Virology Journal (VIROL J)

Publications in this journal

• Article: Risk factors associated with severe manifestations of 2009 pandemic influenza A (H1N1) infection in China: a case--control study.

  Yan-Yan Ren, Yu-Yan Yin, Wen-Qing Li, Yi Lin, Ti Liu, Shuang Wang, Sheng-Yang Zhang, Zhong Li, Xian-Jun Wang, Zhen-Qiang Bi
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  ABSTRACT: BACKGROUND: No studies on the risk factors of 2009 pandemic influenza A (H1N1) in China have been reported. We aimed to investigate the risk factors for severe manifestations of 2009 pandemic H1N1 influenza in China METHODS: A case--control study with 343 severe hospitalized patients and 343 randomly selected mild controls was conducted. The diagnosis was established by assessment of clinical symptoms and confirmed by the real-time reverse-transcriptase-polymerase chain reaction assay. Severe or mild patients were classified by uniform criteria issued by the Ministry of Health in China. RESULTS: The multivariable logistic regression analysis showed that the overweight or obese subjects admitted to hospital with H1N1 influenza were more likely to experience severe manifestations. The ORs were 3.70 (95% CI: 2.04-6.72) and 35.61 (95% CI: 7.96-159.21) respectively. Subjects at age less than 5 years or older than 60 years had an increased risk of severe manifestations (OR = 21.14, 95% CI: 7.79-57.33). We also observed increased risk among subjects with longer time interval from symptom onset to hospital admission (OR = 3.26, 95% CI: 2.08-5.11) or peasants (OR = 9.79, 95% CI: 5.11-18.78). Those with chronic disorders had increased risk of severe manifestations of H1N1 influenza. CONCLUSION: We provide evidence on the risk factors associated with severe manifestations of 2009 pandemic H1N1 influenza in a study of hospitalized subjects in China.
  Virology Journal 05/2013; 10(1):149.
• Article: SF2/ASF binding region within JC virus NCCR limits early gene transcription in glial cells.

  Elena Uleri, Patrick Regan, Antonina Dolei, Ilker Kudret Sariyer
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  ABSTRACT: BACKGROUND: Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the white matter caused by human neurotropic polyomavirus, JC virus. It is now widely accepted that pathologic strains of JCV shows unique rearrangements consist of deletions and insertions within viral NCCR. While these kinds of rearrangements are related to viral tropism and pathology of the disease, their roles in molecular regulation of JCV gene expression and replication are unclear. We have previously identified SF2/ASF as a negative regulator of JCV gene expression in glial cells. This negative impact of SF2/ASF was dependent on its ability to bind a specific region mapped to the tandem repeat within viral promoter. In this report, functional role of SF2/ASF binding region in viral gene expression and replication was investigated by using deletion mutants of viral regulatory sequences. RESULTS: The second 98-base-pair tandem repeat on Mad1 strain was first mutated by deletion and named Mad1-(1X98). In addition to this mutant, the CR3 region which served the binding side for SF2/ASF was also mutated and named Mad1-DeltaCR3 (1X73). Both mutations were tested for SF2/ASF binding by ChIP assay. While SF2/ASF was associated with Mad1-WT and Mad1-(1X98), its interaction was completely abolished on Mad1-DeltaCR3 (1X73) construct as expected. Surprisingly, reporter gene analysis of Mad1-(1X98) and Mad1-DeltaCR3 (1X73) early promoter sequences showed two and three fold increase in promoter activities, respectively. The impact of "CR3" region on JCV propagation was also tested on the viral background. While replication of Mad1-(1X98) strain in glial cells was similar to Mad1-WT strain, propagation of Mad1-DeltaCR3 (1X73) was less productive. Further analysis of the transcription mediated by Mad1-DeltaCR3 (1X73) NCCR revealed that late gene expression was significantly affected. CONCLUSIONS: The results of this study reveal a differential role of CR3 region within JCV NCCR in expression of JCV early and late genes.
  Virology Journal 05/2013; 10(1):147.
• Article: Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication.

  Tippawan Pissawong, Santi Maneewatch, Kanyarat Thueng-In, Potjanee Srimanote, Fonthip Dong-Din-On, Jeeraphong Thanongsaksrikul, Thaweesak Songserm, Pongsri Tongtawe, Kunan Bangphoomi, Wanpen Chaicumpa
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  ABSTRACT: BACKGROUND: Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies. METHODS: Fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking. RESULTS: HuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages. CONCLUSIONS: HuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.
  Virology Journal 05/2013; 10(1):148.
• Article: Growth characteristics of human parechovirus 1 to 6 on different cell lines and cross- neutralization of human parechovirus antibodies: a comparison of the cytopathic effect and real time PCR.

  Brenda M Westerhuis, Sara Cm Jonker, Sandhia Mattao, Kimberley Sm Benschop, Katja C Wolthers
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  ABSTRACT: BACKGROUND: Human parechoviruses (HPeVs) are among the most frequently detected picornaviruses in humans. HPeVs are usually associated with mild gastrointestinal and respiratory symptoms with the exception of HPeV3 which causes neonatal sepsis and CNS infection. Previous studies showed various results in culturing different HPeV genotypes, inducing only a low cytopathic effect (CPE). METHODS: In vitro growth characteristics of the different HPeV genotypes in a range of 10 different cell lines are scored with CPE and measured in the supernatant by real time PCR. In the optimal cell line for each genotype a standard neutralization assay with the available HPeV antibodies (Abs) was performed and scored by CPE and measured by real time PCR. RESULTS: All six HPeV types were able to replicate on the RD99, A549, and Vero cell lines. HPeV1 was the only genotype able to replicate on all cell lines. Most efficient growth of HPeV1, 2, 4, 5, and 6 was shown on the HT29 cell line, while HPeV3 was unable to replicate on HT29. In all cases viral replication could be measured by real time PCR before CPE appeared. The polyclonal Abs available against HPeV1, 2, 4 and 5 all showed neutralization of their respective genotype after 7 days with inhibition of >60% in real time PCR and full inhibition of CPE, although cross-neutralization is shown. Replication of HPeV3 could only be inhibited by 12% by the anti-HPeV3 (aHPeV3) Ab and no inhibition of CPE was shown after 7 days. CONCLUSION: When replication is monitored by PCR, growth of HPeV genotypes 1 to 6 is supported by most of the cell lines tested, where viral replication is measured before appearance of CPE. A combination of HT29 and Vero cells would therefore support replication of all culturable HPeV types, so viral replication could be detected by PCR within 3 days for all genotypes.In addition, we showed efficient neutralization for HPeV1, 2, 4, 5, while cross- neutralization was shown between these types, indicating possible common neutralizing epitopes. For HPeV3 no efficient (cross-) neutralization was shown, indicating different neutralizing epitopes for HPeV3 compared to the other HPeV genotypes.
  Virology Journal 05/2013; 10(1):146.
• Article: Effects of southern rice black-streaked dwarf virus on the development and fecundity of its vector, Sogatella furcifera.

  Zhi Tu, Bing Ling, Donglin Xu, Maoxin Zhang, Guohui Zhou
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  ABSTRACT: BACKGROUND: Southern rice black-streaked dwarf virus (SRBSDV) threatens rice production in China and Vietnam. The virus is vectored by the migrating white-backed planthopper (WBPH, Sogatella furcifera) in a circulative, propagative, and persistent manner. A persistently-transmitted plant virus might affect its vector's development and fecundity directly by infecting the vector itself and/or indirectly altering the host plant. This study evaluated the direct and indirect effects of SRBSDV on WBPH performance to better understand the virus--vector--host plant relationship in terms of its effects on the biological parameters of the vector. METHODS: Three experimental WBPH populations were established. Viruliferous and non-viruliferous populations were fed on SRBSDV-infected rice seedlings for 48 h as first-instar nymphs; infection status was confirmed by RT--PCR after they died. The control population was fed on healthy rice. Each insect was individually transferred to a healthy rice plant grown in a glass tube at 20[degree sign]C, 25[degree sign]C, or 28[degree sign]C. Life parameters, including nymphal duration, survival rate, adult sex ratio, macropterous proportion, longevity, and oviposition amounts, of each population were measured at each temperature. RESULTS: The life parameter data indicated that SRBSDV and infected rice plants adversely influenced WBPH; the effects were temperature dependent. Compared with the control population, viruliferous populations showed significant changes, including prolonged nymphal stages and reduced survival rates at 20[degree sign]C, while the non-viruliferous population had higher survival rates at 20[degree sign]C and lower rates at 28[degree sign]C compared with the control. Both populations had significantly shorter adult life spans at 25[degree sign]C and lower oviposition amounts at 28[degree sign]C relative to the control. CONCLUSIONS: Both SRBSDV-infection and feeding on infected rice plants affected vector performance. Although a longer nymphal period benefits viral acquisition and transmission by nymphs and might increase rice infection rate, in general, SRBSDV infection of the vectors and host plants was unfavorable to WBPH population expansion.
  Virology Journal 05/2013; 10(1):145.
• Article: TH1 cytokine response to HCV peptides in Egyptian health care workers: a pilot study.

  Mona M Rafik, Alaa El-Dien Ms Hosny, Khaled O Abdallah, Amal A Abbas, Rania A Abo Shady, Dina A Soliman, Khaled M Nasr El-Din Rakha, Shahira F Alfedawy
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  ABSTRACT: Our objective was to elucidate the effects of different HCV peptides on TH1 cytokine synthesis (interleukin 2(IL2), gamma interferon (INFgamma) and tumor necrosis factor alpha (TNF alpha)), in a proliferative response in a high risk population of HCV seronegative aviremic Egyptian healthcare workers (HCW). We studied the TH1 cytokine response to different HCV peptides among 47 HCW with and without evidence of HCV infection. Participants were classified according to the proliferation index (PI) in a CFSE proliferation assay as an indicator of previous exposure to HCV. Cytokines were analyzed using Luminex xMAP technology. Results showed that positive PI HCW produced a higher IL2 in response to all HCV peptides except NS4, a higher IFNgamma response to NS3 and NS4 and no difference in TNFalpha response when compared to the negative PI HCWs. When compared to chronic HCV HCW, positive PI HCW showed no difference in the IL2 response, a higher IFNgamma response to NS4 and NS5 HCV peptides and a higher TNFalpha response to all peptides. In conclusion the magnitude and type of cytokines produced in HCV infection is critical in determining the outcome of infection. NS4 & NS5 HCV peptides induce a protective TH1 response in positive PI HCW.
  Virology Journal 05/2013; 10(1):144.
• Article: Mosquito-borne arbovirus surveillance at selected sites in diverse ecological zones of Kenya; 2007 -- 2012.

  Caroline Ochieng, Joel Lutomiah, Albina Makio, Hellen Koka, Edith Chepkorir, Santos Yalwala, James Mutisya, Lillian Musila, Samoel Khamadi, Jason Richardson, Joshua Bast, David Schnabel, Eyako Wurapa, Rosemary Sang
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  ABSTRACT: BACKGROUND: Increased frequency of arbovirus outbreaks in East Africa necessitated the determination of distribution of risk by entomologic arbovirus surveillance. A systematic vector surveillance programme spanning 5 years and covering 11 sites representing seven of the eight provinces in Kenya and located in diverse ecological zones was carried out. METHODS: Mosquitoes were sampled bi-annually during the wet seasons and screened for arboviruses. Mosquitoes were identified to species, pooled by species, collection date and site and screened for arboviruses by isolation in cell culture and/or RT-PCR screening and sequencing. RESULTS: Over 450,000 mosquitoes in 15,890 pools were screened with 83 viruses being detected/isolated that include members of the alphavirus, flavivirus and orthobunyavirus genera many of which are known to be of significant public health importance in the East African region. These include West Nile, Ndumu, Sindbis, Bunyamwera, Pongola and Usutu viruses detected from diverse sites. Ngari virus, which was associated with hemorrhagic fever in northern Kenya in 1997/98 was isolated from a pool of Anopheles funestus sampled from Tana-delta and from Aedes mcintoshi from Garissa. Insect only flaviviruses previously undescribed in Kenya were also isolated in the coastal site of Rabai. A flavivirus most closely related to the Chaoyang virus, a new virus recently identified in China and two isolates closely related to Quang Binh virus previously unreported in Kenya were also detected. CONCLUSION: Active transmission of arboviruses of public health significance continues in various parts of the country with possible undetermined human impact. Arbovirus activity was highest in the pastoralist dominated semi-arid to arid zones sites of the country where 49% of the viruses were isolated suggesting a role of animals as amplifiers and indicating the need for improved arbovirus disease diagnosis among pastoral communities.
  Virology Journal 05/2013; 10(1):140.
• Article: Epidemiological analysis of respiratory viral etiology for influenza-like illness during 2010 in Zhuhai, China.

  Hongxia Li, Quande Wei, Aijun Tan, Leyi Wang
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  ABSTRACT: BACKGROUND: Influenza-like illnesses (ILI), a subset of acute respiratory infections (ARI), are a significant source of morbidity and mortality worldwide. ILI can be caused by numerous pathogens, however; there is limited information on the etiology and epidemiology of ILI in China. METHODS: We performed a one-year surveillance study (2010) of viral etiology causing ILI and investigated the influence of climate on outbreaks of ILI attributed to viruses at the Outpatient Department of Zhuhai Municipal People's Hospital in Zhuhai, China. RESULTS: Of the 337,272 outpatients who sought attention in the Outpatient Department of Zhuhai Municipal People's Hospital in 2010, 3,747 (1.11%) presented with ILI. Of these patients presenting with ILI, 24.66% (924/3,747) had available samples and were enrolled in this study. At least one respiratory virus was identified in 411 patients (44.48%) and 42 (4.55%) were co-infected with two viruses. In patients co-infected with two viruses, respiratory syncytial virus (RSV) was detected in 50% (21/42). Among common viral pathogens detected, significant differences in age distributions were observed in seasonal influenza virus A (sFulA, H3N2) and B (sFluB), pandemic H1N1 2009 influenza viruses (H1N1pdm09), RSV, and adenovirus (ADV). Infections with sFluA (H3N2), sFluB, RSV, and human metapneumovirus (HMPV) had characteristic seasonal patterns. The incidences of sFluA (H3N2), ADV, and RSV correlated with air temperature. Alternatively, the incidence of sFluB correlated with relative air humidity. CONCLUSIONS: These results demonstrate that a wide range of respiratory viral pathogens are circulating in Zhuhai city. This information needs to be considered by clinicians when treating patients presenting with ILI.
  Virology Journal 05/2013; 10(1):143.
• Article: Highly sensitive serological methods for detecting tomato yellow leaf curl virus in tomato plants and whiteflies.

  Yan Xie, Xiaoyang Jiao, Xueping Zhou, Huan Liu, Yuequn Ni, Jianxiang Wu
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  ABSTRACT: BACKGROUND: Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus in the family Geminiviridae, which causes severe losses in tomato production in tropic and subtropic regions. METHODS: The purified TYLCV virions were used as the immunogen to produce monoclonal antibodies (MAbs) using the hybridoma technology. MAb-based dot enzyme-linked immunosorbent assay (dot-ELISA) and direct tissue blot immunoassay (DTBIA) were developed for sensitive, simple, and rapid detection of TYLCV in field tomato and whitefly (Bemisia tabaci) samples collected from TYLCV prevalent provinces in China. RESULTS: Using the hybridoma technology, six murine MAbs (1C4, 8D10, 6E3, 2F2, 3F4 and 4G3) against TYLCV were prepared. Using the MAb 1C4, dot-ELISA and DTBIA were then established for detecting TYLCV in field tomato and whitefly samples collected form only TYLCV prevalent provinces in China. The dot-ELISA could detect TYLCV in infected tissue crude extract diluted at 1:5,120 (w/v, g mL-1), and in viruliferous whitefly homogenate diluted at 1:128 (individual whitefly/muL), respectively. Field tomato samples (n=487) and whitefly samples (n=110) from TYLCV prevalent districts in China were screened for the presence of TYLCV using the two developed methods, and the results were further confirmed by PCR and nucleotide sequencing. The survey revealed that TYLCV is widespread on tomato plants in Zhejiang, Shandong and Henan provinces in China. CONCLUSIONS: The developed dot-ELISA is very suitable for the routine detection of TYLCV in field tomato and whitefly samples, and the DTBIA is more suitable for the routine detection of TYLCV in large-scale tomato plant samples collected from only TYLCV prevalent areas.
  Virology Journal 05/2013; 10(1):142.
• Article: Analytical technologies for influenza virus-like particle candidate vaccines: challenges and emerging approaches.

  Christine M Thompson, Emma Petiot, Alexandre Lennaertz, Olivier Henry, Amine A Kamen
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  ABSTRACT: Influenza virus-like particle vaccines are one of the most promising ways to respond to the threat of future influenza pandemics. VLPs are composed of viral antigens but lack nucleic acids making them non-infectious which limit the risk of recombination with wild-type strains. By taking advantage of the advancements in cell culture technologies, the process from strain identification to manufacturing has the potential to be completed rapidly and easily at large scales. After closely reviewing the current research done on influenza VLPs, it is evident that the development of quantification methods has been consistently overlooked. VLP quantification at all stages of the production process has been left to rely on current influenza quantification methods (i.e. -- Hemagglutination assay (HA), Single Radial Immunodiffusion assay (SRID), NA enzymatic activity assays, Western blot, Electron Microscopy). These are analytical methods developed decades ago for influenza virions and final bulk influenza vaccines. Although these methods are time-consuming and cumbersome they have been sufficient for the characterization of final purified material. Nevertheless, these analytical methods are impractical for in-line process monitoring because VLP concentration in crude samples generally falls out of the range of detection for these methods. This consequently impedes the development of robust influenza-VLP production and purification processes. Thus, development of functional process analytical techniques, applicable at every stage during production, that are compatible with different production platforms is in great need to assess, optimize and exploit the full potential of novel manufacturing platforms.
  Virology Journal 05/2013; 10(1):141.
• Article: Detection of specific HPV subtypes responsible for the pathogenesis of condylomata acuminata.

  Matthew G Hawkins, David M Winder, Siolian L Ball, Katie Vaughan, Christopher Sonnex, Margaret A Stanley, Jane C Sterling, Peter K Goon
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  ABSTRACT: BACKGROUND: The low-risk human papillomavirus types 6 and 11 are responsible for approximately 90% of anogenital wart cases, with approximately 190,000 new and recurrent cases reported in the UK in 2010. The UK has recently selected the quadrivalent HPV vaccine, which conveys protection against both HPV6 and HPV 11, as part of its immunisation programme for 2012 and it is expected that this will reduce disease burden in the UK. The aims of the study were to evaluate current strategies used for the monitoring of HPV infection in genital warts and to assess the suitability of laser-capture microdissection (LCM) as a technique to improve the understanding of the natural history of HPV types associated with genital wart lesions. METHODS: DNA and RNA were extracted from whole wart, surface swabs and LCM sections from 23 patients. HPV types present were determined using the Linear Array HPV Genotyping Test (Roche), with HPV DNA viral load and mRNA expression investigated using qPCR and qRT-PCR, respectively. RESULTS: Results indicated that swabbing the surface of warts does not accurately reflect potential causative HPV types present within a wart lesion, multiple HPV types being present on the surface of the wart that are absent in the lower layers of tissue isolated by LCM. Although it was shown that HPV DNA viral load does not directly correlate with HPV mRNA load, the presence of both DNA and mRNA from a single HPV type suggested a causative role in lesion development in 8/12 (66.6%) of patients analysed, with dual infections seen in 4/12 (33.3%) cases. HPV 6 and HPV 11 were present in more than 90% of the lesions examined. CONCLUSIONS: Surface swabbing of warts does not necessarily reflect the causative HPV types. HPV type specific DNA and mRNA loads do not correlate. HPV 6 and 11 were likely to be causally involved in over 90% of the lesions. Dual infections were also found, and further studies are required to determine the biological and clinical nature of dual/multiple infections and to establish the relationship of multiple HPV types within a single lesion.
  Virology Journal 05/2013; 10(1):137.
• Article: Quantitative detection of relative expression levels of the whole genome of Southern rice black-streaked dwarf virus and its replication in different hosts.

  Peng He, Jia-Ju Liu, Ming He, Zhen-Chao Wang, Zhuo Chen, Rong Guo, James C Correll, Song Yang, Bao-An Song
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  ABSTRACT: BACKGROUND: In recent years, a disease caused by Southern rice black-streaked dwarf virus (SRBSDV) has resulted in significant loss in rice production in Southern China and has spread quickly throughout East and Southeast Asia. This virus is transmitted by an insect vector, white-backed planthopper (WBPH) Sogatella furcifera (Hemiptera: Delphacidae), in a persistent propagative manner. Aside from rice, SRBSDV can also infect numerous Poaceae plants. However, the molecular mechanism of interaction between SRBSDV and its plant or insect vector remains unclear. In order to address this, we investigated the whole viral genome relative mRNA expression level in distinct hosts and monitored their expression level in real-time in rice plants. METHODS: In this study, a reliable, rapid, and sensitive method for detecting viral gene expression transcripts is reported. A SYBR Green I based real-time polymerase chain reaction (PCR) method was adopted for the quantitative detection of SRBSDV gene expression in different hosts and real-time changes in gene expression in rice. RESULTS: Compared to the relative mRNA expression level of the whole genome of SRBSDV, P3, P7-1, and P9-2 were dominantly expressed in rice and WBPH. Similarly, these genes also exhibited high expression levels in corn, suggesting that they have more important functions than other viral genes in the interaction between SRBSDV and hosts, and that they could be used as molecular detection target genes of SRBSDV. In contrast, the levels of P6 and P10 were relative low. Western blotting analysis partially was also verified our qPCR results at the level of protein expression. Analysis of the real-time changes in SRBSDV-infected rice plants revealed four distinct temporal expression patterns of the thirteen genes. Moreover, expression levels of P1 and other genes were significantly down-regulated on days 14 and 20, respectively. CONCLUSION: SRBSDV genes showed similar expression patterns in distinct hosts (rice, corn, and WBPH), indicating that SRBSDV uses the same infection strategy in plant and insect hosts. P3, P7-1, and P9-2 were the dominantly expressed genes in the three tested hosts. Therefore, they are likely to be genes with the most crucial function and could be used as sensitive molecular detection targets for SRBSDV. Furthermore, real-time changes in SRBSDV genes provided a basis for understanding the mechanism of interaction between SRBSDV and its hosts.
  Virology Journal 05/2013; 10(1):136.
• Article: Comparison of a loop-mediated isothermal amplification for orf virus with quantitative real-time PCR.

  Guangxiang Wang, Youjun Shang, Yanhua Wang, Hong Tian, Xiangtao Liu
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  ABSTRACT: BACKGROUND: Orf virus (ORFV) causes orf (also known as contagious ecthyma or contagious papular dermatitis), a severe infectious skin disease in goats, sheep and other ruminants. Therefore, a rapid, highly specific and accurate method for the diagnosis of ORFV infections is essential to ensure that the appropriate treatments are administered and to reduce economic losses. METHODS: A loop-mediated isothermal amplification (LAMP) assay based on the identification of the F1L gene was developed for the specific detection of ORFV infections. The sensitivity and specificity of the LAMP assay were evaluated, and the effectiveness of this method was compared with that of real-time PCR. RESULTS: The sensitivity of this assay was determined to be 10 copies of a standard plasmid. Furthermore, no cross-reactivity was found with either capripox virus or FMDV. The LAMP and real-time PCR assays were both able to detect intracutaneous- and cohabitation-infection samples, with a concordance of 97.83%. LAMP demonstrated a sensitivity of 89.13%. CONCLUSION: The LAMP assay is a highly efficient and practical method for detecting ORFV infection. This LAMP method shows great potential for monitoring the prevalence of orf, and it could prove to be a powerful supplemental tool for current diagnostic methods.
  Virology Journal 05/2013; 10(1):138.
• Article: The highly pathogenic H7N3 avian influenza strain from July 2012 in Mexico acquired an extended cleavage site through recombination with host 28S rRNA.

  Sebastian Maurer-Stroh, Raphael Tc Lee, Vithiagaran Gunalan, Frank Eisenhaber
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  ABSTRACT: BACKGROUND: A characteristic difference between highly and non-highly pathogenic avian influenza strains is the presence of an extended, often multibasic, cleavage motif insertion in the hemagglutinin protein. Such motif is found in H7N3 strains from chicken farm outbreaks in 2012 in Mexico. METHODS: Through phylogenetic, sequence and structural analysis, we try to shed light on the role, prevalence, likelihood of appearance and origin of the inserted cleavage motifs in these H7N3 avian influenza strains. RESULTS: The H7N3 avian influenza strain which caused outbreaks in chicken farms in June/July 2012 in Mexico has a new extended cleavage site which is the likely reason for its high pathogenicity in these birds. This cleavage site appears to have been naturally acquired and was not present in the closest low pathogenic precursors. Structural modeling shows that insertion of a productive cleavage site is quite flexible to accept insertions of different length and with sequences from different possible origins. Different from recent cleavage site insertions, the origin of the insert here is not from the viral genome but from host 28S ribosomal RNA (rRNA) instead. This is a novelty for a natural acquisition as a similar insertion has so far only been observed in a laboratory strain before. Given the abundance of viral and host RNA in infected cells, the acquisition of a pathogenicity-enhancing extended cleavage site through a similar route by other low-pathogenic avian strains in future does not seem unlikely. Important for surveillance of these H7N3 strains, the structural sites known to enhance mammalian airborne transmission are dominated by the characteristic avian residues and the risk of human to human transmission should currently be low but should be monitored for future changes accordingly. CONCLUSIONS: This highly pathogenic H7N3 avian influenza strain acquired a novel extended cleavage site which likely originated from recombination with 28S rRNA from the avian host. Notably, this new virus can infect humans but currently lacks critical host receptor adaptations that would facilitate human to human transmission.
  Virology Journal 05/2013; 10(1):139.
• Article: Comparative expression of Toll-like receptors and inflammatory cytokines in pigs infected with different virulent porcine reproductive and respiratory syndrome virus isolates.

  Lili Zhang, Jie Liu, Juan Bai, Xiaoye Wang, Yufeng Li, Ping Jiang
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  ABSTRACT: BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is largely responsible for heavy economic losses in the swine industry worldwide because of its high mutation rate and subsequent emergence of virulent strains. However, the immunological and pathological responses of pigs to PRRSV strains with different virulence have not been completely elucidated. METHODS: Twenty-four piglets were divided into 4 groups (n = 6 each) and inoculated with highly pathogenic PRRSV isolate BB0907 (HP), low pathogenic PRRSV NT0801 (LP), LP derivative strain NT0801-F70 (LP-der), and DMEM medium (control), respectively. The changes in TLR2, 3, 7, and 8 gene expression and TNF-alpha, IL-1beta, IL-6, IFN-gamma, and IL-10 secretion were evaluated using real-time PCR and ELISA at 6, 9, and 15 days post inoculation (d.p.i.). The cytokine levels were evaluated in the supernatants of porcine alveolar macrophages (PAMs) and peripheral blood mononuclear cells (PBMCs) following stimulation with LTA, poly(I:C), CL097, and PRRSV individually. RESULTS: HP caused more severe clinical signs and pathological lesions in swine than LP and LP-der had almost no virulence compared with LP. The serum levels of IL-1beta, IL-6, TNF-alpha, and IFN-gamma were increased in HP-infected piglets, which were greater than in those infected with LP or LP-der. The mRNA levels of TLR3, 7, and 8 were significantly up-regulated in PAMs in HP-infected pigs compared to those in groups LP and LP-der. Furthermore, TNF-alpha and IL-1beta secretion in PAMs from group LP was statistically greater than those from the control group after stimulation with either poly(I:C) or CL097. Meanwhile, TNF-alpha, IL-1beta, and IL-6 levels in CL097-stimulated PBMCs from HP-infected pigs were markedly higher than those from the LP- and LP-der-infected groups. CONCLUSIONS: We found that HP was a stronger inducer of TLR 3, 7, and 8 expression and IL-1beta, IL-6, TNF-alpha, and IFN-gamma production compared to LP and LP-der. HP enhanced production of TNF-alpha, IL-1beta, and IL-6 in PBMCs following CL097-stimulation more than LP and LP-der, whereas LP enhanced the secretion of TNF-alpha and IL-1beta in poly(I:C)- and CL097-stimulated PAMs. Our data regarding cellular reactivity to different isolates should be useful in the development of more efficacious vaccines.
  Virology Journal 04/2013; 10(1):135.
• Article: Meta-analysis of the short-term effects of lamivudine treatment for severe chronic hepatitis B.

  Lin Zhang, Jiang-Fu Liu, Meng Wang, Chun-Qiu Hao
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  ABSTRACT: PurposeTo evaluate the short-term effect of lamivudine (LMV) treatment for severe chronic hepatitis B. METHOD: Patient data related to the safety and efficacy of using lamivudine (LMV) to treat hepatitis B virus (HBV)-induced liver failure or severe hepatitis were acquired from previous literature. These studies were retrieved from PubMed, Ovid, SpringerLink, Biosis Previews, Academic Search Premier, ProQuest Medical Library, Cochrane Library, China National Knowledge Infrastructure Full-text Database, VIP Chinese Scientific Journal Database, and Chinese Biomedicine. Relative risk and weighted mean difference were used to measure the effects. The major predictors observed included total bilirubin (TBIL), prothrombin activity (PTA), survival rate, and HBV-DNA negative change rate. Groups were further divided according to the clinical course and disease staging. RESULTS: A total of 242 studies were retrieved from the databases. At weeks 4, 8, and 12 of the treatment course, the survival rates and PTA of the test group were distinctively higher than those of the control group. However, TBIL concentrations in the test group were lower than the control group. The HBV-DNA negative change rate was distinctively higher throughout the 12 weeks of LMV treatment. For patients who started LMV treatment in the middle stage, the mortality rate of the test group was lower. For patients who started LMV treatment during the advanced stage, no significant difference was observed between the test and control groups. CONCLUSION: LMV decreased HBV-DNA levels in the serum, improved liver function in patients, and enhanced survival rate during the early and medium stages of severe chronic hepatitis B.
  Virology Journal 04/2013; 10(1):134.
• Article: Aquareovirus protein VP6 colocalizes with NS80 protein in infected and transfected cells.

  Dawei Wen, Liming Yan, Ling Shao, Hong Guo, Xiaoming Li, Qin Fang
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  ABSTRACT: BACKGROUND: Aquareovirus particle is comprised of central core and outer capsid, which is built by seven structural proteins (VP1-VP7). The protein VP6 has been identified to be a clamp protein of stabilizing inner core frame VP3, and bridging outer shell protein VP5. However, the biological properties of VP6 in viral life cycle remain unknown. RESULTS: The recombinant VP6 (rVP6) of aquareovirus was expressed in E. coli, and the polyclonal antibody against VP6 was generated by using purified rVP6 in this study. Following the preparation of VP6 antibody, the VP6 component in aquareovirus infected cells and purified viral particles was detected by Immunoblotting (IB) assay. Furthermore, using Immunofluorescence (IF) microscopy, singly transfected VP6 protein was observed to exhibit a diffuse distribution mainly in the cytoplasm, while it appeared inclusion phenotype in infected cells. Meanwhile, inclusion structures were also identified when VP6 was coexpressed with nonstructural protein NS80 in cotransfected cells. CONCLUSIONS: VP6 can be recruited by NS80 to its inclusions in both infected and transfected cells. The colocalization of VP6 and NS80 is corresponding to their homologous proteins sigma2 and muNS in MRV. Our results suggest that VP6 may play a significant role in viral replication and particle assembly.
  Virology Journal 04/2013; 10(1):133.
• Article: A mobile genetic element with unknown function found in distantly related viruses.

  Torstein Tengs, Anja Bråthen Kristoffersen, Tsvetan R Bachvaroff, Christine Monceyron Jonassen
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  ABSTRACT: BACKGROUND: The genetic element s2m seems to represent one of very few examples of mobile genetic elements in viruses. The function remains obscure and a scattered taxonomical distribution has been reported by numerous groups. METHODS: We have searched GenBank in order to identify all viral accessions that have s2m(-like) sequence motifs. Rigorous phylogenetic analyses and constrained tree topology testing were also performed in order to investigate the apparently mobile nature of s2m. RESULTS: The stem-loop s2m structure can be found in four families of + ssRNA viruses; Astroviridae, Caliciviridae, Picornaviridae and Coronaviridae. In all of these virus families, with the possible exception of Caliciviridae, multiple gains and/or losses of s2m would have to be postulated in order to explain the distribution of this character. CONCLUSIONS: s2m appears to be a mobile genetic element with a unique evolutionary history in all of the four virus families where it can be found. Based on our findings and a review of the current literature on s2m, a hypothesis implying an RNAi-like function for the s2m element is also outlined.
  Virology Journal 04/2013; 10(1):132.
• Article: Use of immuno-dominant epitope derived from genotype 4 as a diagnostic reagent for detecting the antibodies against Hepatitis E Virus.

  Xiu Bing-Shui, Feng Xiao-Yan, He Jing, Chen Kun, Liu Jing, Dai Zhen-Hua, Yang Xi-Qin, Wang Guo-Hua, Wang You-Chun, Zhang He-Qiu, Song Xiao-Guo, Zhu Cui-Xia
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  ABSTRACT: BACKGROUND: Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered. METHODS: We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4. RESULTS: The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014). CONCLUSIONS: The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples.
  Virology Journal 04/2013; 10(1):131.
• Article: ZASC1 knockout mice exhibit an early bone marrow-specific defect in murine leukemia virus replication.

  Shannon Seidel, James Bruce, Mathias Leblanc, Kuo-Fen Lee, Hung Fan, Paul Ahlquist, John At Young
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  ABSTRACT: BACKGROUND: ZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010) METHODS: To determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies. RESULTS: Mice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection. CONCLUSIONS: These studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection.
  Virology Journal 04/2013; 10(1):130.