2012 Vol.27(5)


Research Article

Rapid Detection of Filoviruses by Real-time TaqMan Polymerase Chain Reaction Assays

Yi Huang, Hongping Wei, Yunpeng Wang, Zhengli Shi, Herve Raoul, Zhiming Yuan

2012, 27(5): 273 doi: 10.1007/s12250-012-3252-y

Received: 10 April 2012 Accepted: 06 August 2012
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Ebola virus (EBOV) and Marburg virus (MARV) are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics. To date, there is no specific laboratory diagnostic test in China. There is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. In this study, the TaqMan RT-PCR assays targeting the nucleoprotein gene of the Zaire Ebolavirus (ZEBOV) and MARV were developed and their sensitivities and specificities were investigated. Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109, corresponding to the threshold of a standard RNA transcript. The results showed that there were about 1010 RNA copies per milliliter of virus culture supernatant, equivalent to 10000 RNA molecules per infectious virion, suggesting the presence of many non-infectious particles. These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable for future surveillance and specific diagnosis of ZEBOV and MARV in China.

Regulation of Hepatitis C Virus Replication and Gene Expression by the MAPK-ERK Pathway

Rongjuan Pei, Xiaoyong Zhang, Song Xu, Zhongji Meng, Michael Roggendorf, Mengji Lu, Xinwen Chen

2012, 27(5): 278 doi: 10.1007/s12250-012-3257-6

Received: 26 April 2012 Accepted: 17 August 2012
Abstract [PDF 557 KB] Springerlink
The mitogen activated protein kinases-extracellular signal regulated kinases (MAPK-ERK) pathway is involved in regulation of multiple cellular processes including the cell cycle. In the present study using a Huh7 cell line Con1 with an HCV replicon, we have shown that the MAPK-ERK pathway plays a significant role in the modulation of HCV replication and protein expression and might influence IFN-? signalling. Epithelial growth factor (EGF) was able to stimulate ERK activation and decreased HCV RNA load while a MAPK-ERK pathway inhibitor U0126 led to an elevated HCV RNA load and higher NS5A protein amounts in Con1 cells. It could be demonstrated that the inhibition of the MAPK-ERK pathway facilitated the translation directed by the HCV internal ribosome entry site. Consistently, a U0126 treatment enhanced activity of the HCV reporter replicon in transient transfection assays. Thus, the MAPK-ERK pathway plays an important role in the regulation of HCV gene expression and replication. In addition, cyclin-dependent kinases (CDKs) downstream of ERK may be involved in the modulation of HCV replication since roscovitine, an inhibitor of CDKs had a similar effect to that of U0126. Modulation of the cell cycle progression by cell cycle inhibitor or RNAi resulted consistently in changes of HCV RNA levels. Further, the replication of HCV replicon in Con1 cells was inhibited by IFN-?. The inhibitory effect of IFN-? could be partly reversed by pre-incubation of Con-1 cells with inhibitors of the MAPK-ERK pathway and CDKs. It could be shown that the MAPK-ERK inhibitors are able to partially modulate the expression of interferon-stimulated genes.

Comparison of Immune Responses against FMD by a DNA Vaccine Encoding the FMDV/O/IRN/2007 VP1 Gene and the Conventional Inactivated Vaccine in an Animal Model

Farahnaz Motamedi Sedeh, Hoorieh Soleimanjahi, AmirReza Jalilian, Homayoon Mahravani

2012, 27(5): 286 doi: 10.1007/s12250-012-3258-5

Received: 28 April 2012 Accepted: 16 July 2012
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Foot-and-mouth disease virus (FMDV) is highly contagious and responsible for huge outbreaks among cloven hoofed animals.The aim of the present study is to evaluate a plasmid DNA immunization system that expresses the FMDV/O/IRN/2007 VP1 gene and compare it with the conventional inactivated vaccine in an animal model. The VP1 gene was sub-cloned into the unique Kpn I and BamH I cloning sites of the pcDNA3.1+ and pEGFP-N1 vectors to construct the VP1 gene cassettes. The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector expressed GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector, which solely expressed the GFP protein.Six mice groups were respectively immunized by the sub-cloned pcDNA3.1+-VP1 gene cassette as the DNA vaccine, DNA vaccine and PCMV-SPORT-GMCSF vector (as molecular adjuvant) together, conventional vaccine, PBS (as negative control),pcDNA3.1+ vector (as control group) and PCMV-SPORT vector that contained the GMCSF gene (as control group).Significant neutralizing antibody responses were induced in the mice which were immunized using plasmid vectors expressing the VP1 and GMCSF genes together, the DNA vaccine alone and the conventional inactivated vaccine (P<0.05).Co-administration of DNA vaccine and GMCSF gene improved neutralizing antibody response in comparison with administration of the DNA vaccine alone, but this response was the most for the conventional vaccine group. However, induction of humeral immunity response in the conventional vaccine group was more protective than for the DNA vaccine,but T-cell proliferation and IFN-γ concentration were the most in DNA vaccine with the GMCSF gene. Therefore the group that was vaccinated by DNA vaccine with the GMCSF gene, showed protective neutralizing antibody response and the most Th1 cellular immunity.

Molecular Characterization of Avian-like H1N1 Swine Influenza A Viruses Isolated in Eastern China, 2011

Xian Qi, Yuning Pan, Yuanfang Qin, Rongqiang Zu, Fengyang Tang, Minghao Zhou, Hua Wang, Yongchun Song

2012, 27(5): 292 doi: 10.1007/s12250-012-3262-9

Received: 03 June 2012 Accepted: 06 September 2012
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Currently, three predominant subtypes of influenza virus are prevalent in pig populations worldwide: H1N1, H3N2, and H1N2. European avian-like H1N1 viruses, which were initially detected in European pig populations in 1979, have been circulating in pigs in eastern China since 2007. In this study, six influenza A viruses were isolated from 60 swine lung samples collected from January to April 2011 in eastern China. Based on whole genome sequencing, molecular characteristics of two isolates were determined. Phylogenetic analysis showed the eight genes of the two isolates were closely related to those of the avian-like H1N1 viruses circulating in pig populations, especially similar to those found in China. Four potential glycosylation sites were observed at positions 13, 26, 198, 277 in the HA1 proteins of the two isolates. Due to the presence of a stop codon at codon 12, the isolates contained truncated PB1-F2 proteins. In this study, the isolates contained 591Q, 627E and 701N in the polymerase subunit PB2, which had been shown to be determinants of virulence and host adaptation. The isolates also had a D rather than E at position 92 of the NS1, a marker of mammalian adaptation. Both isolates contained the GPKV motif at the PDZ ligand domain of the 3’ end of the NS1, a characteristic marker of the European avian-like swine viruses since about 1999, which is distinct from those of avian, human and classical swine viruses. The M2 proteins of the isolates have the mutation (S31N), a characteristic marker of the European avian-like swine viruses since about 1987, which may confer resistance to amantadine and rimantadine antivirals. Our findings further emphasize the importance of surveillance on the genetic diversity of influenza A viruses in pigs, and raise more concerns about the occurrence of cross-species transmission events.

Evaluation of a Direct Rapid Immunohistochemical Test (dRIT) for Rapid Diagnosis of Rabies in Animals and Humans

Shampur Narayan Madhusudana, Sundaramurthy Subha, Ullas Thankappan, Yajaman Belludi Ashwin

2012, 27(5): 299 doi: 10.1007/s12250-012-3265-6

Received: 14 June 2012 Accepted: 07 September 2012
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Presently the gold standard diagnostic technique for rabies is the direct immunofluorescence assay ( dFA) which is very expensive and requires a high level of expertise. There is a need for more economical and user friendly tests, particularly for use in developing countries. We have established one such test called the direct rapid immunohistochemical test (dRIT) for diagnosis of rabies using brain tissue. The test is based on capture of rabies nucleoprotein (N) antigen in brain smears using a cocktail of biotinylated monoclonal antibodies specific for the N protein and color development by streptavidin peroxidase-amino ethyl carbazole and counter staining with haematoxollin. The test was done in parallel with standard FAT dFA using 400 brain samples from different animals and humans. The rabies virus N protein appears under light microscope as reddish brown particles against a light blue background. There was 100 % correlation between the results obtained by the two tests. Also, interpretation of results by dRIT was easier and only required a light microscope. To conclude, this newly developed dRIT technique promises to be a simple, cost effective diagnostic tool for rabies and will have applicability in field conditions prevalent in developing countries.

Characterization of Synonymous Codon Usage Bias in the Pseudorabies Virus US1 Gene

Meili Li, Zhiyao Zhao, Jianhong Chen, Bingyun Wang, Zi Li, Jian Li, Mingsheng Cai

2012, 27(5): 303 doi: 10.1007/s12250-012-3270-9

Received: 10 July 2012 Accepted: 12 September 2012
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In the present study, we examined the codon usage bias between pseudorabies virus (PRV) US1 gene and the US1-like genes of 20 reference alphaherpesviruses. Comparative analysis showed noticeable disparities of the synonymous codon usage bias in the 21 alphaherpesviruses, indicated by codon adaptation index, effective number of codons (ENc) and GC3s value. The codon usage pattern of PRV US1 gene was phylogenetically conserved and similar to that of the US1-like genes of the genus Varicellovirus of alphaherpesvirus, with a strong bias towards the codons with C and G at the third codon position. Cluster analysis of codon usage pattern of PRV US1 gene with its reference alphaherpesviruses demonstrated that the codon usage bias of US1-like genes of 21 alphaherpesviruses had a very close relation with their gene functions. ENc-plot revealed that the genetic heterogeneity in PRV US1 gene and the 20 reference alphaherpesviruses was constrained by G+C content, as well as the gene length. In addition, comparison of codon preferences in the US1 gene of PRV with those of E. coli, yeast and human revealed that there were 50 codons showing distinct usage differences between PRV and yeast, 49 between PRV and human, but 48 between PRV and E. coli. Although there were slightly fewer differences in codon usages between E.coli and PRV, the difference is unlikely to be statistically significant, and experimental studies are necessary to establish the most suitable expression system for PRV US1. In conclusion, these results may improve our understanding of the evolution, pathogenesis and functional studies of PRV, as well as contributing to the area of herpesvirus research or even studies with other viruses.
Brief Report

Generation of Monoclonal Antibodies against Non-structural Protein 3AB of Foot-and-Mouth Disease Virus

Tong Lin, Junjun Shao, Huiyun Chang, Shandian Gao, Guozheng Cong, Junzheng Du

2012, 27(5): 316 doi: 10.1007/s12250-012-3261-x

Received: 31 May 2012 Accepted: 12 September 2012
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To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV), BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained, named C6 and E7 respectively . The microneutralization titer was 1:1024 for mAb C6, and 1:512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens. The titers in abdomen liquor were 1:5×106 for C6 and 1:2×106 for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.

Spatial Trend of Foot and Mouth Disease Virus (FMDV) Serotypes in Cattle and Buffaloes, Pakistan

Muhammad Abubakar, Muhammad Javed Arshed, Qurban Ali, Manzoor Hussain

2012, 27(5): 320 doi: 10.1007/s12250-012-3271-8

Received: 12 July 2012 Accepted: 06 September 2012
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The present study describes the frequency of Foot and Mouth Disease (FMD) virus serotypes (O, A and Asia-1) in major regions (all provinces) of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution of various FMD serotypes and their comparison is discussed. A total of 590 samples (Epithelial tissue) have been analyzed during a period of five years (2005–2009). Out of 590 samples, 180 were found positive, giving an overall confirmation of FMDV about 33.2 %. Of the prevalent serotypes, FMDV ‘O’ serotype caused most outbreaks (20.7 %), followed by serotype A (6.6 %) and serotype Asia-1 (4.6 %) while there was no positive case of type ‘C’. The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas. Based on the data of 590 samples (>50 outbreaks), the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %, while in cattle alone, it was 37.1 %, higher than in buffalo (28.7 %). There were eight cases of mixed serotypes infection, indicating the presence of endemic state of disease. Another significant feature was the change over time. In phase-I (2005–2007), there was an overall prevalence of 29.4 %, while the occurrence of the serotype O, A and Asia-1 was 20.4 %, 2.9 % and 4.7 %, respectively. During phase-II (2008–2009), the overall prevalence was 59.21 %, while those of serotype O, A and Asia-1 were 22.4 %, 31.6 % and 4.0 %, respectively. This clearly indicated a shift from serotype O to A, which may help to explain the occurrence of more severe outbreaks, despite vaccination.