Isolation and Identification of a New Strain of Porcine Transmissible Gastroenteritis Virus from Chongqing, Southwestern China

December 9, 2015 — admin
AttachmentSize
zhenhui.pdf3.48 MB
Embedded Scribd iPaper - Requires Javascript and Flash Player

Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Zhenhui, S. 22
Isolation and Identifcation of a New Strain of Porcine Transmissible
Gastroenteritis Virus from Chongqing, Southwestern China
Zhenhui, S.,* Xianjin, D., Xinzhi, C.,Yue, Z., Yuntian, L., and Yong, L.
Department of Veterinary Medicine, Southwest University, Rongchang Campus, Chongqing 402460, P.R. China.
Zhenhui S. and Xianjin D. have made an equal contribution to this article.
*
Corresponding Author: Assistant Professor Zhenhui SONG, PhD., Department of Veterinary Medicine, Southwest University, Rongchang Campus,
Chongqing 402460, P.R.China. Tel: 00-86-23-46751087, Mobile: 00-86-15736158518, Fax: 00-86-23-46751732. E-mail: szh7678@126.com
ABSTRACT
Porcine transmissible gastroenteritis virus (TGEV) infections continue to cause signifcant losses in the
swine industry. Te objective of the study was to document the isolation a new strain of TGEV, information
which may also contribute signifcantly to understanding molecular epidemiology and evolution of TGEV in
China. In this study, a suspected transmissible gastroenteritis virus (TGEV) strain was isolated from faeces
of sick piglets in Chongqing (China) using swine testis (ST) cell lines. With the total RNA extracted from
infected ST cells, the M gene of TGEV was amplifed by polymerase chain reaction (PCR). Te virus was
named as the CQ strain, and was also identifed by homology analysis, cell cytopathic efects (CPE), indirect
fuorescent antibody test (IFA) and electron microscopy (EM). Te results indicated that this virus was TGEV
by a series of identifcation tests. Te membrane proteins M gene was cloned and sequenced. Alignment
with other 21 reference strains of TGEV and 1 strain PRCV in GenBank, showed that the homology was
94.3-99.6% for nucleotide sequence, 92.4-98.9% for amino sequence, respectively. Te phylogenetic analysis
has indicated that TGEV strains can be classifed into two clusters, the CQ strain belonged to cluster I, and
shared closest relationship with Purdue-P115.
Keywords: Identifcation; Isolation; Southwestern China; Transmissible gastroenteritis virus;
Sick piglets
INTRODUCTION
Transmissible gastroenteritis virus (TGEV) is the etiologi-
cal agent of transmissible gastroenteritis (TGE), which is a
condition associated with high morbidity in pigs of all ages
and high mortality in sucking piglets. TGEV is a member
of the coronaviridae family, which possesses a large 28.5 kb
single-stranded sense RNA genome, and is comprised of four
structural proteins encoded by the spike (S), membrane (M),
envelope (sM), and nucleoprotein (N) genes. Te S protein
forms the peplomers on the virion envelope and carries major
antigenic sites which induce neutralizing antibodies (1, 2).
Te M protein is embedded in the lipid envelope, taking part
in virus particle assembly, and the N protein is associated
with the genomic RNA to form the nucleocapsid, inducing
cell immunity to infected animals. Te small sM protein is
localized in the perinuclear region of infected cells, and is
expressed on the cell surface, possibly causing apoptosis (3-6).
TGEV has been reported in many swine producing
countries between the late 1980’s and the 1990’s (7-9). In
China, an outbreak of porcine transmissible gastroenteritis
was frst reported in the 1970’s. Since then, the disease has
been prevalent in the country. Vaccination is an efective
prophylactic measure. Although there are many commercial
vaccines, the traditional inactivated vaccines have many de-
fciencies. Terefore, the disease is still a major problem in
the swine industry in China (10, 11).
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 23 New Strain of Porcine TGEV in China
Te main clinical symptoms of the disease in the infected
piglets are diarrhea and vomiting. TGE occurs in most of
swine-raising farms with death in 100% of suckling pigs less
than 1 week old resulting in severe economic losses (12).
Although TGE occurs frequently in China, the TGEV strains
isolated from Southwest China had rarely been reported (13).
Until now, the characterization of TGEV strains isolated
from Hebei, Sichuan, Fujian, Jilin provinces have been re-
ported in the years of 2010-2013 (14-16). Information about
the history, incidence and prevalence of the TGE occurring
southwestern China is limited. Here, we report the TGEV
strain CQ isolated from infected piglets in Chongqing prov-
ince in southwestern China by using isolation in cell cultures,
fuorescent antibody assay, electron microscopy and PCR
amplifcation. Te key objective of this work was to detect
TGEV form clinically suspected diseased piglets based on
serological and molecular basis, which may help to reveal
the epidemiology, diversity, and evolution of isolated strains
in China, and potentially facilitate the development of more
efective preventive measures for TGEV.
MATERIALS AND METHODS
Virus isolation
Samples: Tirty two samples of faeces were collected from
sick piglets with symptom for diarrhea, vomiting and dehy-
dration in the winter months, which may have been infected
with TGEV according to clinical signs (Figure 1). A total of
1g of faeces was homogenized in 4 ml of phosphate-bufered
saline (PBS, pH 7.2) and then freeze-thawed three times
and then centrifuged at 5000 rpm for 30 min. 1 mL of the
supernatant (1:10 dilution of samples) was fltered and in-
oculated into monolayer cultures of swine testis (ST) cells
for 1 hour. Dulbecco’s modifed eagle medium supplemented
with 10% fetal bovine serum up to 5ml was then added. Te
ST cultures were observed for 5-7 days for the presence of
cytopathic efects (CPE). Te cultures showing cytopathic
efects were frozen and thawed, supernatants were collected
after centrifugation with subsequent storage at -70°C for
virus isolation.
Identifcation of the virus
TGEV was detected by indirect fuorescent antibody test
(IFA) and electron microscopy (EM). For IFA, 100 TCID
50
of TGEV was inoculated to 24-well tissue culture plates
containing confuent ST cell monolayer. Te plates were
incubated at 37°C with 5% CO
2
. When CPE was observed
in these wells, the supernatant was removed and the cell sheet
fxed in cold acetone for 20 min at -20°C and stained by an
IFA technique (17).
For electron microscopy, the virus released from cell
culture was mounted on grids and negatively stained in
2% phosphotungstic acid and observed under transmission
electron microscopy.
RNA extraction, PCR amplifcation and sequencing
Total RNA was isolated from infected ST cells using
TRIZOL reagent (TaKaRa Biotechnology (Dalian Co.,
Ltd., China)) according to the manufacture’s protocol. In
short, 200 µl of infected ST cell was incubated with 1 ml of
TRIZOL for 5 min at room tempera-
ture. 0.2 ml of chloroform was added.
After vortexing, the mixture was incu-
bated for 5 min at room temperatuer.
Te phases were separated by centrifu-
gation (12,000×g at 4°C for 15 min)
and the aqueous phase was transferred
to a new ependorf tubes. 1×volume of
isopropyl alcohol and 0.1×volume of 3
M sodium acetate were added to this
aqueous phase and incubated for 30
min at -20°C.
Te precipitated RNA was pelleted
by centrifugation (12,000×g at 4°C for
15 min), and after the removal of super- Fig 1: Pathological changes from the intestines of a sick piglet with suspected TGEV.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Zhenhui, S. 24
natant, the RNA pellet was washed twice with 70% ethanol.
After drying, the RNA was resuspended in 25 µl of DEPC-
treated water. Using mRNA as template, single-stranded
cDNAs were generated by AMV (TaKaRa Biotechnology
(Dalian Co., Ltd., China)) according to the manufacturer’s di-
rections. Te primers sequences used in this study were as fol-
lows: Sense primer: 5’-GGGGGATCCCCACCATGAAGA
TTTTGTTAAT-3’; Antisense primer: 5’-GGGG
zAATTCTTATACCATATGTAATAATTTTTCTTG-3’.
All primers were synthesized by Sangon Biotech Co., Ltd.
(Shanghai, China). Te PCR conditions were 94°C for 2 min,
followed by 30 cycles of DNA amplifcation (30 s at 94°C,
30 s at 55°C, and 1 min at 72°C) and 10 min incubation at
72°C. Te PCR products were separated by electrophoresis,
purifed and cloned into the pMD18-T vector (TaKaRa
Biotechnology (Dalian Co., Ltd., China)) and sequenced
by using classical dideoxy Sanger sequencing (TaKaRa
Biotechnology (Dalian Co., Ltd., China)).
Analysis of sequence
Homologous comparisons of the M gene nucleotide sequenc-
es and deduced amino acids of the CQ strain with those of
other 23 virus strains (including TGEV 22 strains: 96-1993
(AF104420); 133 (AF481365); attenuated H (EU074218);
AYU (HM776941); DAE ( JQ693054); FJ ( JQ700303); H16
(FJ755618); HN2002 (AY587883); HX (KC962433); KT2
( JQ693055); KT3 ( JQ693056); Miller M6 (DQ811785);
Miller M60 (DQ811786); PUR46-MAD (AJ271965);
Purdue P115 (DQ811788); SC-Y (DQ443743); TFI
(Z35758); TO14 (AF302262); TS (AY335549); Virulent
Purdue (DQ8117890; WH-1 (HQ462571), PRCV 1 strain:
PRCV ISU-1 (DQ811787) were performed with DNAStar
software (http://www.dnastar.com). A phylogenetic tree was
generated based on M protein sequences of reference TGEV
strains, Bovine coronavirus 1 strain 179-07-11 (EU019216),
Feline coronavirus 1 strain 79-1683 (AB086904) and Canine
coronavirus 1 strain HF3 (AY864661) by applying the
distance-based Neighbor-joining method in the software
MegAlign (DNASTAR Inc., Madison, WI, U.S.A).
RESULTS
Isolation and identifcation
Tirty two samples with a clinical diagnosis of suspected
TGE were subjected to TGEV RT-PCR assays. Twenty
samples tested resulted in the expected band sizes of am-
plifed products (data not shown). Te isolation of TGEV
from one sample positive for RT-PCR testing was obtained
by ST cell cultures. Te primarily cytopathic efects (CPE)
were observed in ST cell infected with 10 passages virus. In
comparison to the control cells, cells with cytopathic changes
showed cell rounding, strong refraction, cell aggregation and
coalesence to form clusters after 48 h of post inoculation
which gradually increased to afect 95% of cells which became
completely detached. (Figure 2). Te indirect fuorescent an-
tibody test (IFA) showed that specifc fuorescence appeared
Fig 2: Te CPE of TGEV. A: Te ST cells as negative control; B: Te CPE of CQ strain.
Te primarily cytopathic efects (CPE) were observed in ST cell infected with 10 passages virus. Te ST cells generated CPE cell lesions: cell
rounding, refraction, aggregation and coalescence.
A B

Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 25 New Strain of Porcine TGEV in China
in the cytoplasm of infected ST cells, which suggested that
the isolated virus might be TGEV (Figure 3). Te negatively
stained virus particles extracted from the suspension of cell
infected virus were approximately 149 nm in diameter when
examined by the electron microscope (Figure 4), and ap-
peared with morphology similar to TGEV.
Amplifcation, sequencing and analysis of M gene
Te ST infected cells with the isolate were positive by the
RT-PCR assays, and the expected sizes 789bp of the PCR
products were observed as a clear electrophoretic band
(Figure 5). Te obtained M genes segments by sequencing
have been submitted for GenBank under accession No.
KF273109. Alignment with other 21 reference strains of
TGEV and 1 strain PRCV in GenBank, showed that the
homology was between 94.3-99.6% for nucleotide sequence
(Table 1) and 92.4-98.9% for amino sequence (Table 2),
which shows highly homologous between the isolated strain
and other TGEV strains.
Phylogenetic analysis
To better understand the relationship of the isolate to other
strains of TGEV, genetic sequences of M in GenBank were
used to construct phylogenetic trees. Te multiple sequence
alignments showed that there is 3 nucleotide deviations,
nt19(A→G), nt164(A→G), nt249(T→C), resulting in 2 dif-
ferences (Ile6→Met6, Asn55→Ser55) in the M gene of CQ
strain compared with other strains of TGEV. Genetic and
phylogenetic analysis have shown that transmissible gastro-
enteritis virus have been divided into two clusters (I and II),
cluster I included the American Purdue and cloned strains,
partial Chinese isolates and strains isolated in Korea. Cluster
II included the American Miller and attenuated Miller
strain, partial Chinese strains TS, HN2002, FJ, H16 and
Fig 3: IFA identifcation in ST cells. A: Uninoculated ST control; B: Specifc fuorescence was produced in infected ST cells.
Te indirect fuorescent antibody test (IFA) showed that the specifcity fuorescence appeared in cytoplasmic infected ST cells.

A B
Fig 4: Electron microscopy: Negatively stained TGEV.
Te negatively stained virus particles extracted from suspension cell
infected virus were approximately 149 nm in diameter.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Zhenhui, S. 26
Fig 5: Te amplifed products by RT-PCR and enzyme identifcation of recombinant plasmids.
Lane 1 and Lane 2: M gene from infected ST cell; Lane 3, Lane 4 and Lane 5: pMD18-M digested by BamHI and EcoRI.
Table 1: Homology of M gene sequence of diferent TGEVs
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 27 New Strain of Porcine TGEV in China
Table 2: Homology of M amino acid sequence of diferent TGEVs
Fig 6: Phylogenetic analysis of M gene sequence of diferent TGEV isolates.
Table 1, 2: Alignment with other 21 reference strains of TGEV and 1 strain PRCV in GenBank, showed that the homology were 94.3-99.6% for
nucleotide sequence, 92.4-98.9% for amino sequence, respectively, which shows highly homologous between CQ strain and other TGEV strains.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Zhenhui, S. 28
Chinese vaccine strain attenuated H (18). Multiple sequence
alignment in this study also indicated that the nt
70
, nt
189,
nt
190
in M gene of genotype cluster I TGEV strains were T, C, G,
respectively. However, the cluster II TGEV strains was G,
T, A, respectively. Te isolated strain was found to belong to
cluster I, showing a closer evolutionary relationship with the
American Purdue-P115 strain (19) (Figure 6). Analysis of
the phylogenetic trees revealed that the two clusters (I and
II) of TGEVs were distinct from the non-Chinese TGEVs,
96-1933 (United Kingdom), TFI (Taiwanese), TO14 ( Japan)
and other coronaviruses HF3 (Canine coronavirus), WSU
79-1683 (Feline coronavirus), 179-07-11 (Bovine corona-
virus) isolated previously. Terefore, based on the results
of gene sequencing and homology analysis of M gene, the
TGEV isolate was named as CQ strain, in combination with
the results of CPE, IFA and EM.
DISCUSSION
In this study, faeces samples were collected from sick piglets
showing clinical signs during winter season in Chongqing,
southern China. Twenty clinical samples showed positive
results for RT-PCR assay. Te feld strain of the virus from
one sample positive for RT-PCR testing was isolated from
inoculated ST cells. Te results of IFA, electron microscopic
examination and RT-PCR tests all showed the virus was
TGEV. CPE were observed after 10
th
passage. ST cell culture
showed characteristic cytopathic efect with cell rounding,
strong refraction, cell aggregation, coalesence to form clusters
after 48 h of post inoculation and gradually increasing to
afect 95% of cells which became completely detached. All
the changes are consistent with CPE of TGEV infecting ST
cell, which agrees with our previous reports (20-22).
Te isolated CQ strain was identifed by molecular
cloning, homology analysis, IFA test and EM observation.
Characteristic specifc intracytoplasmic fuorescence ap-
peared in IFA. Electron microscope examination showed
the characteristic morphology of the virion. PCR was the test
of choice for rapid detection and identifcation of the TGE
outbreak causative agent. Te PCR assay used in this work
showed high specifcity as a unique band of the expected
size (~789bp) was obtained for RNA samples derived from
infected ST cells by CQ strain.
Te results of multiple sequence alignments showed that
three nucleotide deviations existed in the M gene of the CQ
strain. Previously reported studies have suggested that amino
acid mutations in the M protein afected its ability to induce
IFN-α and M protein has a potential role in TGEV virulence
(23, 24). Further studies will therefore be needed to ascertain
the roles of the two amino acid mutations in M protein of
the CQ strain. Te M gene has been regarded as a highly
conserved TGEV gene, but comparison of the sequences
of TGEV M genes revealed that the M gene of M60 strain
had a 6 nucleotide insertion, predicting a membrane protein
2 amino acids longer than in the other strains. PRCV ISU-1
had a 3 nucleotide deletion in the M gene compared with
TGEV strains, predicting an M protein of 261 amino acids
in length. Te amino insertion in TGEV is suggestive of the
M protein being subject to environmental selection pressure
(25).
Genotype classifcation and clustering is based on diver-
gence in the viral genome sequences revealed by phylogenetic
analysis (26-30). Wang et al., using phylogenetic analysis
indicated the Chinese TGEVs were divided into three
groups (G1, G2, and G3). Analysis of the phylogenetic trees
revealed that the G3 Chinese TGEVs represented a separate
group that was distinct from the non-Chinese TGEVs and
from Chines TGEVs isolated previously (31). Phylogenetic
analysis based on 3a and 3b genes of TGEV strains showed
that the Chinese strains were more closely related to TGEV
strains H165, H16, Miller M6, Miller M60, TS, and CHV
than other reference strains (32, 33). In this present study,
based on phylogenetic comparison, the TGEV strains can
be classifed into the two main clusters: cluster I and cluster
II. Te isolated TGEV strains from China SC-Y, HX, AYU,
WH-1 and CQ belonged to cluster I, while other Chinese
strains H16, FJ, HN2002, TS and Chinese vaccine strain at-
tenuated H belonged to cluster II. So it appears that there are
two main clusters prevailing in China. Genetic and phyloge-
netic analysis based on M protein sequences showed that CQ
strain shared the closest relationship with Purdue-P115 than
other TGEV strains or PRCV ISU-1. Trough comparing
the obtained sequences with the known sequences listed in
the GenBank database, we also found that three characteristic
nucleotides including nt
70
, nt
189,
and nt
190
in the M gene of
TGEV may be used to diferentiate the genotype cluster I
from cluster II.
In conclusion, the present study described the isolation
and identifcation of TGEV isolated from sick piglets for the
frst time in Southwest China and named as the CQ strain.
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 29 New Strain of Porcine TGEV in China
According to the results of gene sequencing and homology
analysis of M gene, this study has identifed and analyzed a
new strain of the TGEV isolated from southwest in China,
which may be helpful to enrich the genetic information for
molecular epidemiological investigation and evolution of
TGEV. Furthermore it will be important to perform further
studies to select appropriate vaccine virus for local TGE
prevention.
ACKNOWLEDGMENTS
Financial support for this work was provided by grants from
the Chongqing Basic Research Program (2008bb5243), the
Fundamental Research Funds for the Central Universities
(XDJK2010C093), the Fundamental Research Funds
for the xinjianguighur autonomous region universi-
ties (XJENU2012118), and Urumqi Research Program
(Y121210005).
REFERENCES
1. Bernard, D., Denis, R., Murielle, G., Jacqueine, G. and Hubert,
L.: Four major antigenic site of the coronavirus transmissible gas-
troenteritis virus are located on the amino-terminal half of spike
glycoprotein S. J. Gen. Virol. 71: 1313-1323, 1990.
2. Isabel, C., Gustavo, J., Carlos, S., Maria, J. B. and Luis, E.: Anti-
genic structure of the E2 glycoprotein from transmissible gastro-
enteritis coronavirus. Virus Res. 10: 77-93, 1998.
3. Enjuanes, L., Brian, D. and Cavanagh, K.: Virus Taxonomy. Clas-
sifcation and Nomenclature of Viruses-Coronaviridae. Academic
Press. New York, 2000.
4. Laude, H. and Masters, P.S.: Te coronaviridae-Te Coronavirus
Nucleocapsid Protein. Plenum press. New York, 1995.
5. Murielle, G., Rene, L. H., Jean, F.V. and Hubert, L.: TGEV corona
virus ORF4 encodes a membrane protein that is incorporated in
virions. Virol. 188: 666-675, 1992.
6. Li, D., Huang, Y., Du, Q., Dong, F., Zhao, X.M., Zhang, W.L.,
Xu, X.G. and Tong, D.W.: TGEV nucleocapsid protein induces
cell cycle arrest and apoptosis through activation of p53 signaling.
Biochem. Biophys. Res.Comm. 445: 497-503, 2014.
7. Prichard, G.C.: Transmissible gastroenteritis in endemically in-
fected breeding herds of pigs in East Anglia, 1981-1985. Vet. Rec.
120: 226-230, 1987.
8. Wood, E.N., Pritchard, G.C. and Gibson, E.A.: Transmissible
gastroenteritis of pigs. Vet. Rec. 108: 41, 1981.
9. Woods, R.D. and Wesley, R.D.: Immune response in sows given
transmissible gastroenteritis virus or canine coronavirus. Am. J.
Vet. Res. 47: 1239-1242, 1986.
10. Johnson, M., Fitzgerald, G. and Welter, M.W.: Six most common
pathogens responsible for diarrhea in newborn pigs. Vet. Med. 87:
382-386, 1992.
11. Moxley, R.A. and Olson, L.D.: Clinical evaluation of transmis-
sible gastroenteritis virus vaccines and vaccination procedures
for inducing lactogenic immunity in sows. Vet. Res. 50: 111-118,
1989.
12. Zhou, J.F., Huang, F., Hua X.G., Li, C., Zhang, W., Shen, Y.,
Yan, Y.J., Chen, P.R., Ding, D.Z., Mou, J., Chen, Q., Lan, D.L.
and Yang, Z.B.: Inhibition of porcine transmissible gastroenteritis
virus (TGEV) replication in mini-pigs by shRNA. Virus Res.
149: 51-55, 2010.
13. Fan, J.H., Zuo, Y.Z., Zhao, Y.L., Li, T.Q. and Zhang, X.B.: Clon-
ing and expression of nucleocapsid protein gene of TGEV HBo6
strain. Front. Agric. China. 1: 357-360, 2007.
14. Hou, Y.X., Yue, X.W., Cai, X.H., Wang, S.J., Liu, Y.G., Yuan,
C.L., Cui, L. and Xiu, G.H.: Complete Genome of Transmissible
Gastroenteritis Virus AYU Strain isolated in Shanghai, China. J.
Virol. 86: 11935, 2012.
15. Wang, C., Chen, J., Shi, H., Qiu, H., Xue, F., Liu, C., Zhu, Y., Liu,
S., Almazan, F., Enjuanes, L. and Feng, L.: Molecular characteriza-
tion of a Chinese vaccine strain of transmissible gastroenteritis
virus: mutations that may contribute to attenuation. Virus Genes.
40: 403-409, 2010.
16. Yu, F.S., Wang, S., Chen, S.L., Cheng, X.X., Huang, W.Q., Jiang,
B., Chen, S.Y. and Shao, L.P.: Isolation and identifcation of Por-
cine Transmissible Gastroenteritis Virus FJ strain. Fujian J. Agric.
Sci. 27: 1160-1164, 2012.
17. Zaher, K.S. and Ahmed, W.M.: Investigations on Bovine Ephem-
eral Fever Virus in Egyptian cows and bufaloes with emphasis
on isolation and identifcation of a feld strain. Global Veterinaria.
6: 447-452, 2011.
18. Sanchez, C.M., Jimenez, G., Laviada, M.D., Correa, I., Sune, C.,
Bullido, M.J., Gebauer, F., Smerdou, C., Callebaut, P., Escribano,
J.M. and Enjuanes, L.: Antigenic homology among coronaviruses
related to transmissible gastroenteritis. Virol. 174 : 410-417,
1990.
19. Zhang, X.S., Mustafa, H., David, S., Rebecca, H., Wang, S.L.,
Sarah, S., Daniel, J., Nagesh, H., Tang, Y.X., Elodie, G. and Linda,
S.: Complete genomic sequences, a key residue in the spike protein
and deletions in nonstructural protein 3b of US strains of the
virulent and attenuated corona viruses, transmissible gastroenteritis
virus and porcine respiratory coronavirus. Virol. 358: 424-435,
2007.
20. Song, Z.H. and Guo, W.Z.: Isolation and Genomic Sequence
Analysis of Porcine Transmissible Gastroenteritis Virus. Chin. J.
Virol. 24: 364-368, 2008.
21. Yan, Q.G., Ou, Y., Guo, W.Z., Feng, T., Fan, W. Q., Lai, W.L.,
Cao, H.Z. and Li, B.: Isolation of SC-1 strain of transmissible
gastroenteritis virus of swine and characteristic analysis of gene
7. Chin. J. Vet. Sci. 27: 613-617, 2007.
22. Zhang, X., Hongyan, S., Chen, J.F., Shi, D., Dong, H. and Feng,
F.: Identifcation of the interaction between vimentin and nucle-
ocapsid protein of transmissible gastroenteritis virus. Virus Res.
200: 56-63, 2015.
23. Baudoux, P., Carrat, C., Besnardeau, L., Charley, B. and Laude, H.:
Coronavirus pseudoparticles formed with recombinant M and E
proteins induce alpha interferon synthesis by leukocytes. J. Virol.
72: 8636-8643, 1998.
24. Zuo, H., Dante, S.Z., Karol, S., Siqingaowa, S. and Ren, X.F.:
Research Articles
Israel Journal of Veterinary Medicine  Vol. 70 (4)  December 2015 Zhenhui, S. 30
Transmissible gastroenteritis virus: Identifcation of M protein-
binding peptide ligands with antiviral and diagnostic potential.
Antiviral Res. 99: 383-390, 2013.
25. Li, J. Q., Cheng, J. and Lan, X.: Complete genomic sequence of
transmissible gastroenteritis virus TS and 3’ end sequence char-
acterization following cell culture. Virol. Sin. 25: 213-224, 2010.
26. Evermann, J. and Ridpath, J.: Clinical and epidemiologic observa-
tions of bovine viral diarrhea virus in the northwestern United
States. Vet. Microbiol. 89: 129-139, 2002.
27. Sun, X.H., Fu, S.H., Gong, Z.D., Ge, J.Q., Meng, W.S., Feng, Y.,
Wang, J.L., Zhai, Y.G., Wang, H.Q. and Nasci, R.: Distribution
of arboviruses and mosquitoes in northwestern Yunnan Province,
China. Vector Borne Zoonotic Dis. 9: 623-630, 2009.
28. Vilek, Paton, D., Durkovic, B., Strojny, L., Ibata, G., Moussa, A.,
Loitsch, A., Rossmanith, W., Vega, S. and Scicluna, M.: Bovine
viral diarrhea virus genotype1 can be separated into at least eleven
genetic groups. Arch. Virol. 146: 99-115, 2001.
29. Wang, H.Y., Takasaki, T., Fu, S.H. and Liang, G.D.: Molecular
epidemiological analysis of Japanese encephalitis virus in China.
J. Gen. Virol. 883: 885-894, 2007.
30. Wang, L.H., Fu, S.H., Zhang, H.L., Ye, X.F., Yu, D.S., Deng, Z.,
Yuan, J., Zhai, Y.G., Li, M.H. and Chen, W.X.: Identifcation
and isolation of Genotype-1 Japanese Encephalitis virus from
encephalitis patients. Virol. J. 7: 345-348, 2010.
31. Wang, C.B., Chen, J.F., Shi, H.Y., Qiu, H.J., Xue, F., Liu, S.W.,
Liu, C.M., Zhu, Y.M., Fernando, A., Luis, E. and Feng, L.:
Rapid diferentiation of vaccine strain and Chinese feld strains
of transmissible gastroenteritis virus by restriction fragment length
polymorphism of the N gene. Virus gene. 41: 47-58, 2010.
32. Becher, P., Orlich, M., Kosmidou, A., Baroth, M. and Tiel, H.:
Genetic diversity of pestiviruses: identifcation of novel groups and
implications for classifcation. Virol. 262: 64-71, 1999.
33. Couvreur, B., Letellier, C., Collard, A., Quenon, P., Dehan, P.,
Hamers, C., Pastoret, P. and Kekhofs, P.: Genetic and antigenic
variability in bovine viral diarrhea virus (BVDV) isolates from
Belgium. Virus Res. 85: 17-28, 2002.
Research Articles

Published under a Creative Commons License By attribution, non-commercial