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Prevalence of Three Shrimp Viruses in Zhejiang Province in 2008

Xiaowei YU ; Jianping WANG ; Wei ZHANG ; Zhengli SHI

Virologica Sinica.2011;26(1):67-71. doi:10.1007/s12250-011-3157-6

White spot syndrome virus(WSSV), Taura syndrome virus(TSV)and Infectious hypodermal and haematopoietic necrosis virus(IHHNV)are three shrimp viruses responsible for major pandemics affecting the shrimp farming industry. Shrimps samples were collected from 12 farms in Zhejiang province, China, in 2008 and analyzed by PCR to determine the prevalence of these viruses. From the 12 sampling locations, 8 farms were positive for WSSV, 8 for IHHNV and 6 for both WSSV and IHHNV. An average percentage of 57.4% of shrimp individuals were infected with WSSV, while 49.2% were infected with IHHNV. A high prevalence of co-infection with WSSV and IHHNV among samples was detected from the following samples: Bingjiang(93.3%), liuao(66.7%), Jianshan(46.7%)and Xianxiang(46.7%). No samples exhibited evidence of infection with TSV in collected samples. This study provides comprehensive information of the prevalence of three shrimp viruses in Zhejiang and may be helpful for disease prevention control in this region.

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Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

Yan CHEN ; Hong TIAN ; Jianhui HE ; Jinyin WU ; Youjun SHANG ; Xiangtao LIU

Virologica Sinica.2011;26(1):61-66. doi:10.1007/s12250-011-3154-9

Porcine reproductive and respiratory syndrome is caused by the PRRS virus(PRRSV), which has six structural proteins(GP2, GP3, GP4, GP5, M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay(ELISA)and other methods. Toward this goal, we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7%(266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.

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Close Relationship between the 2009 H1N1 Virus and South Dakota AIV Strains

Cun LI ; Xiaoping AN ; Zhiqiang MI ; Dabin LIU ; Huanhuan JIANG ; Bo PAN ; Sheng WANG ; Bin CHEN ; Yigang TONG

Virologica Sinica.2011;26(1):54-60. doi:10.1007/s12250-011-3149-6

Although previous publications suggest the 2009 pandemic influenza A(H1N1)virus was reassorted from swine viruses of North America and Eurasia, the immediate ancestry still remains elusive due to the big evolutionary distance between the 2009 H1N1 virus and the previously isolated strains. Since the unveiling of the2009 H1N1 influenza, great deal of interest has been drawn to influenza, consequently a large number of influenza virus sequences have been deposited into the public sequence databases. Blast analysis demonstrated that the recently submitted 2007 South Dakota avian influenza virus strains and other North American avian strains contained genetic segments very closely related to the 2009 H1N1 virus, which suggests these avian influenza viruses are very close relatives of the 2009 H1N1 virus. Phylogenetic analyses also indicate that the2009 H1N1 viruses are associated with both avian and swine influenza viruses circulating in North America. Since the migrating wild birds are preferable to pigs as the carrier to spread the influenza viruses across vast distances, it is very likely that birds played an important role in the inter-continental evolution of the 2009 H1N1virus. It is essential to understand the evolutionary route of the emerging influenza virus in order to find a way to prevent further emerging cases. This study suggests the close relationship between 2009 pandemic virus and the North America avian viruses and underscores enhanced surveillance of influenza in birds for understanding the evolution of the 2009 pandemic influenza.

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Molecular Characterization of Segments S7 to S10 of a Southern Rice Black-streaked Dwarf Virus Isolate from Maize in Northern China

Xiao YIN ; Feifei XU ; Fangqiang ZHENG ; Xiangdong LI ; Baoshen LIU ; Chunqing ZHANG

Virologica Sinica.2011;26(1):47-53. doi:10.1007/s12250-011-3170-9

Southern rice black-streaked dwarf virus(SRBSDV)is a novel Fijivirus prevalent in rice in southern and central China, and northern Vietnam. Its genome has 10 segments of double-stranded RNA named S1 to S10according to their size. An isolate of SRBSDV, JNi4, was obtained from naturally infected maize plants from Ji'ning, Shandong province, in the 2008 maize season. Segments S7 to S10 of JNi4 share nucleotide identities of 72.6%-73.1%, 72.3%-73%, 73.9%-74.5% and 77.3%-79%, respectively, with corresponding segments of Rice black-streaked dwarf virus isolates, and identities of 99.7%, 99. 1%-99.7%, 98.9%-99.5%, and 98.6%-99.2% with those of SRBSDV isolates HN and GD. JNi4 forms a separate branch with GD and HN in the phylogenetic trees constructed with genomic sequences of S7 to S10. These results confirm the proposed taxonomic status of SRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4 is an isolate of SRBSDV. Shandong is so far the northernmost region where SRBSDV is found in China.

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Effect of siRNAs on HSV-1 Plaque Formation and Relative Expression Levels of RR mRNA

Zhe REN ; Shen LI ; Qiaoli WANG ; Yangfei XIANG ; Yunxia CUI ; Yifei WANG ; Renbin QI ; Daxiang LU ; Shumin ZHANG ; Peizhuo ZHANG

Virologica Sinica.2011;26(1):40-46. doi:10.1007/s12250-011-3162-9

RNA interference(RNAi)is a process by which introduced small interfering RNA(siRNA)can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1(HSV-1)RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40, respectively. In this study, we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.

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A Simple and Rapid Colloidal Gold-based Immunochromatogarpic Strip Test for Detection of FMDV Serotype A

Tao JIANG ; Zhong LIANG ; Weiwei REN ; Juan CHEN ; Xiaoying ZHI ; Guangyu QI ; Xiangtao LIU ; Xuepeng CAI

Virologica Sinica.2011;26(1):30-39. doi:10.1007/s12250-011-3166-5

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites;no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.

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Construction and Genetic Analysis of Murine Hepatitis Virus Strain A59 Nsp16 Temperature Sensitive Mutant and the Revertant Virus

Guohui CHANG ; Baojun LUO ; Pin LU ; Lei LIN ; Xiaoyan WU ; Jing LI ; Yi HU ; Qingyu ZHU

Virologica Sinica.2011;26(1):19-29. doi:10.1007/s12250-011-3145-x

Coronaviruses (CoVs) are generally associated with respiratory and enteric infections and have long been recognized as important pathogens of livestock and companion animals. Mouse hepatitis virus (MHV) is a widely studied model system for Coronavirus replication and pathogenesis. In this study, we created a MHV-A59 temperature sensitive (ts) mutant Wu"-ts18(cd) using the recombinant vaccinia reverse genetics system. Virus replication assay in 17C1-1 cells showed the plaque phenotype and replication characterization of constructed Wu"-ts18(cd) were indistinguishable from the reported ts mutant Wu"-ts 18. Then we cultured the ts mutant Wu"-ts 18(cd) at non-permissive temperature 39.5℃, which "forced" the ts recombinant virus to use second-site mutation to revert from a ts to a non-ts phenotype. Sequence analysis showed most of the revertants had the same single amino acid mutation at Nsp16 position 43. The single amino acid mutation at Nsp16 position 76 or position 130 could also revert the ts mutant Wu"-ts 18 (cd) to non-ts phenotype, an additional independent mutation in Nsp13 position 115 played an important role on plaque size. The results provided us with genetic information on the functional determinants of Nsp16. This allowed us to build up a more reasonable model of CoVs replication-transcription complex.

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Advances of Bioinformatics Tools Applied in Virus Epitopes Prediction

Ping CHEN ; Simon RAYNER ; Kanghong HU

Virologica Sinica.2011;26(1):1-7. doi:10.1007/s12250-011-3159-4

In recent years, the in silico epitopes prediction tools have facilitated the progress of vaccines development significantly and many have been applied to predict epitopes in viruses successfully. Herein, a general overview of different tools currently available, including T cell and B cell epitopes prediction tools, is presented. And the principles of different prediction algorithms are reviewed briefly. Finally, several examples are present to illustrate the application of the prediction tools.

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Antiviral Activity of Recombinant Cyanovirin-N against HSV-1

Hong YU ; Zongtao LIU ; Rui LV ; Wenqing ZHANG

Virologica Sinica.2010;25(6):432-439. doi:10.1007/s12250-010-3131-3

In this study,a standard strain of HSV-1(strain SM44)was used to investigate the antiviral activity of the recombinant Cyanovirin-N(CV-N)against Herpes simplex virus type 1(HSV-1)in vitro and in vivo.Cytopathic effect(CPE)and MTT assays were used to evaluate the effect of CV-N on HSV-1 in Vero cells.The number of copies of HSV-DNA was detected by real-time fluorescence quantitative PCR(FQ-PCR).The results showed that CV-N had a low cytotoxicity on Vero cells with a CC50 of 359.03±0.56 μg/mL,and that it could not directly inactivate HSV-1 infectivity.CV-N not only reduced the CPE of HSV-1 when added before or after viral infection,with a 50% inhibitory concentration(IC50)with 2.26 and 30.16μg/mL respectively,but it also decreased the copies of HSV-1 DNA in infected host cells.The encephalitis model for HSV-1 infection was conducted in Kunming mice,and treated with three dosages of CV-N(0.5,5 & 10 mg/kg)which was administered intraperitoneally at 2h,3d,5d,7d post infection.The duration for the appearance of symptoms of encephalitis and the survival days were recorded and brain tissue samples were obtained for pathological examination(HEstaining).Compared with the untreated control group,in the 5mg/kg CV-N and 10mg/kg CV-N treated groups,the mice suffered light symptoms and the number of survival days were more than 9d and 14d respectively.HE staining also showed that in 5mg/kg CV-N and 10mg/kg CV-N treated groups,the brain cells did not show visible changes,except for a slight inflammation.Our results demonstrated that CV-N has pronounced antiviral activity against HSV-1 both in vitro and in vivo,and it would be a promising new candidate for anti-HSV therapeutics.

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Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H6N1 AIV

Hua YUE ; Xiaowen LEI ; Falong YANG ; Mingyi LI ; Cheng TANG

Virologica Sinica.2010;25(6):425-431. doi:10.1007/s12250-010-3114-4

Chicken embryo fibroblasts(CEFs)are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus(AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR(QPCR)analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4(RPL4)and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ)are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene(ACTB)and the ribosomal protein L4(RPL4)gene are the best references.

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