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• Expression of endogenous beta retroviruses and Hyal-2 mRNA in immune organs of fetuses and lambs.

  Authors: Jing-Wei Qi, Xiao-Li Wu, Shu-Ying Liu, Gui-Fang Cao

  Virologica Sinica. 27(2):83-92.

  Endogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV), this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). TheEndogenous beta retroviruses (enJSRV) are highly homologous with Jaagsiekte sheep retrovirus (exJSRV), this exogenous retrovirus is the aetiological agent of ovine pulmonary adenocarcinoma (OPA). The aim of this study was to clarify the function of enJSRV and the immunological mechanisms of its corresponding antibody, that is undetectable in JSRV-infected ovine serum. The expression of enJSRV envelope protein and Hyal-2 mRNA in immune organs and lungs of ovine fetuses and lambs were analyzed by Real-Time reverse transcription PCR and In Situ Hybridization using specific probes. In Situ Hybridization results indicated that the enJSRV envelope protein and Hyal-2 mRNA were expressed in thymus, spleen, mesenteric lymph nodes and lungs at different times, while no positive signals were detected in the negative controls. On the other hand, results from Real-Time reverse transcription PCR analysis showed that in 130d fetuses and 3d newborn lambs the enJSRV mRNA levels were much higher in organs associated with the immune system than that in lungs, especially in the thymus and spleen, but levels of Hyal-2 mRNA expression was not significantly different in all collected tissue. These results provided evidence from an immunology point of view to understand why the circulating antibodies against exJSRV are undetectable in JSRV-infected ovine, and will help to unravel the pathogenesis of JSRV-infected ovine.

• Surface display of rice stripe virus NSvc2 and analysis of its membrane fusion activity.

  Authors: Shu-Ling Zhao, Xue-Juan Dai, Jian-Sheng Liang, Chang-Yong Liang

  Virologica Sinica. 27(2):100-8.

  Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood.Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood. Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion. To conveniently study the membrane fusion activity of NSvc2, we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses. Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells. When induced by low pH, the membrane fusion was not observed in the cells that expressed NSvc2. Additionally, the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface. Thus, RSV NSvc2 is probably different from the phlebovirus counterparts, which could suggest different functions. RSV might enter insect cells other than by fusion with plasma or endosome membrane.

• Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex).

  Authors: Zhi-Feng Qin, Jie Sun, Ti-Kang Lu, Shao-Ling Zeng, Qun-Yi Hua, Qing-Yan Ling, Shu-Kun Chen, Jian-Qiang Lv, Cai-Hong Zhang, Bing Cheng, Zhou-Xi Ruan, Ying-Zuo Bi, Joseph J Giambrone, Hong-Zhuan Wu

  Virologica Sinica. 27(2):120-31.

  This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes ofThis study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10(-5)(= 280ELD(50)) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10(-4)(=2800 ELD(50)). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

• Genetic modification of baculovirus expression vectors.

  Authors: Shu-Fen Li, Hua-Lin Wang, Zhi-Hong Hu, Fei Deng

  Virologica Sinica. 27(2):71-82.

  As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified toAs a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications.

• Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in Kazakhstan in 2005.

  Authors: Andrey Bogoyavlenskiy, Vladimir Berezin, Alexey Prilipov, Eugeniy Usachev, Ilya Korotetskiy, Irina Zaitceva, Aydyn Kydyrmanov, Marat Sayatov

  Virologica Sinica. 27(2):93-9.

  Isolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverseIsolates of Newcastle disease virus (NDV) from deceased wild and domestic pigeons in Kazakhstan were obtained from the Almaty region during 2005 and were genotypically analyzed by using reverse transcription polymerase chain reaction (RT-PCR) with primers specific to the viral fusion (F) protein gene. Part of the amplified F protein DNA product (nucleotide sequence 47-422) and the deduced amino acid sequences were compared phylogenetically with those from strains previously reported in other geographic regions. Phylogenetic analysis indicated that the Kazakhstanian pigeon paramyxovirus type 1 (PPMV-1) isolates belong to genotype VI or 4bii. To our knowledge, this is the first reported VI isolates that possess the sequences of (112)GKRQKR(116) (*) F(117) within the F0 protein. The information is fundamental to improving the efficiency of control strategies and vaccine development for NDV.

• Suppression of RNA interference pathway in vitro by Grass carp reovirus.

  Authors: Shuai Guo, Dan Xu, Hong-Xu Xu, Tu Wang, Jia-le Li, Li-Qun Lu

  Virologica Sinica. 27(2):109-19.

  The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carpThe means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

• Analysis on factors related to rabies epidemic in China from 2007-2011.

  Authors: Cui-Ping Yin, Hang Zhou, Hui Wu, Xiao-Yan Tao, Simon Rayner, Shu-Mei Wang, Qing Tang, Guo-Dong Liang

  Virologica Sinica. 27(2):132-43.

  To analyze features of the rabies epidemic in China between 2007 and 2011, identify factors influencing the epidemic and to provide a scientific basis for further control and prevention of rabies,To analyze features of the rabies epidemic in China between 2007 and 2011, identify factors influencing the epidemic and to provide a scientific basis for further control and prevention of rabies, Descriptive epidemiological methods and statistical analysis was used on data collected from the National Disease Reporting Information System between 2007 to 2011 and the National Active Surveillance System between 2007 and 2010. Our analysis shows that while the number of human rabies cases decreased year by year, the number of districts reporting cases did not show significant change. The situations in Guangdong, Guangxi, Guizhou and Hunan provinces clearly improved over the period but they remain provinces with high-incidence, and consequently influence the epidemic situation of surrounding provinces and possibly the whole country. Summer and autumn were high-incidence seasons. Farmers, students and pre-school children represent the high-risk populations, and rates of cases in farmers increased, those for students decreased, and pre-school children remained unchanged. Provinces with active surveillance programs reported a total of 2346 individual cases, of which 88.53% were associated with canines. Postexposure prophylaxis (PEP) of rabies cases was not significantly improved, whereas PEP in post-exposure population was good. In rural regions of China, canine density was reduced somewhat, and the immunization rate increased slightly. Finally we show that while the epidemic decreased 2007 to 2011 in China, cases continued to be diffused in certain regions. Lack of standardization of PEP on rabies cases was the main reason of morbidity. The high density and low immunization of dog in rural areas and the defective situation of PEP are still continuous occurrences in China and remain a cause for concern.

• A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of peste des petits ruminants virus in the clinical samples.

  Authors: Vinayagamurthy Balamurugan, Arnab Sen, Gnanavel Venkatesan, Vinita Yadav, Vandna Bhanot, Veerakyathappa Bhanuprakash, Raj Kumar Singh

  Virologica Sinica. 27(1):1-9.

  A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) usingA sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T(m) of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID(50)). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.

• Inhibitory effects of Ginsenoside Rb1 on apoptosis caused by HSV-1 in human glioma cells.

  Authors: Yuan-Yuan Liang, Bin Wang, Dong-Meng Qian, Ling Li, Zhi-Hao Wang, Ming Hu, Xu-Xia Song

  Virologica Sinica. 27(1):19-25.

  To investigate the inhibitory effects of Ginsenoside Rb1 (GRb1) on apoptosis caused by Herpes Simplex Virus-1(HSV-1) in Human Glioma Cells (U251), U251 cells were infected by HSV-1 at a multiplicityTo investigate the inhibitory effects of Ginsenoside Rb1 (GRb1) on apoptosis caused by Herpes Simplex Virus-1(HSV-1) in Human Glioma Cells (U251), U251 cells were infected by HSV-1 at a multiplicity of infection of 5 and GRb1, GRb1+HSV-1, HSV-1 and control groups. MTT and cell apoptosis assays were used to detect the inhibitory effects of GRb1 on the apoptosis of U251 cells that caused by HSV-1 infection for various concentrations of drug and virus treatments by MTT assay. We found that in the 400 μg/mL GRb1 and 400 μg/mL GRb1+HSV-1 groups, MTT values were higher than control group at all times (P<0. 05). Moreover, the apoptosis rate in the 400 μg/mL GRb1+HSV-1 group was lower than the HSV-1 group (P<0. 05). These results confirmed that, at appropriate concentrations, GRb1 could inhibit nerve cell apoptosis in HSV-1 infections.

• HCV NS5A and NS5B enhance expression of human ceramide glucosyltransferase gene.

  Authors: Jia Guo, Ran Yan, Guo-dong Xu, Cong-yi Zheng

  Virologica Sinica. 27(1):38-47.

  Host genes involved in lipid metabolism are differentially affected during the early stages of hepatitis C virus (HCV) infection. Here we demonstrate that artificial up-regulation of fatty acidHost genes involved in lipid metabolism are differentially affected during the early stages of hepatitis C virus (HCV) infection. Here we demonstrate that artificial up-regulation of fatty acid biosynthesis has a positive effect on the replication of the HCV full-length replicon when cells were treated with nystatin. Conversely, the HCV RNA replication was decreased when fatty acid biosynthesis was inhibited with 25-hydroxycholesterol and PDMP(D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol). In agreement with these results, the expression level of GlcT-1(ceramide glucosyltransferase), a host glucosyltransferase in the first step of GSL (glycosphingolipid) biosynthesis, was found to be closely associated with the expression and replication of HCV RNA. On the other hand, the viral RNA can also activate GlcT-1 in the early stage of viral RNA transfection in vitro. To identify viral factors that are responsible for GlcT-1 activation, we constructed ten stable Vero cell lines that express individual HCV proteins. Based on the analyses of these cell lines and transient transfection assay of the GlcT-1 promoter regions, we conclude that HCV proteins, especially NS5A and NS5B, have positive effects on the expression of GlcT-1. It is possible that NS5A and NS5B stimulate transcription factor(s) to activate the expression of GlcT-1 by increasing its transcription level.

• The protamine-like DNA-binding protein P6.9 epigenetically up-regulates Autographa californica multiple nucleopolyhedrovirus gene transcription in the late infection phase.

  Authors: Ying Peng, Kun Li, Rong-juan Pei, Chun-chen Wu, Chang-yong Liang, Yun Wang, Xin-wen Chen

  Virologica Sinica. 27(1):57-68.

  Protamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription[1]. ItProtamines are a group of highly basic proteins first discovered in spermatozoon that allow for denser packaging of DNA than histones and will result in down-regulation of gene transcription[1]. It is well recognized that the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes P6.9, a protamine-like protein that forms the viral subnucleosome through binding to the viral genome[29]. Previous research demonstrates that P6.9 is essential for viral nucleocapsid assembly, while it has no influence on viral genome replication[31]. In the present study, the role of P6.9 in viral gene transcription regulation is characterized. In contrast to protamines or other protamine-like proteins that usually down-regulate gene transcription, P6.9 appears to up-regulate viral gene transcription at 12-24 hours post infection (hpi), whereas it is non-essential for the basal level of viral gene transcription. Fluorescence microscopy reveals the P6.9's co-localization with DNA is temporally and spatially synchronized with P6.9's impact on viral gene transcription, indicating the P6.9-DNA association contributes to transcription regulation. Chromatin fractionation assay further reveals an unexpected co-existence of P6.9 and host RNA polymerase II in the same transcriptionally active chromatin fraction at 24 hpi, which may probably contribute to viral gene transcription up-regulation in the late infection phase.

• Genetic analysis of the P1 region of human enterovirus 71 strains and expression of the 55 F strainVP1 protein.

  Authors: Jian-qiang Li, Jun-jie Yang, Xiu-juan Fan, Zhen-peng Sun, Yan Sun, Huan Li, Zi-xin Meng, Wei Li

  Virologica Sinica. 27(1):10-8.

  Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand, foot, and mouth disease (HFMD) in children. Different strains from Gansu wereEnterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand, foot, and mouth disease (HFMD) in children. Different strains from Gansu were cloned and the P1 protein was sequenced and analysed. Results indicate that there are three kinds of EV71 infections prevalent in Gansu. The VP1 protein from one of these strains, 55F, was expressed. The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody, the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay. These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.

• Molecular and in vitro characterization of field isolates of bovine herpesvirus-1.

  Authors: Julian Ruiz-Saenz, Jairo Jaime, Gloria Ramirez, Victor Vera

  Virologica Sinica. 27(1):26-37.

  Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and toBovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and to characterize (molecular and biological characterization) BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates, each from a different animal, such as from the respiratory and reproductive tracts. In some cases the cytopathic effect was visible 12 hours post-inoculation, and became characteristic after 36-48 hours. Biological characteristics were evaluated and compared with Iowa and Colorado-1 reference strains, and differences were found in plaque size, virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results showed that some isolates had a highly virulent-like behavior in vitro, compared to the reference strains, with shorter eclipse periods, faster release of virus into the supernatants, and higher burst size and viral titer. There were no differences in glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers. Moreover, using restriction endonucleases analysis, most of the viruses were confirmed as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to develop new monovalent vaccines.

• Development of multiple ELISAs for the detection of antibodies against classical swine fever virus in pig sera.

  Authors: Zhen-hua Yang, Ling Li, Zi-shu Pan

  Virologica Sinica. 27(1):48-56.

  The major immunogenic proteins (E(rns), E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E. coli and purified by affinity chromatography. The recombinant antigensThe major immunogenic proteins (E(rns), E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E. coli and purified by affinity chromatography. The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies in pig sera. Optimum cut-off values were determined by receiver operating characteristic (ROC) analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets. The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results. The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination.

• Ribosome inactivating proteins from plants inhibiting viruses.

  Authors: Inderdeep Kaur, R C Gupta, Munish Puri

  Virologica Sinica. 26(6):357-65.

  Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication ofMany plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

• Phylogenetic analysis of the non-structural (NS) gene of influenza A viruses isolated in Kazakhstan in 2002-2009.

  Authors: Andrey Bogoyavlenskiy, Vladimir Berezin, Alexey Prilipov, Ilya Korotetskiy, Irina Zaitseva, Aydyn Kydyrmanov, Kobey Karamedin, Nailya Ishmukhametova, Saule Asanova, Marat Sayatov, Kainar Zhumatov

  Virologica Sinica. 26(6):376-85.

  Although the important role of the non-structural (NS1 and NEP) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool ofAlthough the important role of the non-structural (NS1 and NEP) gene of influenza A in virulence of the virus is well established, our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Kazakhstan is incomplete. 17 influenza A viruses of different subtypes were studied in this paper. Seven types of haemagglutinin and five different neuraminidase subtypes in eight combinations were found among the isolated viruses. A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. By phylogenetic analysis it was shown that two distinct gene pools, corresponding to both NS allele A with 5 Clades and B, were present at the same time in Kazakhstan. The degree of variation within the alleles was very low. In our study allele A viruses had a maximum of 5% amino acid divergence in Clade while allele B viruses had only 4% amino acid divergence.

• Molecular characterization of Banana streak virus isolate from Musa Acuminata in China.

  Authors: Jun Zhuang, Jian-Hua Wang, Xin Zhang, Zhi-Xin Liu

  Virologica Sinica. 26(6):393-402.

  Banana streak virus (BSV), a member of genus Badnavirus, is a causal agent of banana streak disease throughout the world. The genetic diversity of BSVs from different regions of banana plantationsBanana streak virus (BSV), a member of genus Badnavirus, is a causal agent of banana streak disease throughout the world. The genetic diversity of BSVs from different regions of banana plantations has previously been investigated, but there are relatively few reports of the genetic characteristic of episomal (non-integrated) BSV genomes isolated from China. Here, the complete genome, a total of 7722bp (GenBank accession number DQ092436), of an isolate of Banana streak virus (BSV) on cultivar Cavendish (BSAcYNV) in Yunnan, China was determined. The genome organises in the typical manner of badnaviruses. The intergenic region of genomic DNA contains a large stem-loop, which may contribute to the ribosome shift into the following open reading frames (ORFs). The coding region of BSAcYNV consists of three overlapping ORFs, ORF1 with a non-AUG start codon and ORF2 encoding two small proteins are individually involved in viral movement and ORF3 encodes a polyprotein. Besides the complete genome, a defective genome lacking the whole RNA leader region and a majority of ORF1 and which encompasses 6525bp was also isolated and sequenced from this BSV DNA reservoir in infected banana plants. Sequence analyses showed that BSAcYNV has closest similarity in terms of genome organization and the coding assignments with an BSV isolate from Vietnam (BSAcVNV). The corresponding coding regions shared identities of 88% and -95% at nucleotide and amino acid levels, respectively. Phylogenetic analysis also indicated BSAcYNV shared the closest geographical evolutionary relationship to BSAcVNV among sequenced banana streak badnaviruses.

• Surface display of domain III of Japanese encephalitis virus E protein on Salmonella typhimurium by using an ice nucleation protein.

  Authors: Jian-Lin Dou, Tao Jing, Jing-Jing Fan, Zhi-Ming Yuan

  Virologica Sinica. 26(6):409-17.

  A bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invadeA bacterial cell surface display technique based on an ice nucleation protein has been employed for the development of live vaccine against viral infection. Due to its ubiquitous ability to invade host cells, Salmonella typhimurium might be a good candidate for displaying viral antigens. We demonstrated the surface display of domain III of Japanese encephalitis virus E protein and the enhanced green fluorescent protein on S. typhimurium BRD509 using the ice nucleation protein. The effects of the motif in the ice nucleation protein on the effective display of integral protein were also investigated. The results showed that display motifs in the protein can target integral foreign protein on the surface of S. typhimurium BRD509. Moreover, recombinant strains with surface displayed viral proteins retained their invasiveness, suggesting that the recombinant S. typhimurium can be used as live vaccine vector for eliciting complete immunogenicity. The data may yield better understanding of the mechanism by which ice nucleation protein displays foreign proteins in the Salmonella strain.

• Caspase work model during pathogen infection.

  Authors: Yan-Bin Ma, Hui-Yun Chang

  Virologica Sinica. 26(6):366-75.

  Caspases are an evolutionarily conserved family of aspartate-specific cystein-dependent proteases with essential functions in apoptosis and normally exist in cells as inactive proenzymes. In additionCaspases are an evolutionarily conserved family of aspartate-specific cystein-dependent proteases with essential functions in apoptosis and normally exist in cells as inactive proenzymes. In addition to the inflammatory caspases, the initiator and effector caspases have been shown to have an important role in regulating the immune response, but are involved in different ways. We give a brief introduction on the benefit of apoptosis on the clearance of invasive pathogens, and the caspase functions involved in the immune response. Then we construct a working model of caspases during pathogen invasion. A detailed description of the three modes is given in the discussion. These three modes are regulated by different inhibitors, and there may be a novel way to treat intracellular pathogen and autoimmune diseases based on the specific inhibitors.

• Emerging trends of drug-resistant HIV-1 among drug-treated patients in former blood donors in Hubei, China: a three-year surveillance from 2004 to 2006.

  Authors: Jian Gong, Xiao-Qiong Wang, Xiao Tong, Xi-Hui Shen, Rong-Ge Yang

  Virologica Sinica. 26(6):386-92.

  This study aimed to evaluate emerging trends of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) among 290 formerThis study aimed to evaluate emerging trends of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) among 290 former blood donor HIV-1 infected patients in Hubei, China, from 2004 to 2006, all of whom had received anti-HIV-1 therapy. The presence of NRTI- and NNRTI-associated mutations were established by sequencing; genotypic and predicted phenotypic drug resistance were evaluated using HIVdb Program version 5.0.1 (http://hivdb.stanford.edu/pages/algs/HIVdb.html ). Genotypic drug resistance analysis showed significant increases in percentages of patients carrying HIV-1 strains with M41L, T215Y/F, D67N, K103N, G190A/S, Y181C/F or L210W mutations. Of the variants' predicted phenotypic drug resistance, highly significant increases were detected in percentages of patients carrying HIV-1 with high resistance to zidovudine (AZT) or stavudine (D4T) in NRTIs, and to delavirdine (DLV), efavirenz (EFV) or nevirapine (NVP) in NNRTIs; intermediate resistance to abacavir (ABC), AZT, D4T, didanosine (DDI) or tenofovir disoproxil fumarate (TDF) in NRTIs, and to etravirine (ETR) in NNRTIs; and low and potential low resistance to lamivudine (3TC), ABC, emtricitabine (FTC) or TDF in NRTIs, and to ETR in NNRTIs.

• Bovine herpesvirus 1 protein bICP0 represses the transcription of bISG15 in fetal bovine lung cells.

  Authors: Chang Liu, Xiao-Hong Kong, Wen-Tao Qiao, Yun-Qi Geng

  Virologica Sinica. 26(6):403-8.

  The ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of cattle, can infect FBL cells and induceThe ubiquitin-like modifier bISG15 is an antiviral protein found in fetal bovine lung (FBL) cells. Bovine Herpesvirus 1(BHV-1), which is a viral pathogen of cattle, can infect FBL cells and induce cytopathic effects. Real-time PCR assays showed that BHV-1's infection could repress the basal or inducible transcription of bISG15 in FBL cells. It demonstrates that this repression effect depends on BHV-1 viral infection and new protein synthesis. Our previous work showed that bIRF-3 was the key factor in the stimulation of bISG15 in FBL cells, so the effect of BHV-1 viral protein on bIRF-3 activating the promoter of bISG15 was confirmed. The luciferase assay showed the BHV-1 viral protein bICP0 inhibited the activation of bISG15 promoter stimulated by bIRF-3. Taken together, our work suggested that BHV-1 had some molecular mechanism to resist the cellular bISG15's antiviral functions.

• Genetic characteristics of 2009 pandemic H1N1 influenza a viruses isolated from Mainland China.

  Authors: Jiu-Ru Zhao, Yong-Dong Li, Li-Min Pan, Na Zhu, Hong-Xia Ni, Guo-Zhang Xu, Yong-Zhong Jiang, Xi-Xiang Huo, Jun-Qiang Xu, Han Xia, Na Han, Shuang Tang, Zhong Zhang, Zheng Kou, Simon Rayner, Tian-Xian Li

  Virologica Sinica. 26(6):418-27.

  A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories.A total of 100 H1N1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang, Hubei and Guangdong between June and November 2009, were provided by local CDC laboratories. After MDCK cell culture, 57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing. A total of 39 HA sequences, 52 NA sequences, 36 PB2 sequences, 31 PB1 sequences, 40 PA sequences, 48 NP sequences, 51 MP sequences and 36 NS sequences were obtained, including 20 whole genome sequences. Sequence comparison revealed they shared a high degree of homology (96%-99%) with known epidemic strains (A/California/04/2009(H1N1). Phylogenetic analysis showed that although the sequences were highly conserved, they clustered into a small number of groups with only a few distinct strains. Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences: A/Hubei/86/2009 PKVRDQEG → PKVRDQEA, A/Zhejiang/08/2009 PKVRDQEG → PKVRDQER, A/Hubei/75/2009 PKVRDQEG → PKVRDQGG, the A/Hubei/75/2009 was isolated from an acute case, while the other two were from patients with mild symptoms. Other key sites such as 119, 274, 292 and 294 amino acids of NA protein, 627 of PB2 protein were conserved. Meanwhile, all the M2 protein sequences possessed the Ser32Asn mutation, suggesting that these viruses were resistant to adamantanes. Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.

• Susceptibility to AcMNPV and expression of recombinant proteins by a novel cell clone derived from a Trichoplusia ni QAU-BTI-Tn9-4s cell line.

  Authors: Ming Shan, Shi-Ying Zhang, Lei Jiang, Ming Ma, Guo-Xun Li

  Virologica Sinica. 26(5):297-305.

  It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to otherIt is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines. But the characteristics of the cell line do not always remain stable and may change upon continuous passage. Recently an alphanodavirus, named Tn5 Cell Line Virus (or TNCL Virus), was identified in High Five cells in particular. Therefore, we established a new cell line, QB-Tn9-4s, from Trichoplusia ni, which was determined to be free of TNCL virus by RT-PCR analysis. In this paper, we describe the development of a novel cell clone, QB-CL-B, from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV, and the level of recombinant protein production. This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate; although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies, there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.

• A new indicator cell line established to monitor bovine foamy virus infection.

  Authors: Hong-Yan Guo, Zhi-Bin Liang, Yue Li, Juan Tan, Qi-Min Chen, Wen-Tao Qiao

  Virologica Sinica. 26(5):315-23.

  In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodesIn order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro, we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR, from -7 to 1012). The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR. One clone, designated BFVL, was selected from ten neomycin-resistant clones. BFVL showed a specific and inducible dose- and time-dependent luciferase activity in response to BFV infection. Although the changes in luciferase activity of BFVL peaked at 84 h post infection, it was possible to differentiate infected and uninfected cells at 48 h post infection. A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL. Moreover, the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection. These findings suggest that the BFVL-based assay is rapid, easy, sensitive, quantitative and specific for detection of BFV infection.

• Production of CCHF virus-like particle by a baculovirus-insect cell expression system.

  Authors: Zhao-Rui Zhou, Man-Li Wang, Fei Deng, Tian-Xian Li, Zhi-Hong Hu, Hua-Lin Wang

  Virologica Sinica. 26(5):338-46.

  Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family, which is widespread and causes high fatality. The nucleocapsid of CCHFV isCrimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family, which is widespread and causes high fatality. The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment. In this research, the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus. Under an electron microscope, Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells. Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation, which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).

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