Jäckel [99].
5.3.3. Immunization of BALB/c mice and hybridoma cell preparation
Four BALB/c mice per antigen were immunized intraperitoneally with 100µg of
recombinant NSs or NSm protein on days 0, 30, 60 and 90. A final boost with 100µg
protein solution was carried out three days before spleen cells were fused with SP2/0
myeloma cells in a ratio of 1:4 in the presence of polyethylene glycol 1500 (PEG,
Sigma-Aldrich). Hybridoma cells were selected in RPMI 1640 media (Gibco, life
technologies, Thermo fisher, Germany) with hypoxanthine-aminopterin-thymidine
(HAT) selective medium (Sigma-Aldrich) and 10% foetal calf serum (life
technologies), BM Condimed H1 (Hybridoma cloning supplement, Sigma-Aldrich)
non-essential amino acids (life technologies), L-glutamine (200mM, life technologies),
penicillin (10000 units, life technologies), streptomycin (10mg/ml, life technologies),
and sodium pyruvate (100mM, life technologies). Stepwise the HAT medium was
replaced by hypoxanthine-thymidine (HT) medium (Sigma-Aldrich) followed by
maintenance media. Supernatants of hybridoma clones were screened for anti-NSs
and anti-NSm antibodies by indirect ELISA followed by characterization by western
blotting and indirect immunofluorescence. The antibody classes were determined
using the commercial Pierce Rapid ELISA Mouse mAb Isotyping Kit (Thermo Fisher)
according to the manufacturer’s instructions.
5.3.4. Enzyme-linked immunosorbent assay (ELISA)
Mouse polyclonal serum and hybridoma supernatants were assessed with an indirect
ELISA previously published by Jäckel [100]. In short, Maxisorb immunoplates (Nunc,
Denmark) were coated overnight at 4°C with 1µg/ml recombinant NSs or NSm
28
protein. After blocking with 10% skim milk (Difco, BD, USA) the plates were washed
three times with PBS containing 0.1% Tween 20 and the hybridoma supernatants,
solved 1:1 in PBS were added for 1h at 37°C. Mouse polyclonal serum was diluted
1:25 in PBS. After washing as before a horseradish peroxidase-conjugated goat-anti-
mouse second antibody (Jackson Immunoresearch, USA) was added and incubated
equally. After washing the substrate ABTS (2,2’-azino di ethylbenzothiazoline sulfonic
acid, Roche) was added, incubated in the dark for 30 minutes and the reaction was
stopped with 1% SDS. The optimal density was measured at 405nm with a Tecan
ELISA reader. Hybridoma clones with antibody titers higher than 0.5 (OD405nm) were
selected for further use.
5.3.5. SDS Page and western blotting
Recombinant viral proteins (Gn, Gc, NP, NSs and NSm), and proteins of the
supernatant and cell lysate of RVFV MP12-infected Vero 76 and C6/36 cells were
analyzed by SDS gel electrophoresis in a 16% acrylamide gel. 20µl of each protein
fraction was mixed with 20µl loading buffer (1% SDS, 25 mM Tris/HCl pH 7.4, 0.5%
mercaptoethanol, 0.001% bromophenol blue) and incubated at 95°C for 5 min before
loading the acrylamide gel. Staining was performed with a staining solution
containing 0.25% Coomassie Blue, 10% acetic acid, and 50% methanol for 30 min
and discoloration with 10% acetic acid plus 50% methanol.
Gn, Gc, NP, NSs and NSm recombinant proteins, as well as the proteins of the
supernatant and cell lysate of RVFV MP12-infected Vero 76 and C6/36 cells were
further analyzed by Western Blot analysis. The proteins were transferred from an
acrylamide gel onto PVDF membranes by semi-dry electroblotting. After blocking 30
minutes at room temperature (RT) with 5% skim milk (Difco) in PBS/0.1% Tween 20
the membranes were incubated for 1h at RT with corresponding monoclonal
antibodies. The membranes were than washed three times for 10 min in PBS/0.1%
Tween 20 and a second antibody, an alkaline phosphatase-conjugated goat-anti-
mouse antibody (Jackson Immunoresearch) diluted 1:2000 was incubated for 1 hour.
Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) (Thermo Fisher) served as
loading control for the infected cell lysates and was detected with an anti-rabbit
alkaline phosphatase-conjugated secondary antibody (diluted 1:2500; Jackson
Immunoresearch). After a second washing step, the proteins were visualized by
29
chemiluminescence detection with CDP-Star substrate (Tropix, Sigma-Aldrich) and
Versadoc Imaging system and Image Lab Software (both Bio Rad, USA),
respectively.
5.3.6. Infections
1x105 cells of Vero 76 or C6/36 cells were respectively seeded on 10mm glass
coverslips in 10cm cell culture dishes with an amount of 10ml per dish or in 96 well
cell culture plates with an amount of 100µl per well and were infected at a confluency
of 80% with the RVFV MP12 strain. For is purpose supernatants were removed and
RVFV MP12 was added to the wells at a multiplicity of infection (MOI) of 1 in
minimum essential medium containing 2% FBS, penicillin (100U/ml) and
streptomycin (100µg/ml). Cell culture dishes were filled with an amount of 5ml virus
suspension whereas the 96 cell plates were filled with 100µl per well. Control cells
(MOK) were filled with maintenance medium (minimum essential medium containing
2% FBS, penicillin (100U/ml) and streptomycin (100µg/ml)). After 1h incubation at
37°C/28°C and 5% CO2, 50µl maintenance medium was added to each well followed
by an incubation for 24h at 37°C/28°C and 5% CO2. After the incubation,
supernatants were discarded and wells were covered with 4% PFA
(Paraformaldehyde, Sigma-Aldrich). Slight modifications were made when handling
dishes: the virus was discarded after one hour of infection, the dishes were washed
twice with PBS and the coverslips were transferred into 4% PFA after indicated time
points. Residual cells were collected and centrifuged at 1600 rpm, 12°C.
Supernatants were harvested and stored at -20°C. The cells were lysed with lysis
buffer containing 50mM Tris, 10mM NaCl, 0.1% Tween-20 and 5mM EDTA in a ratio
of 1:1 and also stored at -20°C.
5.3.7. Indirect immunofluorescence assay (IFA)
For IFA, fixed cells were washed three times with PBS and permeabilized with PBS
containing 0.1% Triton X (Sigma-Aldrich) for 10 minutes at RT. After washing, 0.1M
Glycine in PBS (Carl Roth, Germany) was added to the cells for 10 min at RT. After
the incubation, cells were washed and incubated in blocking reagent (PBS containing
20% bovine serum albumin, Merck Millipore, UK; 0.2% Tween-20, Sigma-Aldrich;
30% Glycerine, Carl Roth and 0.02% sodium azide, Carl Roth) for 30 min.
30
Subsequently, hybridoma cell supernatants dissolved in blocking reagent were added
and incubated for 1h at RT followed by washing three times for 5 minutes with PBS.
The secondary antibody Cy3(indocarbocyanine)-labeled goat anti-mouse IgG
antibody (1:500, Jackson Immunoresearch) and DAPI (1:20000, Carl Roth) diluted in
blocking reagent were added to the cells and incubated for one hour. After the final
washing step, 50µl PBS was added to each well of the 96 well plates and the
fluorescence staining was evaluated by fluorescence microscopy (Nikon, Japan). For
the coverslips, each cover slip was mounted on slides using ProLong Antifade
Mounting medium (life technologies, Thermo Fisher), dried overnight and read on the
next day.
5.4. Results
5.4.1. Generation of monoclonal antibodies against NSs and NSm
BALB/c mice were immunized with recombinant, bacterial expressed NSs and NSm
proteins, which displayed a molecular mass of 29 kDa and of 14 kDa,
correspondingly (Fig.5.S1.). Subsequently, 125 days after the first immunization, the
spleens were fused and hybridoma cells were generated. Hybridoma cell
supernatants were screened for the production of monoclonal antibodies against
RVFV NSs or NSm protein using recombinant NSs or NSm as antigen in an indirect
ELISA.
Further characterization of ELISA-positive clones was run by Western Blot analysis
with bacterial expressed NSs/NSm as antigen as well as with lysates of RVFV MP12-
infected Vero cells. Finally, the ELISA-positive cell clones were inspected by IFA in
RVFV MP12-infected Vero cells followed by isotyping (Tab.5.1.). In total eight specific
anti-NSs hybridoma cell clones and seven specific anti-NSm hybridoma cell clones
were identified (Tab.5.1.).
5.4.2. Comparative viral protein expression in Vero 76 cells and C6/36 cells
Mammalian Vero 76 cells (Chlorocebus sabaeus) and mosquito C6/36 cells (Aedes
albopictus) were infected with RVFV MP12 vaccine strain at a MOI of 1. Monitoring of
viral protein expression was conducted using three mabs directed against structural
proteins (Gn_3, Gc_5B10 and NP_9) from previous studies [99, 100] as well as two
31
mabs directed against the nonstructural proteins (NSs_5B7A1B2 and NSm_1E9A2)
from this study (described above).
To analyze early protein expression, Vero 76 cells were infected with RVFV MP12
and were checked regularly from 2 hours post infection (hpi) up to 48 hpi. The first
infected cells showing cytopathic effects (CPEs) were observed after 24 hpi and CPE
was extensive at 48 hpi. In contrast CPE was not observed in infected mosquito cells
at any time point even when the incubation time was extended to 96 hpi.
Using immunofluorescence analysis, the Gn protein was detected as early as two hpi
forming small spots close to the nucleus of the infected Vero 76 cells (Fig.5.1.). At the
same time point, Gc appeared in a diffuse distribution. The nucleoprotein was first
observed homogenously distributed throughout the cytoplasm at 3 hpi. After 4 hpi the
nonstructural protein NSm was detected accumulated in the cytoplasm around the
nucleus, while the NSs protein accumulated in small dots in the nucleus. Figure 1
gives an overview about initial of viral protein expression in RVFV MP12 infected
Vero 76 as well as infected C6/36 cells. At later time points increased expression
levels were observed for all five viral proteins using IFA. Proteins were best visible in
staining at 10 hpi and 48 hpi (Fig.5.2.). Especially the NSs protein was expressed at
high levels and formed specified filamentous structures within the nucleus of infected
Vero 76 cells (Fig.5.3.).
In total the viral protein expression of the glycoproteins could be detected after two
hpi of RVFV MP12 infected Vero 76 cells followed by the expression of the
nucleoprotein and the two nonstructural proteins NSs and NSm.
In RVFV-infected mosquito cells expression of structural and nonstructural proteins
were found as early as 6 hpi using IFA (Fig.5.1.). Glycoproteins Gn and Gc, the
nucleoprotein and the nonstructural proteins NSm and NSs accumulated in the
cytoplasm (Fig.5.2.). The NSm protein was found predominantly close to the nucleus
of infected cells whereas the nucleoprotein was distributed more homogenously in
the cytoplasm at all time points during infection. Interestingly, in infected mosquito
cells the NSs protein was detected scattered throughout the cytoplasm at 10 hpi. This
distribution patterns in mosquito and mammalian cells were distinct since the NSs
protein was detected predominantly in the nucleus of Vero cells (Fig.5.3.). At later
stages of mosquito cell infection (up to 72h), visualization of the NSs protein signals
became weaker and it was found accumulated in small, discrete dots (Fig.5.3.).
32
Taken together positive staining was observed as early as 6 hpi in RVFV MP12-
infected C6/36 cells when viral proteins accumulated in the cytoplasm. In contrast to
mammalian cell infection NSs protein formed only small, discrete dots at 16 hpi
without formation of comparatively filamentous structures at any time point of
mosquito cell infection.
During the course of infection, an increase in viral protein expression was detected in
both, infected mammalian cells and mosquito cells using Western Blot analysis
(Fig.5.4.). In Vero 76 cells, detection of the viral nucleoprotein was possible from the
beginning at 2 hpi and peaked at 48 hpi. Respective detection of Gn, Gc, NSs and
NSm was possible after 6-8 hours. However, in C6/36 cells the detection of these
proteins was only possible at later times, starting at 10 hpi for NP and Gn. Detection
of Gc, NSs and NSm in infected mosquito cell lysate started at 24 hpi.
In Vero 76 cell lysates, the viral proteins were detected at early time points after
infection. NP showed the most intense signals followed by NSs and Gn. In contrast,
detection of viral proteins in mosquito cell lysates was possible beginning at 10 hpi.
Likewise, NP and Gn were the proteins with the most intense signals at the end of
the time course (96 hpi in case of mosquito cells).
The corresponding immunofluorescence staining of all time points is shown in Fig
5.S2, indicating that all cells were infected. Taken together these results highlight the
differences of MP12 virus replication and interaction within the mammalian host and
the mosquito vector. Greatest differences in protein expression, distribution and
accumulation are related to proteins with major significance for the replication
strategy of the virus as demonstrated for NSs in Vero 76 cells and for NSm in C6/36
cells.
Lastly, to assess virus secretion in these two cell lines, the supernatants of infected
Vero 76 and C6/36 cells were analyzed by Western Blot analysis. In mammalian
cells, the structural proteins NP, Gn and Gc were detected in the supernatant at 24
hpi. In contrast, in mosquito cells these structural proteins were detected in
supernatant at 48 hpi (Fig.5.5.).
5.5. Discussion
Although mosquitoes are crucial for transmission and maintenance of the RVFV, little
is known about the virus replication cycle in arthropod cells and the differences within
33
mammalian cells. We therefore analyzed RVFV distribution, accumulation as well as
dissemination of structural and nonstructural viral proteins in both cell types by using
a newly established immunofluorescence assay (IFA) and Western Blot analysis.
Infection studies of mammalian host cells, Vero76, as well as the Aedes albopictus
derived cell line C6/36 were carried out with an attenuated RVFV strain, RVFVF
MP12. For these assays previously described mabs directed against the
nucleocapsid protein (NP) and both glycoproteins, Gn and Gc were utilized [99, 100]
as well as newly generated mabs directed against the nonstructural proteins NSs and
NSm.
It has been shown, that as soon as 20 minutes post infection viral mRNA can be
detected in Vero E6 cells indicating early translation of viral proteins [53]. In line with
these findings we were able to detect early viral protein expression of the
glycoproteins in infected Vero 76 cells at 2 hpi followed by NP and the nonstructural
proteins NSs and NSm at 3-4 hpi. In mosquito cells, the expression was prolonged by
4 hours which is likely due to the lower incubation temperature.
RVFV-infected mammalian and mosquito cells show high expression levels of
nucleocapsid protein which is a result of its central role in ribonucleoprotein (RNP)
formation and subsequent transport and viral genomic package [101]. High levels of
viral RNPs [102] were visualized by diffuse NP staining throughout the cytoplasm.
During the viral replication cycle, the two glycoproteins are synthesized as large
cytoplasmic precursor polyproteins from the M segment and co-translationally
processed into Gn and Gc during translocation into the endoplasmic reticulum (ER)
[103]. Accordingly, our observations by immunofluorescence analysis showed similar
distribution patterns of both proteins at early stages of infection. These consisted of
punctured structures surrounding the nucleus in Vero 76 and in C6/36 cells. After
dimerization of the two glycoproteins within the ER the proteins are transferred into
the Golgi apparatus for further posttranslational processing. It has been shown that
this is accompanied by an altered distribution pattern of Gn and Gc [48, 104, 105] as
indicated here by homogenous cellular staining. At the 5’ end of the M genomic
segment of RVFV an additional protein, the NSm, is encoded. It is dispensable for
the virus replication in mammalian cells but is essential for the infection and
replication in the insect cells and harbors anti-apoptotic properties [55, 62, 63, 106,
107]. Likewise to Gn and Gc protein the NSm protein displayed a cytoplasmic
34
staining in both cell types due to its location in the ER and its integration into the
adjacent mitochondrial outer membrane [108]. The significance for mosquito cells
correlates with an elevated level of NSm expression in C6/36 cells as shown by
immunoblotting of corresponding cell lysates. Most striking differences, however,
were obtained for NSs protein which represents the main virulence factor of the virus
in mammalian hosts. It has already been reported that different properties are
accompanied by a significant higher expression level of NSs in mammalian
compared to insect derived cell lines [109]. NSs expression in RVFV MP12-infected
Vero cells started as discrete dots in the nucleus from four hours post infection and
gradually assembled into filamentous structure, while in RVFV MP12-infected C6/36
cells no filamentous structures were observed at any time point. Small dots in the
nucleus of infected C6/36 cells emerged at 72 hpi. After 24h around 90% of infected
Vero cells expressed NSs whereas the percentage of NSs in infected C6/36 cells
reached a maximum of around 50% and dropped to approximately 20% of NSs
expressing cells at 72 hpi (data not shown). The high expression and distribution
level of NSs in mammalian cells therefore indicates an efficient mechanism of the
virus to counteract the host anti-viral response [94].
The NSs protein harbors multiple properties to influence the host innate immune
response of vertebrates. This includes down regulation of protein kinase R [110] and
interaction with the basal transcription factor TFIIH [111]. However, main function is
the blocking of the IFN-ß response [58, 59, 94] which plays a central role in the anti-
viral response in mammalian cells but is not a part of the pathogen defense in
mosquitoes [112]. This function is attributed to the filamentous form which might
explain the lack of filamentous structures in C6/36 cells and characterized by the
interaction with SAP 30 (Sin3A Associated Protein 30) and the formation of a
multiprotein repression complex targeting the interferon β promoter [61]. Different
defense mechanisms in insect cells might be the reason to evade fatal consequences
of RVFV infections. An example is the RNA silencing pathway which leads to the
degradation of viral RNAs through viral RNA cleavage by Dicer enzymes [113, 114].
Consistent to previous reports RVFV MP12 related structural proteins were found 24
hpi in supernatant of infected Vero cells [115]. Due to the longer replication time in
mosquito cells, first signs of released virus particles were seen after 48 hours of
infection.
35
5.6. Conclusions
In summary, by monitoring five viral proteins during infection of mammalian and
mosquito cells with RVFV MP12 strain we could determine differences in
dissemination, distribution and filament formation which reflect the discrepancies of
virus replication and interaction with the mammalian host compared to the mosquito
vector. Most pronounced effects were observed for nonstructural proteins NSs and
NSm.
36
5.7. Tables
Table 5.1. Characteristics of produced monoclonal antibodies.
a
mAb NSs name
ELISA Western Blot IFA Ig class
recombinant NSs (E.coli expressed)
recominant E protein of West
Nile Virus (E.coli
expressed)
RVFV proteins
(Gn, Gc, NP, NSm)
recombinant NSs (E.coli expressed)
RVFV - MP12 lysate from
Vero 76 cells
RVFV - MP12 propaged in Vero 76 cells
4C12A2B + - - - - + IgM
2D6C3C + +/- +/- + - ++ IgM
5H8A3B + - - + + +++ IgG1
5H4B1C + - - + + ++ IgA
5C3A1B2 + - - + + +++ IgG1
5B7A1B2 + - - + + +++ IgG1
2A10B1B + - - + + +++ IgG1
5F12C1B3 + - - + + +++ IgG3
b
mAb NSm
name ELISA Western Blot IFA Ig class
recombinant NSm (E.coli expressed)
recominant E protein of West
Nile Virus (E.coli
expressed)
RVFV proteins
(Gn, Gc, NP, NSs)
recombinant NSm (E.coli expressed)
RVFV - MP12 lysate from
Vero 76 cells
RVFV - MP12 propaged in Vero 76 cells
1C11B3C1 + - - + - - IgG2b
1D5A3 + - - + - - IgG2b
3E4A2D + - - + - +++ IgG3
1A4A3A + - - + - + IgG1
1H9B1 + - - + - ++ IgG2b
1E9A2 + - - + - +++ IgG2a
1E4C2 + - - + + +++ IgG1
a anti-NSs hybridoma clones. b anti-NSm hybridoma clones. Cell supernatants from
hybridoma cell clones were screened by indirect ELISA using corresponding recombinant
proteins, a foreign E.coli expressed protein (recombinant West-Nile-Virus envelope protein)
and other RVFV recombinant proteins for analyzing cross reactions among each other. All
ELISA positive clones were tested in western blotting with corresponding recombinant
protein and lysate from RVFV MP12 infected Vero 76 cells. Additional characterization was
carried out with indirect immunofluorescence assay and the determination of the antibody
classes for each clone.
37
5.8. Figures
Figure 5.1. First appearance of RVFV MP12 derived proteins in Vero 76 cells and
C6/36 cells.
Scale bars represent 100µm.
38
Figure 5.2. Distribution of RVFV MP12 derived proteins in Vero 76 cells and C6/36
cells at different time points of infection.
Scale bars represent 100µm.
39
Figure 5.3. Expression of NSs protein in cells infected with RVFV MP12 at a MOI of
1.
Scale bars represent 100µm.
40
Figure 5.4. Western blotting of infected cell lysates on sequential time points.
a from Vero cells. b from C6/36 cells. Lysates of infected cells that were harvested at 10
consecutive time points post infection.
41
Figure 5.5. Protein secretion in supernatants of infected Vero 76 cells (a) and C6/36
cells (b) detected by western blotting.
a b
42
Figure 5.S1. SDS Page analysis and Coomassie blue staining of recombinant NSs
and NSm protein
43
Figure 5.S2. Comparative protein expression in RVFV MP12 infected cells.
Vero 76 cells and C6/36 cells were infected at a MOI of 1 and monitored by
immunofluorescence microscopy course of infection. a Comparative immunofluorescence
staining of MP12 related proteins in infected Vero 76 cells. b Corresponding protein
expression in mosquito C6/36 cells. Scale bars represent 100µm.
44
45
Chapter 6: Comparison of different serological test
systems to detect anti RVFV IgG antibodies
in cattle and camel sera
Manuscript IV
C. Mroz1, K.M. Schmidt1, U. Ziegler1, M. Rissmann1, M. Gwida2, M. El-Ashker3, B.O.
EL Mamy4, K. Isselmou4, B. Doumbia5, Martin Eiden1, Martin H. Groschup1
1 Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Greifswald - Isle of
Riems, Germany
2 Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University,
Mansoura 35516, Egypt
3 Department of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura
University, Mansoura, Egypt
4 Service de Pathologie Infectieuses, Centre National de l'Elevage et de Recherches Vétérinaires
(CNERV), Nouakchott, Mauritania
5 Ministère du développement rural, Nouakchott, Mauritania
46
6.1. Abstract
As a mosquito-borne zoonotic disease the Rift Valley fever (RVF) causes consistently
large outbreaks with great impacts on human and animal health. The introduction of
the Rift Valley fever virus (RVFV) to the Arabian Peninsula in 2000 illustrated the
potential of the virus to spread also to further northern countries. Surveillance
systems with detailed and accurate investigations of the seroprevalence are
necessary to recognize the circulation of the Rift Valley fever virus in endemic and
non-endemic countries. Serological test methods like virus neutralization test,
enzyme linked immunosorbent assays (ELISA) or immunofluorescence assays (IFA)
often show diverging results which are related to the test methods and their different
accuracy. In this study a new in-house immunofluorescence assay based on the
RVFV vaccine strain MP12 was established and compared with the traditional VNT,
the gold standard for RVFV antibody detection and a frequently used commercial
ELISA. This study was aimed at providing the most convenient test for rapid and
easy IgG identification with preferably great test accuracy. Both, the commercial
ELISA and the immunofluorescence test showed results accordant with VNT in
around 80% of tested cattle and camel sera. 71% of the samples showed consistent
results in all three test methods.
47
6.2. Short communication
Rift Valley Fever (RVF), a mosquito-borne disease in humans, ruminants and
camels, is caused by the Rift Valley Fever Virus (RVFV), a Phlebovirus of the family
Phenuiviridae [1, 38, 91]. Infections in livestock are characterized by high fever, acute
hepatitis, abortions and high neonatal mortalities mainly in sheep [1, 32, 38, 116].
Transmissions to humans often result in a flu-like mild febrile illness, however severe
complications including hemorrhagic fever syndrome, encephalitis or ocular disease
may occur [4, 37]. Since its first identification in Kenya in 1931 the RVFV has been
associated with large epidemics throughout south and eastern Africa [3]. In 2000, the
disease was recognized for the first time in Saudi Arabia and Yemen which raised the
concern for a possible spread to northern countries [3]. Due to the great impact of
RVF, surveillance systems were implemented in order to reveal the spread [117].
Detailed and accurate investigations of the seroprevalence based on routine
diagnostic methods are necessary to determine the circulation of RVFV in endemic
countries and during epizootic episodes [118].
Several serological methods are currently used for identification of IgG antibodies in
serum samples. The virus neutralization test (VNT) is highly specific and therefore
still recommended as reference method. Further, the hemagglutination inhibition test
and the enzyme linked immunosorbent assays (ELISA) are frequently used as well
as the immunofluorescence, complement fixation and immunodiffusion assays [82].
This study was aimed to compare the VNT, a frequently used commercial ELISA and
a newly developed immunofluorescence assay in order to find the most reliable and
convenient test for rapid and easy IgG identification.
Cattle serum samples from 493 apparently healthy Holstein Frisian cows as well as
77 camel derived samples were kindly provided by the Mansoura University,
Mansoura, Egypt. Additional camel serum samples (n=26) were kindly provided by
the Centre National de lÉlevage et de Recherches Vétérinaires (CNERV) in
Mauritania. Due to biosafety reasons, all sera were subjected to Gamma irradiation
(irradiation for 24h by 30 kilo gray; Synergy Health, Radeberg) prior to the serological
examinations.
48
All sera were tested by VNT according to the procedure described in the OIE
terrestrial manual, 2015 using Vero 76 cells (Collection of Cell Lines in Veterinary
Medicine, Friedrich-Loeffler-Institut, Germany) and the RVFV MP12 vaccine strain
[82]. Neutralizing antibody titers were expressed according to the Behrens - Kaerber
method as 50% neutralization dose (ND50). Serum samples with ND50 values lower
than 10 were determined as negative while ND50 values above 10 were defined as
positive. Samples with ND50 values higher than 30 were summarized as ≥30 and
were not further diluted.
Moreover, the ID Screen® Rift Valley Fever Competition Multi-species ELISA
(cELISA) was carried out according to the manufacturer’s instructions to detect anti-
RVFV specific antibodies. Samples with a percentage of inhibition lower than 40 were
defined as positive, between 40-50% as questionable and results higher than a
percentage of inhibition of 50 were defined as negative.
A newly developed in-house indirect immunofluorescence assay (IFA) was also
carried out as follows: Vero 76 (1x105 cells) were seeded in 96 well cell culture plates
and were infected at a confluence of 80% with the RVFV MP12 strain at a MOI of 1 in
minimal essential media containing 2% FBS, penicillin (100U/ml) and streptomycin
(100µg/ml) for 24 hours at 37°C and 5% CO2. Control cells were filled with
maintenance media. Afterwards supernatants were discarded and cells were fixed
with 4% PFA (Paraformaldehyde, Sigma-Aldrich) for 30 minutes. Fixed cells were
washed three times with PBS and permeabilized with 0.1% Triton X (Sigma-Aldrich)
in PBS for ten minutes at room temperature (RT). After washing as before 0.1M
Glycine, solved in PBS (Carl Roth, Germany) was added for ten minutes at RT.
Again, the cells were washed three times with PBS and incubated in blocking reagent
(PBS containing 20% bovine serum albumin, Merck Millipore, UK; 0.2% Tween-20,
Sigma-Aldrich; 30% Glycerine, Carl Roth and 0.02% sodium azide, Carl Roth) for 30
minutes. Cattle sera were dissolved in a ratio of 1:50 in blocking buffer and were
added to infected and control cells and incubated for 1h at RT followed by washing
three times for five minutes with PBS. The second antibody, Cy3-labeled donkey anti-
bovine IgG antibody (1:500, Jackson Immunoresearch) diluted in blocking buffer
were added for one hour. After final washing step, 50µl PBS was added to each well
and the fluorescence staining was evaluated with a fluorescence microscope (Nikon,
Japan). For camel sera slight modifications were necessary. The sera were dissolved
49
1:100 and the polyclonal anti-camel antiserum produced in rabbit (Bethyl
laboratories, Montgomery, TX, USA) was used (dilution 1:75 in blocking buffer) as a
second antibody followed by a Cy3-conjugated goat anti-rabbit antibody (Dianova)
diluted 1:1500 in blocking buffer, each incubated for one hour at RT and intercalated
by washing steps as described for cattle sera.
By VNT, neutralizing antibodies were detected in 39.8% of the samples (197 out of
493 bovine sera, 40 out of 103 camel sera). Table 6.1. shows the total numbers of
positive, negative and inclusive samples for each test.
Disadvantages of performing VNT are the lengthy and laborious testing procedure (at
least one week). In comparison, the IFA needs only about three days, including cell
growth, infection and the IFA assay itself, and allows testing of at least 40 field
samples per plate. Much faster and less laborious is the commercial ID Screen®
cELISA, which provides results in less than two hours [119]. Together with its easy
handling and high number of tested samples per plate, this ELISA is most suitable for
testing large serum numbers. The comparison of different ELISAs by ring trail
revealed also the high diagnostic sensitivity and specificity of the ID Screen® cELISA
[120]. Therefore, several seroepidemiological studies were carried out using this test
[121-124]. Surprisingly, our results indicated lower test accuracy since we found only
an agreement of around 80% between cELISA and VNT (479 out of 596 samples
with consistent results, Tab. 6.2.). Likewise, similar sensitivity results were observed
also for the IFA (83.9%). The cELISA and the newly developed IFA showed an
accordance of 78% (465/596 samples with consistent results and 71.2% of cattle
sera and 75.3% of the camel sera displayed consistent results in all of three test
methods (Tab.6.2.).
In 2011 Ergünay demonstrated a slight agreement between VNT, ELISA and IFA
respectively, by detecting antibodies against Sandfly virus, another Phlebovirus out of
the family Phenuiviridae. He pointed out that phleboviral neutralization is a complex
phenomenon and neutralizing epitopes may not necessarily be detected in diagnostic
assays which result in a lower agreement between VNT and other serological assays
[125]. The cELISA detected 82.3% and the IFA 83.5% of VNT positive samples which
could be a result of missed detection of neutralizing antibodies in these tests.
Furthermore, ELISA or IFA may be sensitive to cross reactive antibodies from other
Phleboviridae [126]. It could be assumed that both tests detected more positive
50
samples than the VNT due to cross reactive antibodies. Another reason could be the
appearance of non-neutralizing antibodies.
IFA and ELISA are therefore suitable assays for large scale screening purposes.
However, the combination of different serological test methods may provide a better
insight in the antibody status of the given animal population.
6.3. Tables
Table 6.1. Overview of positive, negative and inconclusive results in each of the
three test methods with calculation of the diagnostic sensitivity and specificity of the
ELISA and the IFA.
The VNT was used as the standard, therefore the SE and SP were set to 100%. a) Results
of bovine sera b) results of camel sera, c) results of all tested sera
a) bovine sera
Assay positive negative
incon-
clusive
Sensitivity
(%)
Specificity
(%)
VNT 197 296 0 100 100
cELISA 227 250 16 84.4 77.5
IFA 214 279
86.8 85.5
b) camel sera
Assay positive negative
incon-
clusive
Sensitivity
(%)
Specificity
(%)
VNT 40 63 0 100 100
cELISA 33 68 2 89.1 100
IFA 41 62 0 67.5 77.8
c) total sera
Assay positive negative
incon-
clusive
Sensitivity
(%)
Specificity
(%)
VNT 237 359 0 100 100
cELISA 260 318 18 84.8 81.6
IFA 255 341 0 83.5 84.1
51
Table 6.2. Combination of the results from different methods.
71.5% of the samples showed consistent results in all three test methods. VNT and ID
Screen® cELISA showed a general agreement of 80.4%, VNT and IFA of 83.9% and ID
Screen® cELISA and IFA accorded in 78.02% to each other.
test method number of reactive samples
VNT
ID
Screen®
cELISA
IFA cattle camel overall
+ + + 147 25 172
+ + - 15 8 23
- + + 24 / 24
- + - 40 / 40
+ - + 20 1 21
+ - - 10 5 15
+ +/- - 1 / 1
+ +/- + 4 1 5
- +/- + 3 / 3
- - + 16 14 30
- +/- - 8 / 8
- - - 205 49 254
52
Chapter 7: General Discussion
Rift Valley fever is the only significant hemorrhagic fever virus in humans which also
has a significant impact on animal health and, as a consequence, may have severe
effects on the ecological balance of a country [127]. Due to the unpredictable and
rapid nature of RVF epidemics a comprehensive control strategy in endemic
countries, such as Egypt, is urgently needed [8]. The virus was introduced in 1977,
affecting hundreds of thousands of humans and domestic animals [4, 21, 32, 34].
After this first severe RVF outbreak control strategies, including the vaccination of all
susceptible animals with locally produced and purchased vaccines, were
implemented. Despite the vaccination program further outbreaks reoccurred in
1993/94 in Damietta, Aswan and Beheira governorates, 1997 in upper Egypt and,
most recently, 2003 in the Kafr el Sheikh and Damietta governorates [8, 66, 128].
Although the latest reported Egyptian RVF outbreak dates back more than 10 years,
serological evidence of the RVFV circulation was found in different Egyptian
governorates during the current inter-epidemic period [129-133].
Seroepidemiological studies for the determination of spatio-temporal dynamics of
RVFV are restricted to the time period and location in which the samples were
collected and the selected species. Furthermore, the transmission cycle of RVFV in
Egypt is insecure and the maintenance of the virus during inter-epidemic periods is
still not completely understood [128, 133]. Due to the important role of cattle and
camels in the amplification cycle of RVFV and the high susceptibility of small
ruminants, especially sheep, [3, 86, 134, 135] this thesis aimed to provide insight
into the current antibody status against RVFV in Egyptian livestock throughout the
country. In addition, the risk of exposure for animals and humans should be further
evaluated.
Manuscript I provides an overview of the situation in buffalos, camels and small
ruminants, whereas manuscript II focuses on the seroprevalence in dairy cattle.
Recent publications described the epidemiological situation in about two-thirds of the
Egyptian governorates with partially diverging results [129-131, 133]. In the study
described in this thesis ruminant sera were collected from the governorates Port
Said, Dakahlia, Gharbia and for the first time from the Damietta and Ismailia
53
governorates which completes the availability of serological data for Egypt. Moreover,
additional sera derived from camels were provided from an abattoir near Cairo and
from camels in the Red Sea governorate. These animals had been imported from
Sudan.
In contrast to previous studies which reported prevalence rates in non-vaccinated
ruminants to be around 15%, 13%, 21% and 25% respectively [129-131, 133], the
results in this study (manuscript I and II) showed only low prevalence levels in the
investigated animals. Non-immunized ruminants including goats, sheep, buffalos as
well as small holding cattle, showed a total prevalence of 0%, 0.5%, 6% and 8%,
respectively. Especially the new findings in small ruminants contrast strongly with
previous reports, e.g. investigations from Selim et al., 2015 who found a prevalence
in 15% of the sheep and goats from the Dakahlia governorate [133]. The
considerably higher prevalence might be due to an unknown vaccination status in the
aforementioned study or to a low transmission rate of virus during inter-epidemic
periods. According to the farmers’ statements the animals in question in our study
had never been vaccinated against RVFV. All animals were born after the last
reported outbreak in 2003 and had not been imported from other endemic countries.
Furthermore, the ruminants moved only in a restricted area. Taking this into account,
the results indicate a low level of virus transmission during the current inter-epidemic
period in the Dakahlia governorate as well as in the Damietta and Port Said
governorates. Cattle with a prevalence of 8% and buffalos with a prevalence of 6%
showed the highest antibody titers in non-immunized animals and were most
probably infected naturally. Although the prevalence rates were lower in contrast to
previous reports, the results stated here underline the important role of cattle and
buffalos for the replication cycle of RVFV in Egypt. Low prevalence levels without any
evidence of acute infections after testing a subset of sera with RT qPCR or IgM
capture ELISA confirmed the low level of virus activity during the sampling period.
It was suggested that the origin of previous Egyptian RVF outbreaks was the import
of infected animals like camels from other endemic countries [8, 27]. With regard to a
relatively high prevalence of about 10% in camels from the abattoir in Cairo and
substantial neutralizing antibody titers, the role of camels for virus amplification even
during inter-epidemic periods in Egypt should not be ignored. Interestingly, none of
the imported animals from Sudan harbored antibodies against RVFV. Unfortunately,
54
there was no data available regarding the origin of the abattoir camels. Therefore, it
was impossible to determine whether these camels were infected in Egypt or the
seroconversion derived from a natural infection which had occurred already prior to
their importation.
Summarizing all data, there is evidence for a persistent presence of RVFV in Egypt
and for an active virus circulation even during the current inter-epidemic period. This
is consistent with latest findings in infected mosquitos and the sporadic detections of
IgM positive animals in the current inter-epidemic period [31, 131].
The vaccination of livestock is one of the most effective methods to control RVF as
well as to save animal and human lifes and minimize economic losses [127]. Egypt is
the only country world-wide which implemented a continuous vaccination program for
all susceptible livestock animals. So far, little was known about the effectiveness of
this vaccination program. Therefore, the antibody status of vaccinated versus non-
vaccinated cattle was compared in manuscript II.
Contrary to previous reports from Taha et al., 2001 [136] and Eweis et al., 2008
[137], who observed a vaccination efficacy of 70% and 65% respectively, the findings
presented here indicate a significantly lower seroprevalence level of only about 15%
in immunized cattle. Byomi et al., 2015 [129] determined similar rates of antibodies in
immunized cattle. Missed vaccinations (incl. boostering) and individual mistakes in
handling and using the vaccine may be the reasons for the low seroprevalences in
vaccinated animals. The findings of Byomi et al. in the Menoufia, Beheira and Kafr el
Sheikh governorates in conjunction with our results from the Dakahlia, Damietta and
Port Said governorates demonstrate that the low incidence of seroconversion in
immunized cattle should be of substantial concern. Apart from vaccine application
mistakes an insufficient immunogenicity of the Egyptian inactivated vaccine might
also be a reason for its insufficient efficacy. Efficacy studies of the Egyptian vaccine
have focused largely on the vaccination of sheep instead of cattle [138]. A study from
Eweis et al., 2008 compares the immunization of different species with a high
increase of positive results after booster immunization. However, in this study the
animals showed pre-existing antibodies at day zero of the study which raises
questions about the time of initial timepoint of vaccination [137].
It has been mentioned that, on the one hand, inactivated vaccines are less
immunogen and need frequent booster vaccinations to induce a protective
55
immunogenicity [139]. On the other hand, the immunogenicity might be less strong in
cattle than in small ruminants, or the protection of cattle may be independent of a
detectable seroconversion [78, 139, 140]. However, an immunized population with an
unclear status enhances the risk for animal and public health as RVFV can circulate
without any clinical signs. DIVA vaccines, which allow the discrimination of
vaccinated from infected animals are not yet available. Therefore, to eradicate the
virus, a proportion of 80% of the animal population should be immune, either as a
result of a previous infection or through vaccination [141]. As a basis for disease
control high levels of immunity in individual farms are as important as high levels of
immunity in the population. The findings in this study are also striking in this respect
as prevalences varied between 0% and 48% for individual farms.
In summary, the results of the present study highlight the need for a thorough re-
evaluation of the Egyptian RVF disease control strategy, paying particular attention to
the vaccination program.
In general, serological studies yielded diverse results which is most likely due to the
test methods used and to the respective sample selections. Therefore, we have
compared three different serological assays (i.e. cELISA, IFA and VNT) to eventually
reveal their suitability for seroprevalence studies. The virus neutralization test (VNT)
is considered to be the gold standard assay due to its high specificity and sensitivity.
However, the VNT is a highly labor-intensive method [142, 143].
In contrast to the cattle sera, all samples from buffalos, small ruminants and camels
were analyzed with the commercial ID Screen® RVFV multispecies competition
ELISA as well as with VNT (manuscript I). In this study the overall correspondency
between ELISA and VNT was high, displaying only eleven discrepant results from a
total of 731 analyzed sera (= 80.4%).
To perform seroepidemiological tests independently from commercial test kits a novel
diagnostic in-house immunofluorescence assay based on the RVFV vaccine strain
MP12 was established. Nearly 500 cattle samples and 100 camel-derived sera were
investigated with this assay and the results were compared with the results from ID
Screen® RVFV ELISA and VNT (manuscript IV). Corresponding comparative
serological studies especially for cattle had not been carried out before. Both, ID
Screen® RVFV ELISA and IFA showed results accordant with the VNT in around
80% of the tested cattle and camel sera. Surprisingly, these findings indicated that
56
the ID Screen® RVFV ELISA displayed a sensitivity and specificity rate of about 85%
and 82%, respectively. Due to the complex structure of neutralizing antibodies [125] it
is possible that assays like ELISA and IFA could fail to detect neutralizing antibodies.
Otherwise non-neutralizing antibodies and cross reactive antibodies detected by
ELISA and IFA leads to different results when comparing these tests systems [125].
Regarding the various seroconversions against different viral proteins the IFA
represents an appropriate tool for the detection of antibodies against all viral proteins.
Furthermore, infected cells produce conformational proteins which might help to
detect antibodies with low affinities. As a consequence of the results presented in
manuscript IV a combination of different serological test methods may provide a
better insight in the antibody status of the given animal population.
To get a better insight into the RVFV virus amplification in susceptible hosts it is
necessary to better understand the replication in mosquito vectors. Little is known in
this respect, although mosquitoes are a prerequisite for virus transmission and for the
maintenance of RVFV in an area. Therefore, the replication, protein accumulation
and distribution pattern of structural as well as nonstructural viral proteins in mosquito
and vertebrate cells were compared in this thesis, using newly established
immunofluorescence and Western blotting assays. The assays were carried out with
already existing mabs against the nucleocapsid protein (NP) and both glycoproteins,
Gn and Gc [99, 100] as well as with newly generated mabs against the nonstructural
proteins NSs and NSm (manuscript III).
As a first step recombinant protein variants of NSs and NSm were generated, using a
bacterial expression system. The proteins displayed molecular masses of about 29
kDa and 14 kDa respectively, and were subsequently used for the immunization of
BALB/c mice. Supernatants of hybridoma cells, generated by standard cell
fusion/hybridoma production protocols, were screened with a NSs and NSm specific
in-house ELISA which resulted in eight NSs and seven NSm derived monoclonal
antibodies. These mabs as well as already existing mabs against the structural
proteins, [99, 100] allowed to carry out a comparative expression kinetic study in
mammalian (Vero 76) and mosquito (C6/36) cells for the first time (as outlined in
manuscript III). In both cell lines replication and expression of all viral proteins were
observed, leading to extensive cytopathic effects (cpe) in Vero cells within 48 hours,
while no cpe was revealed for the C6/36 cells.
57
Using IFA the Gn protein appeared in the cytoplasm as discrete dots two hours after
the infection of Vero cells, one hour later Gn protein was detected jointly with Gc and
NP. In mosquito cells a similar expression pattern of these three structural proteins
was observed six hours after infection. In both cell lines a steady increase of the
corresponding proteins was observed throughout the observation period. Most
prominent staining was related to the Gn protein, which could be found in 100% of
the infected mammalian cells after 24 hours, similar to mosquito cells which showed
a Gn related staining in 100% of infected cells after 96 hours. In both cases NP and
Gc expression was significantly lower. Both glycoproteins showed the same
distribution pattern with a specific accumulation around the nucleus of infected cells
in contrast to NP, which was distributed homogenously throughout the cytoplasm.
Equally, the nonstructural protein NSm displayed a similar cytoplasmic staining in
both cell types and an accumulation around the nucleus, probably at the endoplasmic
reticulum/mitochondrial interface [108]. NSm displays anti-apoptotic properties [108]
and is negligible as far as the viral replication in mosquito cells is concerned but
dispensable for the virus replication in mammalian cells [63]. The mosquito related
relevance of NSm came along with an elevated expression level in C6/36 cells
compared to Vero cells.
Major differences, however, were observed regarding the nonstructural proteins NSs
and NSm. In mammalian cells, NSs was detected four hours after infection by
forming discrete dots within the nucleus and subsequent polymerization into NSs
specific filamentous structures within the nucleus. The expression kinetic revealed a
high level of expression for the NSs protein in mammalian cells (90% after 24 hours
of infection) in contrast to a low level in C6/36 with a maximum of around 50%
followed by a decrease in expression to 20% after 72 hours. Moreover, NSs could be
detected within the cytoplasm and rather late - at 72 hpi - as slight dots within the
nucleus of C6/36 cells. No characteristic filamentous structures as in the Vero cells
were observed. Diverging appearance may arise from the different NSs functions
from being a major virulence factor in vertebrates and still having an unknown role in
the mosquito vector. NSs interferes with the host innate immune response of
vertebrates by blocking interferon-ß (IFN-ß) gene expression, downregulating protein
kinase R and interacting with TFIIH, a basal host cell transcription factor which
results in facilitating the viral replication and the proliferation of infection among the
58
cells [58, 59, 94]. Moreover, NSs targets and modifies the expression of genes
coding for coagulation factors [95] and is responsible for the induction of
chromosome cohesion and segregation defects in infected murine and ovine cells
[96]. This function is mainly attributed to the formation of protein filaments. Therefore,
preventing the formation of NSs filaments would elude the detrimental effects on cell
transcription. This was recently reported in infection studies with virulent RVF strain
ZH548 in Aedes aegypti and Aedes albopictus derived cells lines (Aag2 and U4.4). In
mosquito cells which expressed virus-derived small interfering RNAs like Dicer-2 the
NSs protein was eliminated [114]. Interestingly, in the same study nuclear filaments
were formed within C6/36 cells which lack a Dicer-2 pathway. These findings
indicated an additional pathogen defense mechanism in insects. Since the MP12-
derived NSs sequence harbors three nucleotide exchanges compared to ZH548, the
results from our study highlight an alternative and sequence dependent mechanism
to inhibit filament formation and to reduce the expression level of NSs. This
corresponds with previous MP12 expression studies using different arthropod cell
lines in which NSs was expressed at significantly lower levels compared to vertebrate
cells [109].
All proteins could be also detected by immunoblotting, analyzing cell lysates
harvested at different stages of infection. Again, viral proteins were observed later in
mosquito cells compared to mammalian Vero cells. In addition, virus release into the
supernatant was observed after 24h in Vero 76 and after 48h in C6/36 cells by
detecting NP, Gn and Gc protein.
In summary, the comparative expression kinetic highlighted for the first time the
differences in protein expression and accumulation of five RVFV related proteins in
mammalian and insect cells. In this study we were able to demonstrate that most
differences in protein expression, distribution and accumulation are related to high
expression levels of proteins which have an important role in the replication of the
virus. This is demonstrated by NSs in Vero 76 cells and by NSm in C6/36 cells.
59
Chapter 8: Summary
Seroprevalence of Rift Valley fever virus specific antibodies in livestock in
Egypt and expression studies of virus related proteins in mammalian and
arthropod cells
Claudia Mroz
Rift Valley fever virus, a Phlebovirus of the family Phenuiviridae consistently causes
severe outbreaks and large epidemics in many African countries affecting humans,
ruminants and camels. The virus was introduced into Egypt in 1977 and caused an
extensive epidemic with thousands of infected humans, high mortality rates in
livestock and high economic losses. Up to now, this has been considered as one of
the largest epidemics in the RVF history in Africa. After a long inter-epidemic period,
the RVF re-occurred in the Nile Delta of Egypt in 1993 with subsequent outbreaks in
1994, 1997 and, most recently, in 2003. The causes of the different outbreaks, as
well as the maintenance of the virus during inter-epidemic periods, in which the virus
circulates at a low level between mosquito vectors and wild ruminants, are not
entirely known.
Seroepidemiological studies are useful tools to monitor the current antibody status in
non-immunized livestock in different areas. Moreover, these examinations can
provide an epidemiological risk assessment to evaluate the potential threat of new
RVF outbreaks. Therefore, as one objective of this thesis, 873 field serum samples of
non-immunized animals including sheep, goats, buffalos and camels from different
Egyptian governorates were checked for the presence of anti-RVFV antibodies using
a serological approach, including a commercial ELISA, virus neutralization test and
indirect immunofluorescence assay. The main finding in this analysis was a low
antibody presence of 2.3 per cent in the investigated animals without any signs of an
acute infection. The highest prevalence of about 9.9 per cent was detected in camels
from an abattoir near Cairo. Moreover, an additional analysis of 1,349 non-
immunized cattle sera displayed a seroprevalence of about 7.9 per cent. All in all, the
results indicate a low level of virus transmission during the current inter-epidemic
period and consequently, the maintenance of the virus in the Nile Delta of Egypt. In
addition, these results underline the important role of bovines and camels in the
60
amplification cycle of RVFV in Egypt. It has been suggested that the presence of
unvaccinated susceptible livestock combined with favorable conditions for the
breeding of mosquitos facilitates the persistence and potential emergence of RVFV in
the country. That is why a vaccination program was implemented in Egypt, which is
unique in Africa and even world-wide. According to this program every susceptible
livestock has to be vaccinated twice a year with a locally produced inactivated anti-
RVFV vaccine. Although this program was already established in 1980, there is little
information about the effectiveness of the vaccination and the subsequent
seroconversion in immunized livestock. On account of this, a further aim of this thesis
was a comparative serological survey of immunized and non-immunized dairy cattle
from four Nile Delta governorates. Analysis of 2,818 cattle sera from 26 different
farms that had participated in the official vaccination program were compared to
1,349 cattle-derived sera from 27 small holdings without any vaccination against
RVFV. The serological testing was carried out in a three-step approach, including a
commercial ELISA followed by the re-assessment of positive and questionable
results by virus neutralization test and indirect immunofluorescence assay.
Surprisingly, the results demonstrated a low seroprevalence of about 15 per cent in
immunized cattle, which was not significantly higher than those in non-vaccinated
animals (7.9 per cent). In addition, we could determine highly divergent
seroprevalence rates between individual farms. Such inadequate levels of
immunization in conjunction with the lack of DIVA vaccines oppose the beneficial
effects of the vaccination program and raise serious concerns about public and
animal health policies in Egypt. As a result, re-evaluation of this vaccination program
is highly recommended.
In order to understand RVFV occurrence, information about virus amplification in
susceptible hosts as well as information about the replication in mosquito vectors
must be present. In both cases attention must be paid to the role of the nonstructural
proteins NSs and NSm, which are the main virulence factors of the virus. NSs and
NSm protein manipulate the mammalian host cell immune defense by deranging the
interferon response and, respectively, by inhibiting the virus induced apoptosis of the
infected cells. However, their impact in mosquito related amplification and
persistence is largely unknown.
61
Therefore, another part of this thesis was a comparative infection study with RVFV-
vaccine strain MP12 to monitor the protein expression in mammalian as wells as in
arthropod cells. For this purpose, monoclonal antibodies against both nonstructural
proteins were generated and validated for their application in immunofluorescence
and Western Blot analysis. The study was complemented by monoclonal antibodies
against the nucleocapsid protein and the two glycoproteins which allowed us to follow
the expression kinetics of five different viral proteins during infection of Vero and
C6/36 cells. Cytopathic effects could only be monitored in Vero cells, accompanied
by NSs derived filamentous structures solely within the mammalian nucleus.
Contrary, NSs was distributed throughout the mosquito cell cytoplasm and formed
small dots within the nucleus 72 hours post infection. Furthermore, the elevated
expression level of the NSm protein in C6/36 cells compared to Vero cells highlights
the mosquito related relevance of this protein. These findings provide additional
evidence for a divergent role of NSs and NSm during replication in the mammalian
host compared to the mosquito vector and highlight different antiviral defense
strategies.
All in all, the present findings of the serological analysis of Egyptian livestock confirm
an ongoing low level of RVFV circulation in the Egyptian Nile Delta. However, the
significant deficiency of seroconversion in cattle highlights the necessity to re-
evaluate the vaccination program. In addition, the expression kinetic of RVFV related
proteins described here allow for the first time the comparison of five RVFV proteins
and provides an insight into the different roles of the nonstructural proteins in vector
and host cell.
62
Chapter 9: Zusammenfassung
Seroprävalenz von Rift Valley Fieber Virus spezifischen Antikörpern in
Nutztieren in Ägypten, sowie Expressionsstudien von Virusproteinen in
Säuger- und Insektenzellen
Claudia Mroz
Rift Valley Fieber Virus, ein Phlebovirus innerhalb der Familie Phenuiviridae
verursacht schwere Ausbrüche sowie große Epidemien in vielen afrikanischen
Ländern bei denen insbesondere Menschen, Wiederkäuer und Kamele betroffen
sind. Das Virus wurde im Jahr 1977 nach Ägypten eingeschleppt, wo es eine
massive Epidemie mit tausenden infizierten Menschen, vielen Verlusten innerhalb
der Nutztierpopulation und großen ökonomischen Einbußen verursachte. Bis jetzt gilt
diese Epidemie als eine der größten in der afrikanischen Geschichte. Nach einer
langen inter-epidemischen Phase trat das Rift Valley Fieber erneut 1993 im
ägyptischen Nildelta auf; weitere Ausbrüche folgten 1994, 1997 und zuletzt 2003. Die
Ursachen der verschiedenen Ausbrüche, sowie das langfristige Überdauern des
Virus während der inter-epidemischen Phasen, in denen das Virus auf niedrigem
Level zwischen Mücken und Wildwiederkäuern zirkuliert, sind nicht gänzlich bekannt.
Seroepidemiologische Studien sind nützlich, um den aktuellen Antikörperstatus von
nicht-immunisierten Tieren in verschiedenen Regionen zu beobachten. Außerdem
können diese Untersuchungen zu einer epidemiologischen Risikoanalyse beitragen,
um die aktuelle Gefahr erneuter Rift Valley Fieber Ausbrüche zu evaluieren. Ein Ziel
dieser Arbeit war es daher, 873 Feldseren von nicht immunisierten Tieren, bestehend
aus Schafen, Ziegen, Büffeln und Kamelen aus verschiedenen ägyptischen
Gouvernements auf das Vorhandensein von anti-RVFV Antikörpern serologisch
mittels kommerziellen ELISA, Virusneutralisationstest und indirekter
Immunfluoreszenz zu untersuchen. Diese Untersuchung zeigte in erster Linie ein
niedriges Antikörpervorkommen von 2,3 Prozent in den untersuchten Tieren, ohne
jegliche Anzeichen akuter Infektionen. Die höchste Prävalenz von 9,9 Prozent wurde
bei Kamelen aus einem Schlachthof in der Nähe von Kairo detektiert. Darüber hinaus
wurde in einer weiteren Studie von 1.349 Seren nicht-immunisierter Rinder eine
Seroprävalenz von 7,9 Prozent festgestellt. Insgesamt deuten diese Ergebnisse auf
63
eine geringe Virusverbreitung innerhalb der aktuellen interepidemischen Phase hin,
folglich aber auch auf das Vorhandensein des Virus im ägyptischen Nildelta.
Zusätzlich unterstreichen diese Resultate die bedeutende Rolle von Boviden und
Kameliden im RVFV-Vermehrungszyklus in Ägypten. Vermutlich ist die
Viruspersistenz und das potenzielle aktive Auftreten des RVFV im Land erst durch
das Vorhandensein nicht geimpfter, empfänglicher Tiere in Kombination mit
günstigen Bedingungen für die Mückenvermehrung möglich. Aus diesem Grund
wurde in Ägypten ein für Afrika und weltweit einzigartiges Impfprogramm etabliert.
Demnach sollen alle empfänglichen Tiere zweimal im Jahr mit einem lokal
produzierten, inaktiven anti-RVFV-Impfstoff geimpft werden. Auch wenn dieses
Programm bereits 1980 eingeführt wurde, sind nur wenige Informationen bezüglich
der Effektivität der Impfung und der verbundenen Serokonversion der immunisierten
Nutztiere verfügbar. Vor diesem Hintergrund führten wir eine vergleichende Studie
von immunisierten und nicht-immunisierten Milchkühen aus vier Gouvernements im
Nildelta durch. Es wurden 2.818 Rinderseren aus 26 verschiedenen Farmen, die an
dem offiziellen Impfprogramm teilnehmen im Vergleich zu 1.349 Rinderseren aus 27
Kleinsthaltungen ohne Impfprogramm gegen RVFV analysiert. Die serologischen
Testungen wurden in einem drei-stufigen Ansatz durchgeführt. Nach Untersuchung
mit einem kommerziellen ELISA folgten Bestätigungstests bei positiven und
fraglichen Resultaten mittels Virusneutralisations- und Immunfluoreszenztest.
Überraschenderweise zeigten die Ergebnisse bei den immunisierten Rindern mit nur
15 Prozent eine geringe Seroprävalenz, die nicht signifikant höher war als die
Prävalenz der nicht geimpften Tiere (7,9%). Darüber hinaus konnten wir große
Unterschiede in der Seroprävalenz zwischen den einzelnen Farmen feststellen.
Derartige inadäquate Impflevel in Verbindung mit fehlenden DIVA-Vakzinen kehren
den nutzbringenden Effekt eines Impfprogramms um und erwecken ernste Bedenken
bezüglich des ägyptischen Gesundheitsmanagements von Mensch und Tier. Eine
erneute Evaluierung des Impfprogramms in Ägypten ist demzufolge dringend
anzuraten.
Ein Verständnis der RVFV-Ausbruchsgeschehen setzt neben Kenntnissen über die
Virusvermehrung in den empfänglichen Tieren auch eine Kenntnis der
Virusvermehrung in den Mückenvektoren voraus. In beiden Fällen sollten die Nicht-
Strukturproteine NSs und NSm Beachtung finden, die die Hauptvirulenzfaktoren des
64
Virus darstellen. Das NSs und NSm Protein manipulieren die Immunabwehr von
Wirtzellen der Säugetiere mittels einer Beeinträchtigung der Interferon-Antwort
beziehungsweise mit der Inhibition der virusinduzierten Apoptose von infizierten
Zellen. Die Bedeutung in der Amplifikation und Persistenz in den Mücken ist jedoch
weitestgehend unbekannt.
Aus diesem Grund war die vergleichende Infektionsstudie mit dem RVF-Impfstamm
MP12, in der die Proteinexpression in Säugetier- und Arthropodenzellen verfolgt
wurde, ein weiterer Teil dieser Arbeit. Zu diesem Zweck wurden monoklonale
Antikörper gegen die beiden Nicht-Strukturproteine generiert und für den Einsatz in
der Immunfluoreszenz und im Western Blot validiert. Die Expressionsstudie wurde
durch monoklonale Antikörper gegen das Nukleokapsidprotein und die beiden
Glykoproteine komplettiert. Dadurch bestand die Möglichkeit, fünf verschiedene virale
Proteine während der Infektion von Vero 76 und C6/36 Zellen zu verfolgen.
Zytopathische Effekte wurden nur bei den Vero-Zellen beobachtet, ebenso zeigten
nur die Säugetierzellen die auf das NSs Protein basierenden filamentösen Strukturen
in den Zellkernen. Im Gegensatz dazu befand sich das NSs Protein im gesamten
Zytoplasma der Mückenzellen verteilt und formte kleine Punkte nach einer
Infektionszeit von 72 Stunden. Des Weiteren wird durch die vermehrte Expression
des NSm Proteins in den C6/36 Zellen im Vergleich zu den Vero-Zellen die Relevanz
dieses Proteins in den Mückenzellen hervorgehoben. Diese Ergebnisse geben
weitere Hinweise für die unterschiedliche Rolle von NSs während der Replikation in
Säugetier- im Vergleich zur Mückenzelle und verdeutlichen die unterschiedlichen
anti-viralen Bekämpfungsstrategien.
Insgesamt unterstützen die vorliegenden Ergebnisse aus den serologischen
Analysen der ägyptischen Nutztiere die These einer momentan geringen
Viruszirkulation im Nildelta. Die defizitäre Serokonversion geimpfter Rinder
unterstreicht jedoch die Notwendigkeit einer erneuten Evaluation des
Impfprogrammes in Ägypten. Die in dieser Arbeit beschriebene Expressionskinetik
der RVFV assoziierten Proteine ermöglicht außerdem zum ersten Mal den Vergleich
der fünf Proteine des RVFV und gibt damit weitere Hinweise auf die besonderen
Rollen der Nicht-Strukturproteine in Vektor- und Wirtszelle.
65
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76
Chapter 11: Acknowledgements
First of all, I would like to thank Prof. Dr. M. H. Groschup and Dr. Martin Eiden for
their academic supervision and scientific encouragement I got from them throughout
the course of my thesis. I am grateful for the opportunity to work at the Institute of
Novel and Emerging Infectious Diseases and for being part of this project. Whenever
issues needed to be discussed they provided me beneficial help and I am thankful for
their trust in me and my work. Thank you for your constructive discussions, helpful
suggestions and for revising my thesis and all contributing manuscripts.
Moreover, I would like to thank the Federal Foreign Office (German Partnership 303
Program for Excellence in Biological and Health Security) and all colleagues from
Egypt for their support. I would especially like to thank Mayada Gwida and Maged El-
Ashker for the fruitful cooperation and especially for sampling thousands of ruminant
sera within the last years.
Great thanks to Dr. Sven Reiche, Evelin Ball and Sven Sander for their great support
in monoclonal antibody production and for sharing their experiences with me.
I am grateful to Dr. Ute Ziegler, Dr. Kristina M. Schmidt and Dr. Timo Homeier-
Bachmann for granting the neutralization test as well as the immunofluorescence
assay and for giving me valuable advice regarding epidemiological questions.
Furthermore, I would like to thank Birke Böttcher for offering excellent technical
support and help in all belongings. Special thanks to Antje Twigg-Flesner and Cora
Holicki for proofreading of my thesis.
Finally, and most of all I would like to thank my family and all dear friends. I would not
have been able to come so far without your outstanding support, your unlimited
optimism and encouragement at all times. Thank you so much!