SZENT ISTVÁN UNIVERSITY

FACULTY OF VETERINARY SCIENCE

DEPARTMENT OF MICROBIOLOGY AND INFECTIOUS DISEASES

RE-EMERGING TRANSMISSIBLE

GASTROENTERITIS IN PIGS

Written by:

Simon Andersson

Supervisors:

Dr. Imre Biksi, PhD

Dr. Márta Lőrincz, PhD student

Dr. Tamás Tuboly, PhD

Budapest

2010

TABLE OF CONTENTS

INTRODUCTION 3

MATERIALS AND METHODS 9

Samples 9

RNA extraction 9

Reverse transcriptase polymerase chain reaction 10

Gel electrophoresis 11

Cloning 12

Sequence analysis 12

RESULTS 13

Reverse transcriptase polymerase chain reaction 13

Cloning 15

Sequence analysis 15

Serology 16

DISCUSSION 17

ACKNOWLEDGEMENTS 21

SUMMARY 22

ÖSSZEFOGLALÁS 23

REFERENCES 24

2

INTRODUCTION

Porcine transmissible gastroenteritis (TGE) is a disease in pigs caused by a

coronavirus belonging into the Coronaviridae family of the order Nidovirales. Coronaviruses

are enveloped (Figure 1.) and the genetic information is coded by a copy of a single stranded

RNA genome of positive polarity, so far known as the largest such stable RNA genome of

animal viruses, ranging in size between 26.4 – 31.7 kilobases (kb). Other families of

Nidovirales, such as Arteriviridae and Roniviridae share some common characteristics with

the Coronaviridae, besides the structural similarities and the nature of the genome. These

virus families are all known for their nested set replication and transcription profile (de Vries

et al., 1997).

Peplomer(Spike, S)

Nucleoprotein(N)

Membrane (M)

Small membraneenvelope (sM, E)

ssRNA

Figure 1. Electron micrograph and a schematic drawing of the structure of a coronavirus.

The family Coronaviridae is further divided into the coronavirus and the torovirus

genera (Fauquet et al., 2005). The genus coronavirus comprises three groups, or most recently

referred to as subgenera, namely Alpha-, Beta and Gamma-coronavirus. Coronaviruses have

some rather prominent members that cause important diseases, both of human and veterinary

interest such as, Severe Acute Respiratory Syndrome Virus (SARS-CoV), Infectious

Bronchitis Virus (IBV), Feline Infectious Peritonitis Virus (FCoV), Porcine Hemagglutinating

Encephalomyelitis (PHEV) and Transmissible Gastroenteritis (TGEV), just to mention a few

of them. All in all the complete genome of some 26 coronaviruses have been described to this

date. But there are probably more out there and this number is in no doubt going to change in

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a not too distant future. After the SARS outbreak in 2002-2003 (Peiris et al., 2004), research

into coronaviruses has really picked up the pace and new interesting things were found out,

among them the zoonotic potential of some members of the genus (Shi and Hu, 2008; Hon et

al., 2008). The ability to break the species barrier is also evident in case of the newly

emerging enteric bovine coronaviruses that share a 98 % RNA sequence homology with the

HECoV-4408 human enteric coronavirus, frequently isolated in diarrhea cases of children

(Han et al., 2006). Also noteworthy in this respect is the fact that the recently emerged canine

respiratory coronavirus (CRCoV), was originally a bovine coronavirus (Decaro et al., 2007).

Coronaviruses are well known for their flexibility in adapting to new species or new

environmental conditions in the host. Their plasticity has mainly been attributed to three

factors, the high mutation rate due to the lack or limited proof reading activity of the RNA

dependent RNA polymerase (Jenkins et al., 2002; Duffy et al., 2008); the polymerase may

move to a different template during replication (Lai, 1992); and also there seems to be a

negative correlation between genome size and mutation-rate. If the genome grows beyond a

certain size the amount of accumulated deleterious mutations will prove fatal to the virus

(Eigen, 1987). All these together make for a potentially fast evolving virus, which is able to

adapt to new circumstances rather fast.

There are three different coronavirus induced diseases of swine, namely Porcine

Epidemic Diarrhea (PED), Porcine Hemagglutinating Encephalomyelitis (PHE) and TGE,

with three different coronavirus species as the causative agents.

PED was initially reported from Belgium and the United Kingdom in 1978 (Pensaert

and de Bouck, 1978) and later from more countries in Europe and Korea (Debouck et al.,

1982; Chae et al., 2000), and is very similar to the clinical picture observed in case of a TGEV

infection, it has sometimes been referred to as a TGE-like disease. There is however a

difference in the severity of the disease in the affected pigs, while the mortality in piglets

bellow 2 weeks of age may rise to 100% in TGE cases, the losses induced by PEDV usually

remain below 50% in the same age group. Although older pigs may also develop serious

clinical signs.

PHE was first observed causing disease in Ontario, Canada in 1957 (Alexander et al.,

1959) and the virus was later isolated and classified by Greig et al. (1962). During the 1957

outbreak the disease mainly affected piglets younger than two weeks. Clinical signs are

characterized by inappetance, shivering, loss of condition and vomiting. Apart from these

clinical signs those of a severe encephalomyelitis can be observed. In these typically younger

piglets the mortality may sometime reach 100%. Older piglets will most of the time only

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show a slight posterior paralysis (Alexander et al., 1959). The disease seems to be limited to a

single farrowing group, disappears by itself and does not recur (Werdin et al., 1976) as

maternal immunity develops.

Transmissible gastroenteritis was first identified in 1946 (Doyle and Hatchings, 1946)

and caused problems in pig herds until the late 1980s when deletion mutants of the virus, the

Porcine Respiratory Coronaviruses (PRCoV) emerged. TGE is characterized by vomiting,

diarrhoea and dehydration (Hooper and Haelterman, 1969). Clinical signs can be experienced

at 18 to 48 hours post exposure. Four days after infection neutralizing antibodies can be found

in the circulation (Norman et al., 1973). During autopsy the stomach content is described as a

solid caseous curd, which can be bile-stained and becomes more solid as the dehydration

progresses. The most prominent lesions are detected in the intestines where a striking feature

is the marked villous atrophy. The loss of villi is more or less uniform along the length of the

small intestine, however there are some exceptions. The initial part of the duodenum seems to

be unaffected, but villous atrophy can be complete in both the jejunum and the ileum These

changes can be observed as early as 24 hours post infection (Hooper and Haelterman, 1969).

Accompanying the villous atrophy is a failure of cells recruited from the crypt epithelium to

differentiate to the normal columnar epithelium. The cells mainly affected by the TGE virus

are those covering the villi and not the ones found in the crypts. At the microscopic level

signs of recovery can be observed from day four but not until the seventh day post infection is

there a complete regeneration of the villous epithelium.

Virus in high titre can be found in cells of the intestinal mucosa. However,

investigating other tissues, the virus can also be isolated from lung, liver and pancreas tissues.

Pigs exposed to virus at 21 days or later do not have virus in the above mentioned organs

(Norman et al., 1973; Underdahl, 1974). It has also been proven that the virus can replicate in

alveolar macrophages in vitro (Laude et al, 1984).

An important factor in predicting the severity of the infection is what age the pig is

when it is exposed to the pathogen. The diarrhoea, vomiting and consequent electrolyte loss is

more pronounced and has a longer duration in younger pigs. Pigs infected later in life may not

even show clinical signs of an infection. As discussed earlier the hallmark of a TGEV

infection on a microscopical level is villous atrophy and crypt cell hyperplasia. These changes

are correlated with the severity of the clinical signs. Alas in younger pigs the

histopathological alterations are more expressed (Moon et al., 1973).

A remarkable thing happened in Belgium in 1984, there was a dramatic increase in

animals being seropositive for TGEV. However, none of the animals displayed any of the

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clinical signs associated with TGEV infection (Pensaert et al., 1986). On top of this TGEV

could not be isolated from the seropositive animals, instead a new virus was found, a close

relative to the virus causing TGE in pigs. Later it was found out that it most probably was a

deletion mutant of TGEV, with deletions in the spike protein and a non-coding region

(Rasschaert et al., 1990, Britton et al., 1991). PRCoV is pneumotropic, replicating mainly in

alveolar cells, but also in epithelial cells of nasal mucosa, trachea, bronchi, bronchioli, in

alveolar macrophages and in tonsils. It can also replicate in the gastrointestinal tract in cells

located underneath the villi. To reach the gut the virus can either be swallowed or after

primary replication in the respiratory tract there might be a viraemia, leading the virus to

disseminate to the gastrointestinal tract. But virus is isolated at a much lower titre in the

gastrointestinal tract compared to the respiratory tract (Cox et al., 1990). PRCoV infections

are usually subclinical.

What struck observers after the emergence of PRCoV was the relative absence of TGE

outbreaks. The spreading of PRCoV in pig herds seemed to correlate with a decline in

importance of TGE. The explanation for this was that the new virus was able to produce

neutralizing antibodies against TGEV. Protective immunity on the antibody level against TGE

is directed to the spike (S) structural protein of the virion. The spike is responsible for the

attachment of the virus to cellular receptors. TGEV-S (Figure 2) uses two distinct receptor

binding sites (RBS) and through them two different receptors.

S protein trimer

Anchor region

RBS-1

RBS-2

Figure 2. Schematic representation of the Spike of TGEV with the approximate location of the receptor binding sites. (RBS-1: between residues 92 and 250, RBS-2: from amino acid 405 to 465).

6

RBS-2 is a motif, binding to the ubiquitous aminopeptidase-N molecules (Delmas et

al., 1992) of cell membranes, whereas RBS-1 is the binding site of an approximately 200 KDa

receptor limited to the small intestinal cells of newborn animals (Weingartl et al., 1994). One

of the differences between TGEV and PRCoV is that TGEV carries both RBSs whereas

PRCoV, the deletion mutant, lacks RBS-1, rendering it incapable to infect intestinal cells in

the way TGEV does. The same deletion is suitable for the differentiation of TGEV and

PRCoV by molecular methods targeting the S gene or by monoclonal antibody based

serological methods both for antigen and antibody detection.

There is a strong antigenic connection between TGEV and PRCoV. In vitro, the

antibodies directed towards the structural proteins cross react. Sows infected with PRCoV

secrete antibodies with the milk that are capable of decreasing the infection rate in the

newborn gut with a dramatic reduction of the impact of TGEV on a litter, reducing clinical

manifestation and consequently mortality (De Diego, 1994). Multiple exposures to PRCoV

(Sestak et al., 1996) increase both the IgG and IgA titre in the milk.

With the appearance and spread of PRCoV the incidence of TGE gradually decreased

and from an OIE A list disease it became an almost forgotten disease throughout the world.

Occasional reports (Elvander et al., 2000, Brendtsson et al., 2006) of TGEV-specific

seropositivity (“singleton reactors”) indicated that the virus is still present in pig herds but

without clinical manifestation.

Recently, outbreaks of vomiting and diarrhea of 5-7 days old suckling piglets were

observed on a large sow farm in Hungary. This PRRS-negative herd was newly established,

so at first all sows were primiparous. Farrowing started in late August, and the problem did

not reach significant magnitude until January the following year. Affected litters were mainly

those of primiparous gilts, but litters of some multiparous sows showed similar clinical signs.

These usually started with vomiting, then liquid, yellowish diarrhea was seen, and emaciation,

dehydration of the piglets developed rapidly. In a given farrowing room signs usually started

in 5-7 days old litters, but the disease spread rapidly to younger piglets, born later in the room.

Morbidity in some groups was estimated at about 50%, mortality stayed low, at about 4-6%.

Piglets treated with commercial electrolyte supplementation and antibiotic injections

(Gentamox, Ceva) were likely to recover. In some instances, only electrolyte supplementation

was used with the same result. Piglets routinely received preventive oral treatment against

coccidiosis at four days of age (Baycox, Bayer), gilts were vaccinated twice during pregnancy

with a commercial E. coli vaccine (Porcilis Coli, Schering-Plough, Intervet), and against

PCV-2 infection (Circovac, Merial) when vaccines became available. Hypogalactia was

7

usually minimal or absent at the beginning of these outbreaks, sows were in good general

condition, liveborn litter sizes and piglet birth weights were within acceptable limits. The

farm was run according to strict hygienic standards and all-in/all-out was practiced in all

production phases. Outbreaks of vomiting and diarrhea were not possible to relate to sow feed

composition or to particular farrowing rooms. The problem was more frequently observed

from late autumn to early spring. Other age groups on the farm did not experience a similar

condition.

Diagnostic investigations were initiated early in the course of this disease in several

institutions. Initially no pathogen was detected that could be connected to the clinical signs in

all the examined cases. Beta-haemolytic E. coli strains (not typed) were detected in a few

instances, Clostridium perfringens A toxins (cpa, cpß2) were occasionally detected by

multiplex polymerase chain reaction (PCR), but in the majority of the submitted cases no

pathogens could be isolated. Fecal samples were consequently negative for coccidia (standard

flotation), rotavirus (PCR), Clostridium difficile (commercial A/B toxin test) and PRRSV

(PCR). Gross pathological lesions of piglets succumbed to the disease or euthanized due to

terminal illness were restricted to signs of weight loss and dehydration, yellowish fluid filled,

distended small intestinal loops and large intestines and to some mesocolonic edema.

Histopathology of the small intestinal tract did not reveal major changes apart from mild

shortening of the villi. Crypt hyperplasia was not evident in the examined cases and the

colonic mucosa appeared normal. Parenchymal organs did not show pathognomonic

alterations. Since the search for common causative agents was unsuccessful, more uncommon

pathogens were also considered and investigations for their presence commenced.

Although clinical signs and presentation of the condition would fit the description of

enzootic TGE, detection of this agent was not attempted initially. This was because the

widespread infection of Porcine Respiratory Coronavirus (PRCoV) thought to provide

protective immunity to piglets, and therefore clinical TGE has not been detected in Hungary

for the last 15 years.

The purpose of the present study was to identify PRCoV and TGEV coronaviruses in

affected animals, and to characterize the potential differences in the spike gene sequences of

their respective genomes.

8

MATERIALS AND METHODS

Samples

In March, 2009, with the aim to detect if TGEV was present in piglets suffering

clinical signs consistent with an enteral disease at the previously mentioned farm, four

untreated piglets aged 4-6 days were sacrificed and dissected on the farm. Portions of their

small and large intestines were placed into sterile plastic containers and transported on ice to

the laboratory at the Department of Microbiology and Infectious Diseases within four hours of

collection. Samples were also collected from the gastrointestinal tract and parenchymal

organs for histopathology and group 1 coronavirus immunohistochemistry (IHC). Samples for

aerobic bacteriological culture and detection of clostridial toxins were also collected from the

small and large intestine. After the detection of the first TGEV positive cases a wider survey

was initiated, where samples of small intestines, faeces and lung tissues were collected from

approximately 150 animals of the most susceptible age group These samples were gathered at

14 different farms including the original farm, throughout the country.

At the farm where the first TGEV positive piglets were found blood samples intended

for serological examination were drawn from 15 primiparous and 15 multiparous sows

nursing affected litters. These samples were tested for the presence of anti-TGEV antibodies

with a TGEV/PRCoV differentiating commercial ELISA (Svanova) at the Large Animal

Clinic of the Faculty of Veterinary Medicine located in Üllő. The technical details of these

tests are not mentioned here, as they were performed by scientists other than the author of this

thesis. The results however will be referred to, with permission of them, when needed

RNA extraction

About 0.1 g of tissue or faecal samples was used for extraction with the Viral Gene-

spin™ Viral DNA/RNA Extraction Kit (iNtRON Biotechnology). Sterile distilled water up to

150 µl was added to the samples in a 1.5 ml microcentrifuge tube and homogenized with a

mortar (Sigma-Aldrich) to break down the cell structures and with this release the nucleic

acids, including the viral RNA, from the cells. 250 µl Lysis Buffer from the kit, heated to

80oC was added to the homogenate and mixed. Subsequently 5 µl 20 mg/ml Proteinase K

(Fermentase) was added to digest proteins of the lysed cells and incubated at 55 oC for 10

9

min. To facilitate the binding of the nucleic acids to the matrix, 350 µl Binding buffer was

added and the mixture loaded into the Spin Columns. The liquid phase was released by

spinning through the cartridges at 12000 g for 1 min. The filter bound DNA/RNA mixture

was washed with the appropriate Wash buffer from the kit twice, by letting 500 µl through at

12000 g, for 1 min each. The filter tubes were dried by centrifuging for an additional 2 min

with the same force. The nucleic acids were then eluted into 30 µl of RNase free sterile

distilled water preheated to 45 oC. The samples were stored at -80 oC until use.

Reverese transcriptase polymerase chain reaction

The following procedure was used for the reverse transcription of the RNA templates

(using reagents from Fermentas, Lithuania). To 5 µl of the purified nucleic acids 1 µl random

hexamer primer (100 µM), 10 U RNase Inhibitor and 6 µl sterile double distilled water were

added, incubated at 65oC for 5 minutes. Following the incubation step the mixture was

supplemented with 4 µl RevertAid™ Reverse Transcriptase buffer, 10 U RNase Inhibitor, 50

U M-MuLV RevertAid™ Reverse Transcriptase, together with 1 µl dNTP (10 mM) and

incubated at 25 oC for 10 min and at 42 oC for 60 min.

The PCR primers used for the specific amplification and differentiation of the

TGEV/PRCoV S genes were designed by the Primer 3 program (Rozen and Skaletsky, 2000)

based on the TGEV Purdue 115 full genome sequence (GenBank Accession Number:

DQ811788), where the S gene is located at 20354-24697 nucleotides. The primers were

TGE2: 5’-AAGGAAGGGTAAGTTGCTCA-3’ (binding at 20282-20301 nt) and TGE3: 5’-

GGTCCATCAGTTACGCCGAA-3’ (21538-21518 nt) flanking 1258 bases of the S gene,

including the RBS-1 (aa: 92-219) and RBS-2 (aa: 405-465) coding sequences. The reactions

were carried out with the TGradient Thermocycler (Biometra) with mixtures of a volume of

50 µl in microcentrifuge tubes. The components of the PCR mixture are listed in Table 1. As

a negative control, 1 µl of water was used instead of cDNA. As a positive control a cloned

TGEV-S gene (Tuboly et al., 1994) was used and it was mixed the same way as the samples.

The Dream Taq Buffer contained KCl diluted in a PCR mix to provide optimal conditions for

the polymerase, MgCl2 was used to increase hybridization strength during primer attachment.

The steps of the PCR were as follows: 5 min at 95 oC, 40 cycles of denaturation at 95 oC,

annealing at 55 oC (both for 30 sec) and elongation at 72 oC 1 min, and a final step at 72 oC for

7 min.

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Table 1. Composition of the PCR reaction mixtures.

Sample (cDNA) 1 µl10X DreamTaq Buffer (Fermentas) 5 µlMgCl2 (25 mM) 3 µldNTP Mix (10 mM diluted 10 times) 1 µlPrimers (25 µM) 1 µlDream Taq DNA Polymerase (5U/µl) 0.2 µlDouble distilled water 38.8 µlEnd volume 50 µl

Gel electrophoresis

2% agarose gels were prepared by mixing agarose (Q-Biogene) powder and TAE

buffer (40 mM Tris acetate, 1 mM EDTA, containing 0.4 µg/ml ethidium bromide, later

replaced by GR Safe DNA Stain I, Life Science Technologies). The mixture was boiled in a

microwave until the agarose was completely dissolved and then poured into a casting box

(closed with casting tape or with stockers) of appropriate size and sample combs according to

how many samples were analyzed at the time. After the gel had solidified, the comb(s) was

removed, the gel inserted into the electrophoresis tank and TAE was added as running buffer.

10 µl of each of the PCR products were thoroughly mixed with 2µl loading buffer and added

to the wells in the agarose gel. For molecular mass standard 2µl of DNA ladder (Fermentas 1

kb and/or 50 bp Ladder) was added to flanking wells. The electrophoresis was done under

constant voltage of 110 V for 20-60 min depending on gel size. The gel was visualized by

ultraviolet light and photographed using the Kodak Electrophoretic Documentation and

Analysis (EDAS 290) System. For GR Safe staining the detection was done with the Dark

ReaderTM system between 420-500 nm wavelengths.

11

Cloning

PCR fragments of sizes characteristic for TGEV or PRCoV S genes were cloned for

long term storage and sequencing. The PCR products were cut off the agarose gel and purified

with the QIAEX Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions.

Which briefly were as follows, the gel slice was dissolved by adding 3 volumes of binding

buffer to 1 volume of gel kept at 50°C for 10 minutes or until completely dissolved. The DNA

was adsorbed to the silica gel solution of the kit, incubated for 5 min at room temperature, and

pelleted by spinning at 12000 g for 2 min. The pellet was washed with 500 µl of the Wash

solution, pelleted again and air dried. The DNA was eluted from the silica gel with 10 µl of

DNAse free distilled water. Cloning was done using the TOPO® TA Cloning® Kit

(Invitrogen) following the instructions of the manufacturer. Which briefly were as follows: 4

μl of the purified PCR product was mixed with 1 μl of Salt Solution and 1 μl of the

topoisomerase carrying TOPO® vector, incubated at room temperature for 5 min. The ligated

product (1 μl) was used for the transformation of electro competent Escherichia coli bacterial

cells (Electroporator 2510, Eppendorf) in 2 mm cuvettes at 2.5 kilo Volt.

Bacteria then were plated onto Luria Broth (LB) plates containing 50-100 μg/ml

ampicillin and grown overnight at 37 oC. Colonies were picked the next day and grown in

liquid LB medium supplemented with ampicillin. The plasmid DNA was extracted using the

Eppendorf mini plasmid isolation kit, according to the manufacturer’s instructions. The DNA

was checked by electrophoresis as described earlier, after digestion with BamHI restriction

endonuclease (Fermentas) with cleavage sites flanking the insert on the cloning vector.

Sequence analysis

The purified DNA clones were sequenced at the Biomi Ltd. (Gödöllő, Hungary) by the

Big Dye terminator cycle sequencing, using the universal M13 reverse and forward primers,

with an ABI310 automated sequencer. The sequence editing, analysis and prediction of amino

acid sequences were conducted using the EditSeq program of the Lasergene package

(DNASTAR Inc., Madison, USA). Sequences were compared to the TGEV Purdue 115 S

gene and other TGEV S gene sequences of the GenBank. Sequence alignments were carried

out with the MegAlign program (Lasergene) by Clustal multiple alignment algorithm.

12

RESULTS

Upon the initial sampling of the first affected farm, TGEV was detected as the only

significant pathogen in two of the four piglets that were sacrificed for the purpose and no

other pathogen was isolated or detected from the two other piglets. Group 1 coronavirus

immunohistochemistry on small intestinal samples gave dubious results, in the two TGEV-

positive cases, mild villous atrophy was seen without any other significant gross or

microscopic alteration in these. As the work done by the author of this thesis was limited to

the molecular biological investigations, and to the evaluation of the serological results, none

of the other results will be presented in details here. They will only be mentioned in the

discussion to underline conclusions.

Reverse transcriptase polymerase chain reaction

The results of the rt-PCR reactions indicated that out of the close to 150 samples

originating from 14 different swine herds only four herds were TGEV positive. The farms,

based on their TGEV/PRCoV profiles could be divided into three groups.

In the first group pigs that suffered from clinical signs associated with enzootic TGE

usually had either PRCoV- or TGEV-S gene sequences in their samples. These gene

fragments were estimated by the electrophoresis to be 550bp and 1250 bp for PRCoV- and

TGEV-S genes respectively. Mixed infections were rarely detected. The PRCoV-S gene

fragments were uniform in size. The typical picture seen after evaluating the rt-PCR by

electrophoresis can be seen in figure 3.

Figure 3. Agarose gel electrophoresis of porcine coronavirus S gene amplicons, samples are from piglets from a herd where clinical signs of enzootic TGEV infection was observed. 1-7: PRCoV-S amplicons with an estimated size of 550bp, 8: TGEV-S amplicon with an estimated size of 1250bp, M: 1kb DNA marker (Fermentas) P: positive control, full size 1285bp TGEV-S gene, N: negative control, sterile water.

13

The second group also consists of animals that were showing clinical signs of enzootic

TGEV infection. Although in this group S genes of a variety of sizes were detected (panel A

of Figure 4). Besides the full size fragment of the original TGEV-S (1258 bp, confirmed by

sequencing) a variety of smaller sized fragments were also detected, dominating among them

one with an approximate size of 600 bp and another with an approximate size of 250 bp, as

judged by the agarose gel electrophoresis. These three fragments were selected for cloning

and sequencing. Besides these three S gene variants, others of intermediate sizes were also

detected, but cloning of these minor fragments was unsuccessful, or when successfully cloned

and sequenced, the sequence did not show any homology with coronavirus genomes (data not

shown). The original full size fragment of the TGEV-S was found together with the smaller

fragments in the same animal, which sets this group apart from the first one. This so because

in the first group mixed infections were rare.

The third and last group consists of animals that did not show clinical signs of

enzootic TGEV infection. In these farms the TGEV-S genes were not detected (panel B of

Figure 4). The S gene amplicons were of approximately 550-600 bp in length, consistent with

that what would be expected in PRCoV positive animals. In this group smaller fragments

were also present.

Figure 4. Electrophoresis of PCR amplicons of samples collected at a TGEV positive (panel A) and a TGEV negative but PRCoV positive (panel B) pig farm. Where fragment size has been indicated it is to be regarded as an estimate and refers to the strongest band(s). 1A: PRCoV-S amplicon 250bp, 2A: PRCoV-S amplicon 550-600bp 3A, 6A: TGEV-S amplicon 1250bp, PRCoV-S amplicon 600-550bp and PRCoV-S amplicon 250bp, 4A: PRCoV-S amplicons, 5A: negative sample, 1B-8B: PRCoV-S amplicons, 550-600bp, M: 1kb DNA marker (Fermentas) P: positive control, full size 1285bp TGEV-S gene, N: negative control, sterile water.

When comparing the presence of TGEV and PRCoV in different organs, like the small

intestine, lungs or lymph nodes, no difference could be observed. Usually lung and gut tissues

or intestinal contents carried the virus. Hence organ preference or tropism could not be

observed.

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Cloning

Cloning of the PCR fragments into the TOPO® TA Cloning vector was successful for

the 1258 bp and the estimated 550-600 bp and 250 bp fragments of the TGEV/PRCoV cases.

Figure 5 shows as an example the results of BamHI digested clones of the 550-600 bp inserts,

together with an intact 1258 bp TGEV-S amplicon.

Figure 5. BamHI digested cloned PCR fragments separated by agarose gel electrophoresis. M: 50 bp molecular mass ladder (Fermentas). The band indicated with a white circle is an intact 1285 bp long S gene amplicon. The bands indicated with black circles are successfully cloned 550-600 bp long fragments.

The cloning of some other fragments was unsuccessful, probably due to the low

amount of DNA amplicons produced by PCR. Where the intermediate sizes were successfully

cloned the sequencing revealed that they were not of coronavirus origin but some unrelated,

mostly bacterial nucleotides. Cloning of individual fragments of the mixed sized amplicons

was necessary, as direct sequencing of the amplified products is not possible in such cases.

Sequence analysis

Sequence analysis determined the exact size of the three amplicon types that were

successfully cloned. The largest fragment was 1258 bp as expected, the fragments originally

estimated to be between 550 and 600 bp had a size of 586 bp. The shortest S gene amplicon

with an estimated size of 250 bp was somewhat larger than expected, reaching 283 bp in

length.

The Genbank search of these nucleotide sequences showed that they were almost

identical with the TGEV Miller M6 strain type viruses (Genbank accession Number:

DQ811785) when considering the appropriate gene portion, with only a few nucleotide

differences. When comparing the fragments to each other it was obvious that the same

15

coronavirus existed in at least three different forms if looking only at the S gene. Namely the

full length TGEV-S type, a shorter one with a 672 nt deletion at the amino terminal half

coding region of the S gene (characteristic to the European type of PRCoV sequences, where

RBS-1 is completely removed), and a further deletion mutant where the deletion was

extended with an additional 303 nt towards the 3’ end of the gene. Still all of these variants

carried the sequence characteristics of the TGEV identified during this study, when only

looking at the non-deleted parts of the gene. All of these deletions retained the functionality of

the remaining portion of the S protein, as the number of nucleotides missing did not alter the

reading frame of the gene.

Serology

Serological tests were done by the scientists at the Large Animal Clinic of the Faculty

of Veterinary Medicine located in Üllő. The results are summarized in Table 2, where the

ELISA positivity of pigs from the original TGEV cases is shown both for TGEV and for

PRCoV.

Table 2. The ELISA results from blood samples collected from 15 primiparous and the 15 multiparous sows in a TGEV-PRCoV differentiating test.

TGEV ELISA PRCoV ELISA

Primiparous Positive Suspect Negative Positive Suspect NegativeNo. 0 6 9 11 0 4% 0 40 60 73,33 0 26,67

Older sows Positive Suspect Negative Positive Suspect NegativeNo. 5 8 2 15 0 0% 33,33 53,33 13,33 100 0 0

16

DISCUSSION

The first appearance of transmissible gastroenteritis (Doyle and Hatchings, 1946)

when pig herds without any immunity encountered the virus was devastating. This epizootic

form of the virus was typical in newly affected herds, with 100% mortality rate of newborn

animals. In older animals the clinical signs were restricted to a mild diarrhea and decreased

production. As the epizootic proceeded worldwide and more and more herds seroconverted,

the mortality rate dropped to 10-50% in newborns, depending on maternal antibody levels.

Still, even with its sporadic epizootic and characteristic enzootic form TGE was one of the

major viral diseases of the swine industry until the appearance and gradual spread of the

mutant PRCoV strains. The origin of PRCoV is not known, it was suspected that some

attenuated vaccine started an individual spread worldwide. But the sequence differences and

differences in the site and size of deletions among the identified strains indicated otherwise

(Figure 6). It seems more likely that changes in the environment, mostly the pig itself, led to

the simultaneous appearance of the mutated viruses (Tuboly, 1995).

Although the protection of the PRCoV induced antibodies is only limited against

TGEV, as an essential receptor binding site of the PRCoV-S protein is usually missing from

these viruses, the constant presence of the new virus and the PRCoV antibodies resulted in a

decrease of the incidence of clinical TGEV in the PRCoV infected herds. By the mid 1990s

only sporadic cases of TGE were reported. Today TGEV is usually considered as a disease of

the past, something that nature itself got rid of. However, there has been occasional reports of

TGEV seropositivity (Elvander et al., 2000, Brendtsson et al., 2006), indicating that the virus

was still present, but at levels below the threshold of clinical manifestation.

17

TGEV-S

100 200 300 4001

1 aa

374

2 aa

374219

1 aa

1447 aa

92 and 94 218

1 aa1-1 aa

21 245224 aa

207 aa

226 aa

227 aa

15

16 242

222

62 289

TGEVwith lower pathogenicity

PRCV in Europe

PRCV in North America

Figure 6. Summary of the mutated TGEV/PRCoV variants detected worldwide, based on GenBank data (Tuboly, unpublished). The upper line indicates the globular amino terminal half of the TGEV-S protein in a linearized form. The boxed areas show deletion mutants of TGEV. Numbers above the lines show the position in the number of amino acids (aa) from the initial part of the gene, those bellow the lines indicate the size of the deletion also expressed as number of aa.

Based on the results of this study, the herd where clinical signs and histopathology

raised the suspicion of a TGEV infection, was indeed proven to be TGEV positive. This is

based on both serological and polymerase chain reaction tests. When the survey was extended

to other herds, the presence of TGEV could be confirmed with PCR in four pig farms. From

the re-emergence one would expect that a new, perhaps more virulent TGEV strain is

emerging, one that is capable of breaking through the immunity induced by PRCoV.

However, the sequencing results indicated that the viruses were very similar to already known

TGE viruses, namely to those of the Miller M6 strain (Zhang et al., 2007). In order to decide

if this truly is a new genetic variant of the virus with higher virulence, other regions of the

genome must be amplified and sequenced.

18

The PRCoV sequences detected in this study can be separated into two different

groups one with a 672 nt deletion and in the other group the deletion was altogether 975 nt

long. The 672 nt deletion (compared to the TGEV-S gene) was identical to what was observed

for the PRCoV strains widespread in Europe, where a 224 aa coding region was deleted

starting at the position of amino acid number 21 of the S gene (Figure 6). It is however

peculiar that after several years of establishing the genetic characteristics of the PRCoV

strains in Europe the same deletion mutant is still present in pig herds, considering the genetic

instability of coronaviruses discussed in the introduction.

The presence of the PRCoV genomes with an even longer deletion within the S gene

was surprising as such large deletions starting at the same site as the previous one and

extending 303 nt further into the 3’ direction has not been reported previously. It raises the

question if such deletion mutants can form infectious particles, or if they can only survive

when packaged into virions produced by the co-existing longer genomes. The possibility that

this deletion mutant remains infective is likely as the altogether 975 nt deletion by itself

should not thwart virus infection. This is so because the RBS for aminopeptidase-N is

encoded from nucleotide 1215 of the gene, which is further downstream on the sequence as

regards to the end of the deletion. Without structural studies of such an S protein variant it is

of course difficult to tell how the lack of such a long protein stretch may change the

conformation of the second receptor binding site.

Our results cannot explain the re-emergence of the virus in clinical conditions, but

from this limited survey it seems that primiparous sows did not completely seroconvert to

TGEV, therefore their piglets probably were not fully protected. PRCoV seronegativity was

detected in a limited number of primiparous sows, indicating that cross-protection might also

be suboptimal. These results indicate that the answer to why TGE is re-emerging lies not

within the genetics of the virus but most likely in the immune response of the pigs. It is

known that porcine circoviruses (PCV) are present worldwide and they are strongly

immunosuppressive (Ramamoorthy and Meng, 2009). Vaccine or infection induced immunity

is generally limited in heavily infected pig herds, even if clinical signs of the circovirus

infection are not apparent. Although we have no direct proof of this assumption, namely that

the presence of PCV is responsible for the TGEV problem, serological results show that

primiparous pigs may remain seronegative both for TGEV or PRCoV. Similar results had

been reported from China (Chen, 2010, personal communication) the world's largest pork

producer, where TGEV again is becoming an important economic threat.

19

Specific prevention of the disease is not possible as a TGEV vaccine is currently

unavailable in Hungary. We have no data on the safety and efficacy of coronavirus vaccines

developed for other species (e.g. cats) used in sows. Back-feeding of intestinal contents of

succumbed or euthanized piglets to pregnant gilts could be a way of exposing gilts to the virus

and boosting maternal antibody levels, however, this technique cannot be recommended as it

may be the source of further infections.

As a final remark it should be noted that no disease can be fully forgotten and

considered a disease of the past. The environment and its inhabitants in which we keep pigs is

subject to constant change. These changes are as unpredictable as the lottery and might within

a short period of time provide an entirely new playing field, where diseases considered

something of the past might re-emerge and again cause problems.

20

ACKNOWLEDGEMENTS

The study was financed in part by the EU SSA-NMSACC-PCVD 518432 grant. The

author is in great debt to his supervisors providing unequaled assistance and guidance in his

work to prepare this thesis. Without their support it would not have been possible to either

perform or finish the work needed. The author thanks Dr. Attila Cságola for her help with

RNA extraction and PCR. The skilled technical help from Irénke Herbák, laboratory

technician, is appreciated. The colleagues at the Large Animal Clinic were very helpful in

providing data about the clinical, pathological and serological results. It must be stated that it

has been an altogether pleasant experience working together with this fantastic group of

people.

Finally I am as always, greatly appreciative of my partner Mia Karlsson for providing

full support when ever needed, making sure that I always land on my feet.

21

SUMMARY

Transmissible gastroenteritis (TGE) of pigs is an enteric disease caused by a porcine

coronavirus. It can affect animals of any age but is most commonly affecting younger

animals. TGEV belongs to the Coronaviridae family, which constitutes enveloped viruses

with an unusually large single stranded RNA genome (up to 28-30 kilobases) of positive

orientation. Although the virus can infect pigs at any age, the clinical signs are less

pronounced in adults. Piglets however, especially during the first week of life develop

devastating intestinal infections. Hallmark clinical signs are mainly vomiting and diarrhea.

Mortality rate in piglets without maternal immunity can reach 100%.

TGE was among one of the most important swine diseases until the mid 1980’s. Then

a deletion mutant, namely the porcine respiratory coronavirus (PRCoV) with no or very

limited pathogenicity emerged and spread around the world. The widespread PRCoV

infection led to the gradual disappearance of TGE, due to the cross-protective immunity

induced by the new virus.

Recently more and more cases of piglet diarrhea with an unclear aetiology have been

reported. This study is a summary of such a case, where TGEV genome was detected by

polymerase chain reaction (PCR) in a swine herd. In this case it was pigs at the age of

weaning that were showing signs of diarrhea. Other viral, bacterial or parasitic infections were

excluded by appropriate laboratory investigations, but a parallel presence of PRCoV in the

affected animals was detected.

The re-emergence of TGEV was confirmed by sequencing the PCR generated

amplicons (targeting the spike, S gene). Apart from the deletion at the site of the S gene the

genome sequences were identical in PRCoV and TGEV cases. The reason for the re-

emergence of the pathogenic coronavirus is not fully understood. But based on the widespread

presence of porcine circoviruses, which are well known for their immunosuppressive nature,

it was speculated and later confirmed by serological tests that there is a decreased cross

protection between PRCoV and TGEV. This decrease in cross protection could be in the

background of the TGEV induced clinical problems.

22

ÖSSZEFOGLALÁS

A transmissible gastroenteritis (TGE) egy sertés coronavírus által okozott

emésztőszervi megbetegedés, amely valamennyi korcsoportot érintheti, leggyakrabban

azonban a fiatal állatokban okoz jelentős klinikai tüneteket. A kórokozó a Coronaviridae

családba tartozik, egy szokatlanul nagy, egyszálú, pozitív irányultságú RNS genomot (28-30

kilobázis) tartalmazó burkos vírus. Bár a vírus minden korcsoportot megbetegíthet, felnőtt

állatokban a klinikai tünetek kevéssé kifejezettek, elsősorban egy hetes kor alatti malacokban

fejlődik ki a súlyos emésztőszervi fertőzés. A tünetek döntően hányás és hasmenés, maternális

immunitás nélkül a mortalitás elérheti a 100%-ot is.

A TGE az 1980-as évek közepéig az egyik legfontosabb vírusos eredetű

sertésbetegség volt világszerte, amíg egy nem, vagy kevéssé patogén deléciós mutáns, név

szerint a sertések légzőszervi coronavírusa (porcine respiratory coronavirus, PRCoV) tűnt fel

és terjedt el a korábban TGE-vel fertőzött állományokban. A PRCoV elterjedése a TGE

fokozatos eltűnéséhez vezetett, mivel az új vírus keresztvédettséget képes indukálni.

Újabban mind gyakrabban jelentkeznek tisztázatlan etiológiájú hasmenéses

megbetegedések sertésekben. Ez a tanulmány egy olyan eset összefoglalása, amelyben TGE

vírus genomot mutattunk ki polimeráz láncreakció (polymerase chain reaction, PCR)

segítségével egy sertéstelepen, választás körüli malacok hasmenéses eseteiből. Más vírusos,

baktériumos és parazitás fertőzést nem sikerült laboratóriumi módszerekkel detektálni, de

párhuzamosan a PRCoV jelenléte kimutatható volt az érintett állatokban.

A TGE vírus megjelenését igazolta a PCR termék (a spike, S gén) szekvencia

elemzése. Az S génen lévő deléciós helyek alapján a PRCoV és a TGE vírus

megkülönböztethető. A patogén coronavírus ismételt felbukkanásának oka még nem teljesen

tisztázott, de a sertés circovírus elterjedésének és immunszuppresszív természetének

köszönhetően felmerült, majd később szerológiai teszttel igazolódott, hogy a TGE és PRCoV

közötti keresztvédettség csökkent. A keresztreakció csökkenése állhatott annak a hátterében,

hogy a TGE vírus idézte elő a klinikai tüneteket.

23

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