characterization of an h3n2 canine influenza virus isolated from tibetan mastiffs in china
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Veterinary Microbiology 162 (2013) 345–352
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aracterization of an H3N2 canine influenza virus isolated from Tibetanastiffs in China
aoyang Teng a,1, Xu Zhang b,1, Dawei Xu a, Jiewen Zhou a, Xiaoguang Dai a, Zhaoguo Chen a,jun Li a,*
nghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, PR China
nzhen Institute of Advanced Technology, Chinese Academy of Science, Shenzhen 518055, PR China
ntroduction
Dogs were not considered a reservoir species foruenza A viruses until January 2004, when H3N8 canineuenza virus was first isolated from racing greyhoundscted with respiratory disease in Florida (Crawford et al.,5). Since then, the H3N8 virus has been identified inst states in the contiguous USA, affecting both racingyhounds and pet dogs (Gibbs and Anderson, 2010). Thesmission of H3N8 equine influenza viruses to dogs has
n reported in Australia and the United Kingdomkland et al., 2010; Newton et al., 2007; Daly et al.,8). In 2007, canine influenza caused by an H3N2uenza virus was reported in South Korea (Song et al.,
2008). Between May 2006 and October 2007, 4 H3N2canine influenza virus isolates were identified from nasalswabs collected from sick dogs at animal clinics inGuangdong province in China (Li et al., 2010).
Phylogenetic analysis indicated that the H3N2 canineinfluenza viruses belonged to a different cluster than thoseof the H3N8 equine and canine influenza viruses (Songet al., 2008). All 8 genes of these H3N2 canine influenzaviruses were �94.7% homologous to Asian H3N2 avianinfluenza viruses (Li et al., 2010). It was hypothesized thatvirus transmission occurred through feeding dogs infectedpoultry products or by aerosol transmission (Gibbs andAnderson, 2010). However, although virus isolates that aregenetically similar to H3N2 canine viruses have beendetected in birds, it is unclear whether the H3N2 canineviruses can re-infect birds, or be transmitted from birds toother dogs.
A serologic survey of H3N2 canine influenza virus wasconducted in farmed and pet dogs in South Korea from Juneto December 2007, and the results indicated that the virus
T I C L E I N F O
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A B S T R A C T
Ten 3-month-old Tibetan mastiffs became ill 2 days after they were bought from a Tibetan
mastiff exhibition, and 4 of them died 2 weeks later. A canine influenza virus (ZJ0110) was
isolated from the lung of a deceased Tibetan mastiff and was characterized in detail.
Sequence analysis indicated that the 8 genes of the canine isolate were most similar to
those of avian-origin canine influenza viruses (H3N2) isolated in South Korea in 2007, with
which they shared >98% sequence identity. ZJ0110 could experimentally infect 6-month-
old beagles by intranasal inoculation and by airborne transmission, causing severe
respiratory syndrome. Moreover, ZJ0110 could replicate in the upper respiratory tracts of
mice and guinea pigs, and the virus titer was comparable to that in the upper respiratory
tracts of dogs. Although the virus was genetically of avian origin, ZJ0110 could not
experimentally infect chicken or ducks by intranasal inoculation. These results suggest
that dogs might be an intermediary host in which avian influenza viruses adapt to
replicate in mammals.
� 2012 Elsevier B.V. All rights reserved.
Corresponding author. Tel.: +86 2 134 293 446;
+86 2 134 293 446.
E-mail address: [email protected] (Z. Li).
These authors contributed equally to this paper.
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology
jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic
8-1135/$ – see front matter � 2012 Elsevier B.V. All rights reserved.
://dx.doi.org/10.1016/j.vetmic.2012.10.006
Q. Teng et al. / Veterinary Microbiology 162 (2013) 345–352346
had spread rapidly through the local dog population (Leeet al., 2009). The transmission of H3N2 canine viruses fromdog to dog by contact was proven through experimentallyinfected and contact-exposed dogs (Song et al., 2009).Airborne transmission through aerosols and large dropletsplays an important role in the spread of human influenza,but it is unknown whether airborne transmission of H3N2canine influenza virus occurs between dogs.
In 2010, we isolated an H3N2 canine influenza virusfrom the lung of a Tibetan mastiff bought from a recent dogexhibition in eastern China. Herein, we describe thebiological properties of this canine influenza virus isolate.
2. Materials and methods
2.1. Outbreaks
In March 2010, ten 3-month-old Tibetan mastiffsstarted to have fever, cough, purulent nasal discharge,and loss of appetite 2 days after they were bought from 3different exhibit areas in a Tibetan mastiff exhibition inZhejiang province of China. Two weeks later, 4 of themdied; the others recovered. Nasal swabs were collectedfrom these Tibetan mastiffs 7 days after they became ill.Lung samples were collected from the dogs that died.
2.2. Virus isolation
The nasal swabs and homogenated lung samples wereused to isolate the influenza A virus by inoculation into 10-day-old specific pathogen-free (SPF) embryonated chickeneggs. After 3 days of inoculation, the allantoic fluids werecollected, and the hemagglutination (HA) test wasperformed using chicken erythrocytes (Rivailler et al.,2010). The fluids shown to agglutinate chicken erythro-cytes were stored at �80 8C until use for virologic andmolecular analyses.
The allantoic fluid from samples capable of agglutinat-ing chicken erythrocytes was tested using a commercialrapid influenza virus antigen detection kit (QuickingBiotech Co., Ltd, Shanghai, China). Hemagglutinin inhibi-tion (HI) tests were performed according to the recom-mendations of the World Organization for Animal Health.
2.3. RT-PCR and sequencing
Viral RNAs were extracted from allantoic fluid using theTrizol reagent (Invitrogen, Carlsbad, CA) according to themanufacturer’s instructions. Reverse transcription wasperformed under standard conditions using 12 bp primers(AGCAAAAGCAGG). PCR amplification was performedusing universal primers for influenza A virus (Hoffmannet al., 2001). PCR products were directly sequenced usingthe BigDye1 Terminator v3.1 Cycle Sequencing Kit (ABI,Foster, CA, USA) and an ABI 3500 genetic analyzer. Tocompile and analyze the sequences, we used the SEQMANprogram (DNASTAR, Madison, WI). The sequences ofreference viruses were obtained from GenBank using thebasic local alignment search tool (BLAST) at the NationalCenter for Biotechnology Information (NCBI). The nucleo-tide sequences were compared and phylogenetic trees
were generated using the MEGALIGN program (DNASTAR)with the Clustal alignment algorithm. The sequences ofcanine, avian, equine, human, and swine influenza virusesselected from GenBank were used as references (data notshown). The reliability of phylogenetic inference at eachtree node was estimated by the bootstrap method with1000 replications, using the MEGA package. In addition,the signal peptide was predicted using the signalP version2.0 (SignalP-NN and SignalP-HMM software) of SignalPWorld Wide Web server (Bendtsen et al., 2004).
2.4. Animal experiments
All the animal experiments were approved by theReview Board of Shanghai Veterinary Research Instituteand by the Animal Care and Use Committee of ShanghaiVeterinary Research Institute. All the data was analyzedusing the PASW Statistics 18.0 software.
2.5. Dogs
To determine the pathogenicity of the virus isolate, six10-week-old beagle puppies (Pet Kennel Breeding Center,Suzhou) free of influenza virus infection (group I) wereinoculated intranasally with 107 50% infectious doses foreggs (EID50) ZJ0110 virus in a volume of 1 mL of allantoicfluids. Animals were housed in a very large cage in abiosafety level II room. Three beagles inoculated intrana-sally with PBS in a volume of 1 mL were used as negativecontrols (group II) and housed separately. Before inocula-tion, the beagles were sedated by intramuscular injectionof 0.1 mg/kg acepromazine malate (Shengda AnimalMedicine Company, Jilin, China). Four days after inocula-tion, 3 beagles in group I were euthanized. Samples of nasaltissue, trachea, lung, kidney, brain, liver, pancreas, spleen,and blood were collected and homogenized in 1 mL of coldphosphate-buffered saline per gram of organs. Solid debriswas pelleted by centrifugation. Virus titers of undilutedand 10-fold serially diluted supernatants were determinedin 9–11-day-old embryonated eggs, and 3 eggs were usedper dilution. The HA titer of allantoic fluid was tested 72 hpost inoculation, and eggs with an HA titer of 8 or morewere considered positive. On days 2, 4, and 6 afterinoculation, the other 3 beagles in group I and all 3 beaglesin group II were sedated, and their nasal tracts werewashed using 5 mL PBS. The nasal washes were collected,and virus titers were determined using 9-day-old SPFembryonated chicken eggs. The beagles in groups I and IIwere monitored daily for 14 days for signs of disease, andthese beagles were euthanized on day 14 post inoculation.Samples of nasal tissue, trachea, and lung were collectedfor virus titration and blood samples were collected for HItest.
To test the airborne transmissibility of the virus, 3 naı̈vedogs (group III) were put into a cage that was placed 0.5 maway from the cage in which the 6 intranasally inoculatedbeagles were housed. This exposure took place on the daythat the 6 beagles were inoculated with ZJ0110. On days 2,4, and 6 after exposure, the beagles in group III weresedated, and their nasal tracts were washed using 5 mLPBS. The nasal washes were collected, and virus titers were
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Q. Teng et al. / Veterinary Microbiology 162 (2013) 345–352 347
ermined using 9-day-old SPF embryonated chickens. Six days after exposure, all 3 beagles in group III werehanized. Samples were collected, and virus titers wereermined. For histopathological study, samples wered in 10% neutral buffered formalin solution, sectioned, stained with hematoxylin and eosin. Alternatively,unohistochemical analysis was conducted with a
noclonal antibody against the NP protein of influenzaviruses. To avoid transmission of the virus fromculated dogs to naı̈ve dogs via investigators, the beaglesgroup III were treated first each time, and all thesonal protective equipment (PPE) was autoclaved afterestigators had contacted with the inoculated dogs.To verify the airborne transmissibility of ZJ0110, 2gles were inoculated intranasally with 107 EID50
110 viruses in a volume of 1 mL of allantoic fluids housed in a large cage. Two days later, 3 naı̈ve beagles
re housed in another cage, which was placed 0.5 may from the cage in which the 2 intranasally inoculatedgles were housed. The beagles were monitored forical symptoms, and seroconversions were tested bylyzing hemagglutination inhibition against ZJ0110 on 21 after exposure. To avoid transmission of the virus
inoculated dogs to naı̈ve dogs via investigators,asures were taken as described previously.
2.6. Mice and guinea pigs
To test the infectivity of ZJ0110 virus in mice, eleven 6-week-old SPF female BALB/c mice (Shanghai Slac Labora-tory Animal Co. Ltd, Shanghai) were lightly anesthetizedwith CO2 and inoculated intranasally with 106 EID50 ofZJ0110 virus in a volume of 50 mL. Three mice wereeuthanized on days 4 and 6 for virus titration of the lung,nasal turbinate, kidneys, spleen, and brain. Organs werecollected, homogenized, and the virus titrated as pre-viously described. The remaining 5 mice were monitoreddaily for 14 days for weight loss and mortality. As anegative control, 5 mice were inoculated with PBS tocompare weight loss and mortality with infected mice.
To investigate the infectivity of ZJ0110 virus in guineapigs, six 8-week-old guinea pigs free of influenza virus(Shanghai Slac Laboratory Animal Co. Ltd) were lightlyanesthetized by intramuscular injection of 0.1 mg/kgacepromazine malate (Shengda Animal Medicine Com-pany, Jilin, China) and inoculated intranasally with106 EID50 of ZJ0110 virus in a volume of 100 mL. Threeguinea pigs were euthanized on days 4 and 14 fordetermination of virus titers in the lungs, nasal turbinate,kidneys, spleen, and brain. As a negative control, 3 guineapigs were inoculated with PBS and euthanized on day 14.
le 1
no acids specific to H3N2 canine influenza viruses.
nes Bird H3N2
consensus
ZJ0110 Canine H3N2
consensus
Equine H3N8
consensus
Canine H3N8
consensus
Human H3N2
consensus
Swine H3N2
consensus
T26 A26 A26 T25 T25 T26 T26
D97 N97 N97 Y96 Y96 N97 N97
L127 I127 I or V127 I127 I127 L127 L127
D188 N188 N188 K187 K187 E or D188 E or D188
W238 L238 L238 W238 L238 R238 W238
H451 N451 N or K451 H450 H450 H451 H451
D505 N505 N505 Y504 Y504 D505 D505
M24 L24 L24 L24 L24 M24 M24
E or D54 K54 K54 N54 N54 E54 E or K54
P156 S156 S156 L156 L156 P156 P156
R432 G432 G432 E432 E432 R432 R432
M105 V105 V105 M105 M105 M105 M or I105
T or A373 K373 K373 T373 T373 N373 A, V or N373
A428 T428 T428 A428 A428 A428 A428
N or S473 K473 K473 N473 N473 N473 N or S473
S65 Y65 Y65 S65 S65 L65 S65
T208 A208 A208 T208 T208 S/T208 T208
A369 V369 V369 A369 A369 A369 A369
M441 K441 K441 M441 M441 M441 M441
K615 R615 R615 K615 K615 K615 K615
1 L or F108 I108 I108 L108 L108 L108 L108
S or R361 N361 N361 S361 S361 R361 R or K361
D377 N377 N377 E377 D377 D377 D377
I397 T397 T397 V397 V397 I397 I397
M or I744 V744 V744 M744 M744 M744 M744
2 I147 T147 T147 V147 V147 I147 T147
M365 I365 I365 M365 M365 M365 M365
M570 V570 V570 M570 M570 M570 M570
2 S22 L22 L22 S22 S22 S22 S22
2 M or Q14 I14 I14 M14 M14 L14 M14
no acid residue (single-letter code) and position in the mature proteins of H3 subtype influenza viruses.
Q. Teng et al. / Veterinary Microbiology 162 (2013) 345–352348
Organs were collected, homogenized, and virus titersdetermined as mentioned above.
2.7. Chickens and ducks
To investigate the infectivity of ZJ0110 virus in chickensand ducks, eleven 6-week-old SPF chickens and 9-week-old ducks (a local outbreed strain of shelducks), whichwere proved to be free of either the influenza A virus or theantibody against the virus, were inoculated intranasallywith 106 EID50 of ZJ0110 virus in a volume of 100 mL andhoused in different isolators. Three chickens and 3 duckswere euthanized on days 4 and 6 for determination of virustiters in the lungs, trachea, liver, pancreas, kidneys, spleen,and brain. In addition, as a negative control, 5 chickens and5 ducks were inoculated with PBS. The remaining infectedand control chickens and ducks were continually mon-itored for 14 days for clinical symptoms and seroconver-sions were analyzed on day 14 after inoculation. Duringthese days, pharyngeal and cloacal swabs were obtainedfor virus titer determination on days 3, 5, 7, 10, and 14 afterinfection.
3. Results
3.1. Virus isolation
Influenza A virus was isolated from nasal swabs of sickTibetan mastiffs and homogenated lungs of deceased
Tibetan mastiffs by inoculation into SPF embryonatedchicken eggs. The allantoic fluids of the eggs inoculatedwith virus induced agglutination of chicken erythrocytes.Allantoic fluids were positive for influenza A virus in assaysusing a commercial rapid influenza virus antigen detectionkit. The virus was identified as H3 subtype influenza virusby the HI test. The virus isolate from homogenated lungtissue of a deceased dog was named as A/canine/Zhejiang/01/2010 (ZJ0110) and was selected for further character-ization.
3.2. Sequence analysis
Eight gene segments of ZJ0110 were amplified by RT-PCR using the universal primers for influenza A viruses,and the PCR products were sequenced. The sequences(GenBank accession JF714149–JF714156) were submittedto GenBank at NCBI. The HA and NA genes of the virusisolate shared a high degree of homology with avian-originH3N2 canine influenza viruses when analyzed using BLASTat the NCBI. The influenza virus isolate was designated A/canine/Zhejiang/01/2010(H3N2) (ZJ0110).
The critical determinants of host species specificity ofinfluenza virus are distributed over several genes (Suzukiet al., 2000). To identify residues within the 8 genes thatmay be associated with adaptation of a bird virus to thecanine host, we compared the amino acid sequences ofcanine H3N2 influenza virus with those of other H3viruses. Thirty amino acid substitutions differentiate the
Nucleotid e Substitu tions (x100)
0
17.3
246810121416
A/canine /Korea /1/2010 (H3 N1)
A/canine /Korea /GCVP01/2007 (H3 N2)
A/canine /Korea /LBM412/2008(H3N2)
A/canine/Zhej iang/1/2010( H3N2)
A/feline/Kor ea/01/2010 (H3 N2)
A/canine /Ji angsu/02/2010(H3 N2)
A/canine /Ji angsu /01/2009 (H3 N2)
A/canine /Guangdong/1 /2006(H3N2)
A/canine /Guangdong/2 /2006(H3N2)
A/canine /Guangdong/2 /2007(H3N2)
A/canine /Guangdong/1 /2007(H3N2)
A/chicken/Korea /LPM88/2006(H3N2)
A/duck/Korea/D42/2007(H3N2)
A/duck/Korea/D130/2007(H3N8)
A/chicken/Korea /LPM17/2004(H3N2)
A/duck/Korea/D126/2007(H3N6)
A/duck/Korea/GJ108/2007 (H3 N2)
A/wild bird/Korea /8G50/2005(H3N8)
A/wild bird/Korea /6D1/2005(H3N8)
A/Chicken/Nanchang /7-010/2000 (H3 N6)
A/swine/Korea /CY04/2007 (H3 N2)
A/swine/Minneso ta/1145/2007 (H3 N2)
A/Boston/1/2008(H3N2)
A/Canterbury /200/2004 (H3 N2)
A/canine /Colorado/30604/2006 (H3 N8)
A/canine /Florid a/242 /2003(H3N8)
A/equine /Gansu/7 /2008(H3N8)
A/equine /Xinj iang/1/2007 (H3 N8)
Fig. 1. Phylogenetic trees for the HA genes of H3N2 influenza A viruses. The tree was generated using MEGALIGN software (DNASTAR) on the basis of the HA
gene sequences: nucleotides 30–1731 (1702 bp) of HA. The length of each pair of branches represents the distance between sequence pairs, and the units at
the bottom of the tree indicate the number of substitution events. Virus sequences referred to in the tree were obtained from GenBank.
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Q. Teng et al. / Veterinary Microbiology 162 (2013) 345–352 349
canine influenza viruses from avian H3N2 influenzaviruses (Table 1). In the HA protein, leucine was foundat position 238 only in the canine influenza viruses; thisobservation was true for both the H3N2 and H3N8subtypes.
3.3. Phylogenetic analysis
Phylogenetic comparison of the H3 nucleotidesequences showed that ZJ0110 was most closely relatedto the H3N2 influenza virus isolates found in South Korearecently (Fig. 1). ZJ0110 and other canine H3N2 virusisolates formed a sub-clade that was separated from theclades of bird H3 influenza viruses. This clade was 1 of 3that together included all H3 subtype influenza viruses(Fig. 1). The phylogenetic comparisons of ZJ0110 and A/canine/Korea/GCVP01/2007 (H3N2; one of the canineinfluenza virus isolates in South Korea in 2007) withrespect to the PA, PB1, PB2, HA, NP, NA, M and NS genesshowed 99.0%, 99.2%, 99.0%, 99.2%, 99.2%, 98.7%, 99.1%, and98.4% identity, respectively.
3.4. Pathogenicity of the ZJ0110 virus in canines
To assess the pathogenicity of the ZJ0110 virus indogs, 6 beagles were inoculated intranasally withZJ0110. The dogs presented with sneezing, coughing,and a runny nose 2 days after inoculation; fever began 3days after inoculation. The body temperature of dogsreturned to normal 7 days after inoculation. However,sneezing and cough continued until the dogs wereeuthanized on day 14 after inoculation (Fig. 2A). Thebody temperatures of puppies inoculated with CIV weresignificant higher than these of control puppies(p < 0.05) on days 3, 4 and 5 post inoculation. Virusesin nasal washes collected from 3 intranasally inoculatedbeagles were titrated in 9-day-old SPF embryonatedchicken eggs (Fig. 2B). The virus titers were 102.75–103 EID50/mL on day 2 after inoculation and increased to103.5–103.75 EID50/mL on day 4 after inoculation. On day6 after inoculation, the virus titer of nasal washes from 1beagle decreased to 103 EID50/mL, and those from theother 2 beagles remained at 103.5 EID50/mL (Fig. 2B). Thevirus titers in the nasal tract, trachea, and lung tissues ofthe intranasally inoculated beagles euthanized on days 4and 14 after inoculation were also determined aspreviously described. The virus titers were 101.5–102.5 EID50/g in nasal tissues, 102.5–103.5 EID50/g intracheal tissues, and 102.5–102.75 EID50/g in lung tissuesof the beagles euthanized on day 4 after inoculation.Surprisingly, even on day 14 after inoculation, virus
2. The pathogenicity of ZJ0110 in puppies. Six 10-week-old influenza
s infection-free beagle puppies were inoculated intranasally with
EID50 ZJ0110 virus. Three naı̈ve puppies were introduced and placed
away from the puppies previously inoculated with ZJ0110 virus.
control puppies were inoculated intranasally with PBS. (A) The body
peratures of 3 puppies inoculated with ZJ0110 virus, 3 indirect-
sure puppies, and 3 control puppies were recorded daily. The body
peratures of puppies inoculated with CIV were significant higher than
e of control puppies (p < 0.05) on days 3, 4 and 5 post inoculation. The
y temperatures of indirect-exposure puppies raised and were
ificant higher than these of control puppies (p < 0.05) on day 6
exposure. (B) Nasal washes were collected from 3 puppies inoculated
ZJ0110 virus, 3 indirect-exposure and 3 control puppies on days 2, 4
6 after infection, and the virus titers were determined using 9-day-
embryonated chicken eggs. The virus titers of the nasal washes from
pies inoculated with ZJ0110 virus were significantly higher than those
e indirect-exposure puppies (p < 0.01) on days 2 and 4 post infection.
s titers from nasal washes of puppies inoculated with ZJ0110 virus
e not significantly different to those from the indirect-exposure
pies (p > 0.05) at 6 days post-infection. (C) Three puppies inoculated
ZJ0110 virus were euthanized on day 4 after inoculation, and 3
rect-exposure puppies were euthanized 2 days later. Samples of
rent tissues were collected and titrated for virus infection in eggs. The
s titers of nasal and lung tissue of inoculated puppies euthanized on
4 post infection were significantly lower than those of indirect-
sure puppies euthanized on day 6 post exposure (p < 0.01). The titers
tracheal tissue of inoculated puppies euthanized on day 4 post
ction were not significantly different from those of indirect-exposure
pies euthanized on day 6 post exposure (p < 0.01). The virus titers of
infection were significantly lower than the titers in both the inoculated
puppies euthanized on day 4 and the indirect-exposure puppies
euthanized on day 6 post exposure (p < 0.05). However, the titers of
lung tissue from inoculated puppies euthanized on day 14 post infection
were significantly higher than those from inoculated puppies euthanized
on day 4 post infection (p < 0.01) and the indirect-exposure puppies
euthanized on day 6 post exposure. The titers of nasal tissue from
inoculated puppies euthanized on day 14 post infection were not
decreased compared to those of puppies euthanized on day 4 post
heal tissue from inoculated puppies euthanized on day 14 post infection (p > 0.05).
Q. Teng et al. / Veterinary Microbiology 162 (2013) 345–352350
titers in lungs were high, at 104.25–104.75 EID50/g. Virustiters in nasal and tracheal tissues decreased to 101.25–101.5 and 0–101.75 EID50/g, respectively (Fig. 2C). Novirus was detected in the brain, kidney, liver, or spleen ofthe infected beagles. Among the blood samples collectedon day 14 post inoculation, sera collected from 1 beaglein group I had HI activity and was associated with an HItiter of 1:80. The sera collected from the remaining 2beagles in group I and the beagles in group II did notshow any HI activity.
To test the transmissibility of the virus, 3 naı̈ve beagleswere exposed to the virus by indirect contact on the daywhen the beagles were inoculated with ZJ0110. On days 2,4, and 6 after contact, nasal washes were collected fromthese beagles. The virus in nasal washes was not detectableon day 2 after exposure; however, on day 4, virus titersincreased to 102.75–103.0 EID50/mL, and on day 6, virustiters reached 103.0–104.5 EID50/mL (Fig. 2B). On day 6 afterexposure, virus titers in the nasal tract, trachea, and lungtissues of the beagles with indirect exposure were 103.25–104.5, 102.25–102.5, and 103.25–103.5 EID50/g, respectively(Fig. 2C). To verify the airborne transmissibility of ZJ0110, 3naı̈ve beagles were exposed indirectly to the dogs thatwere intranasally inoculated with ZJ0110 before 2 days.The beagles started to present with sneezing, coughing,and a runny nose on day 6 after exposure; fever wasobserved 7 days after exposure. The body temperature ofthe dogs returned to normal on day 10 after exposure, andthe cough lasted for 2 weeks. Serum antibody titers were
measured by HI test against ZJ0110 on day 21 afterexposure. The HI titers of the 3 dogs reached to 320, 640,and 160, respectively.
Histopathological analysis revealed heterophil granu-locyte infiltration, exudative inflammation, and pulmon-ary congestion in all of the beagles that were inoculated orhad indirect exposure (Fig. 3A). Immunohistochemicalanalysis showed large amounts of viral antigens in the lungtissues of these beagles (Fig. 3C).
3.5. Pathogenicity and replication of ZJ0110 virus in mice
Mice inoculated intranasally with 106 EID50 of ZJ0110virus lost weight on days 1 and 2 after inoculation,followed by a slow increase in weight and recovery on day10 after inoculation (Fig. 4A). In mice inoculated intrana-sally with 106 EID50 of ZJ0110 virus, virus titers in lungsand nasal turbinates were 104.5–105.75 and 102.5–103.5 EID50/mL, respectively, on day 4 after inoculation.Even on day 6 after inoculation, the virus titers were still ashigh as 103.25–105.25 EID50/g in lungs and 102.25–102.5 EID50/g in nasal turbinates (Fig. 4B). ZJ0110 viruswas not detected in other tissues, including the brain,spleen, liver, and kidneys.
3.6. Replication of the ZJ0110 virus in guinea pigs
The virus replicated well in the nasal turbinates ofguinea pigs inoculated intranasally with 106 EID50 of
Fig. 3. The Image of lesion found on the lung of puppies infected by ZJ0110. Histopathological studies showed (A) severe pneumonia in the lung tissue of
infected puppies and (B) normal lung section in uninfected puppies. The lung sections of (C) infected or (D) uninfected puppies were subjected to
immunohistochemistry with a monoclonal antibody against NP of influenza A virus. Viral antigens were detected in the lung tissue of infected puppies, but
no antigens were detected in lung tissue of uninfected puppies.
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Q. Teng et al. / Veterinary Microbiology 162 (2013) 345–352 351
110 virus. The virus titers ranged from 104.25 to 104.5 on 4 after inoculation. Surprisingly, on day 14 afterculation, the virus titers remained at 102.75–.75 EID50/g in nasal turbinates of guinea pigs (Fig. 5).
ever, the virus was not detected in the lungs or otherans.
3.7. Pathogenicity of the ZJ0110 virus in chickens and ducks
To investigate the infectivity of ZJ0110 virus in chickensand ducks, the virus titers in the lung, trachea, liver,pancreas, kidneys, spleen, and brain of chickens and ducksinoculated intranasally with 106 EID50 of ZJ0110 virus wereanalyzed. ZJ0110 virus was not found in any of theseorgans, neither on day 4 nor day 6 after inoculation. Inaddition, no virus was detected in the pharyngeal or cloacalswabs collected on days 3, 5, 7, 10, and 14 after infection,and no seroconversion was observed in the samples fromthe inoculated chickens or ducks by performing the HI test.These results suggested that the ZJ0110 virus had lost theability to replicate in chickens and ducks.
4. Discussion
Influenza viruses that cause illness in canines are aconcern for canine health worldwide (Gibbs and Anderson,2010). Since the isolation of H3N8 canine influenza virusfrom racing greyhounds in 2004, the virus has reportedlycaused respiratory outbreaks among pet dogs in the USA(Crawford et al., 2005). The transmission of H3N8 equineinfluenza viruses to dogs has been reported in Australia andthe United Kingdom (Kirkland et al., 2010; Newton et al.,
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5
10
15
20
25
30
0 1 2 3 4 5 6 7 8 9 10
PBS control
10^6EID5 0 ZJ011 0
A
wei
gh
t ch
an
ges
(%
)
days post- inocula tion
0
1
2
3
4
5
6
7
Mouse 1 Mouse 2 Mouse 3 Mouse 4 Mouse 5 Mouse 6
Day 4 post-infection Day 6 post-infection
)g/))
05
DIE(
01
gol( retit s
uriV
Lun g Nasal turbinate
B
4. The pathogenicity of ZJ0110 in mice. BALB/c mice were inoculated intranasally with 106 EID50 of ZJ0110 virus in a 50 mL volume or with PBS as
rol. (A) The weight of infected mice was significantly lower than that of the control mice on each day after intranasal inoculation (p < 0.05). (B) The
ns of mice inoculated with 106 EID50 of ZJ0110 virus or with PBS were collected on days 4 and 6 post inoculation, and clarified homogenates were
ted for virus infectivity in eggs at initial dilutions of 1:10 and undiluted if negative at the lowest dilution. The virus titers in lungs or in nasal tissue were
significantly different between days 4 and 6 after inoculation (p > 0.05).
0
1
2
3
4
5
Guineapig
1
Guineapig
2
Guineapig
3
Guineapig
4
Guineapig
5
Guineapig
6
Day 4 post-infection Day 14 post-infection
5. The pathogenicity of ZJ0110 in guinea pigs. Guinea pigs were
ulated intranasally with 106 EID50 of ZJ0110 virus in a 100 mL volume.
gs were collected on days 4 and 14 after inoculation, and clarified
ogenates were titrated for virus infectivity in eggs. The virus titers in
s were not significantly different between these days post
ulation (p > 0.05).
Q. Teng et al. / Veterinary Microbiology 162 (2013) 345–352352
2007). In 2007, the H3N2 influenza virus was first isolatedfrom miniature schnauzer, cocker spaniel, and Jindo dogs inSouth Korea (Song et al., 2008). In this study, we isolated theH3N2 influenza virus from the Tibetan mastiff, which is anancient breed of domestic dog (Canis lupus familiaris) thatoriginated with nomadic cultures of Central Asia.
Host species specificity of influenza virus has beenattributed to multiple amino acids in several genes (Suzukiet al., 2000). The receptor-binding sites on HA and sites 627and 701 on PB2 have proven to be important in interspeciestransmission of avian influenza virus to mammals (Hattaet al., 2001; Li et al., 2005; Skehel and Wiley, 2000). In thisstudy, we identified 30 amino acids that distinguish thecanine H3N2 influenza viruses from other H3 viruses (Table1). Inthe HA protein, leucine was presentat position 238 onlyin the canine influenza viruses, including both the H3N2 andH3N8 subtypes. Whether these amino acids play animportant role in the adaptation of avian influenza virusto dogs is unclear.
Dogs infected with canine influenza virus excrete virusin nasal discharge but not in feces (Song et al., 2008), andthe virus can be transmitted to susceptible dogs throughdirect contact with dogs who are experimentally infected(Song et al., 2009). These findings suggested that dog-to-dog transmission of subtype H3N2 occurs through thenasal route. In the present study, we clearly demonstratedthat the H3N2 canine influenza virus is transmissible tosusceptible dogs through airborne transmission. InfluenzaA viruses are reported to be shed for 2–4 weeks by ducks,and 45 days by pheasants (Humberd et al., 2007; Websteret al., 1992), but are rarely reported to be shed for morethan 10 days post-infection in mammals. In this study, wefound that ZJ0110 could exist at a high titer for up to 14days in the lungs of infected dogs. The reason for this viralpersistence in the lungs should be investigated in futurestudies.
Influenza viruses are transmitted from the avianreservoir to other birds and mammals relatively fre-quently, yet they do not typically establish permanentlineages in these new hosts (Webster, 2002). However,we know little about transmission of canine influenzaviruses to birds and mammals. Experimental inductionof the disease caused by this isolate resulted in severepathologic changes and showed that the virus titersremained high from 4 to 14 days after infection in thelungs of infected dogs. These findings indicate that theinfected dogs excreted influenza virus (H3N2) for a longperiod of time. Influenza viruses of the H3 subtype haveproven to be highly adaptable and are able to recruitavian, mammalian, and human hosts (Harder andVahlenkamp, 2009). In this study, we showed thatthe canine H3N2 influenza virus was able to infectmice and guinea pigs but not ducks or chickens. Thisfinding suggests that canine H3N2 influenza virus hasadapted to replicate in different mammal species.Although the canine H3N2 virus is genetically andantigenically different from strains currently circulatingin humans, we should remain aware of the potential ofthis virus to be transmitted to the human populationfrom pet dogs.
Acknowledgments
This study was supported by the National NaturalScience Foundation of China (no. 31172332), theMinistry of Agriculture’s Major Projects for TransgenicResearch Program, China (no. 2009ZX08010-022B), theSpecial Fund for Agro-scientific Research in the PublicInterest, China (no. 201003012), the Special Fund forInternational Communication and Cooperation (no.S2011ZR0429), and the National High TechnologyResearch and Development Program of China (863Program) (no. 2011AA10A200).
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