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2009 Vol.24(1)


Research Article

Functional Analyses of Mammalian Reovirus Nonstructural Protein μNS*

Chao FAN, Qin FANG

2009, 24(1): 1 doi: 10.1007/s12250-009-3016-5

Received: 10 December 2008 Accepted: 18 December 2008
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Genome replication of reovirus occurs in cytoplasmic inclusion bodies called viral factories or viroplasms. The viral nonstructural protein μNS, encoded by genome segment M3, is not a component of mature virions, but is expressed to high levels in infected cells and is concentrated in the infected cell factory matrix. Recent studies have demonstrated that μNS plays a central role in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly.

Molecular Characterization of a Highly Pathogenetic Porcine Reproductive and Respiratory Syndrome Virus Variant in Hubei, China *

Yi HUANG, Bing ZHANG, Zhen-fang FU, Simon Rayner, Fang-liang ZHENG, Wang-wang LIANG, Ke-li YANG, Di-ping XU, Han-zhong WANG

2009, 24(1): 9 doi: 10.1007/s12250-009-3012-9

Received: 01 December 2008 Accepted: 10 December 2008
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Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized as one of the most important pathogens of pigs throughout the world. In 2006, more than 10 provinces of China have experienced an epizootic outbreak of pig diseases characterized by high fever, reddened skin and high morbidity and mortality. From June 2006 to April 2007, we have investigated some clinical samples in Hubei province by RT-PCR and cloned several major genes, N, GP5 and NSP2 gene, shown in this study. Phylogenetic analysis of these genes revealed that the highly pathogenic PRRSV variant, ZB, was responsible for 2006 emergent outbreak of pig disease in Hubei province similar with those variants isolated from other provinces in China in 2006, and belongs to the NA-type PRRSV. In the PRRSV variants, the N and GP5 shear about 90% identity with prototypic ATCC VR-2332 and some typical NA-type Chinese isolates, except the 2850bp NSP2 gene (only shares 65% identity with ATCC VR-2332). But they all shear more than and 97% identity with other highly pathogenetic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and 2 deletions in the Nsp2 protein when compared with the previous isolates. Most of the variants found in 2006 epizootic outbreak of pig diseases in China were the farthest variants from the typical NA-type PRRSV in phylogenetic distance, and these diversities may be responsible for the differences in the pathogenicity observed between these variants and original Chinese PRRSV strains.

Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract *

Xiu-ying PU, Jian-ping LIANG, Xue-hong WANG, Tao XU, Lan-ying HUA, Ruo-feng SHANG, Yu LIU, Yan-mei XING

2009, 24(1): 19 doi: 10.1007/s12250-009-2983-x

Received: 31 July 2008 Accepted: 28 November 2008
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To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus(IAV)(H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney(MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin’s minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.

Complete Nucleotide Sequence of a Mumps Virus SP Strain Isolated in China*

Shao-hui MA, Jian-sheng LIU, Hai-jing SHI, Li-chun WANG, Jing-jing WANG, Long-ding LIU, Qi-han LI

2009, 24(1): 28 doi: 10.1007/s12250-009-2984-9

Received: 04 August 2008 Accepted: 18 December 2008
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The complete nucleotide sequence of the mumps virus SP, which was isolated in China, was determined. As with other mumps viruses, its genome was 15 384 nucleotides (nts) in length and encoded seven proteins. The full-length nucleotide sequence of the SP isolate differed from other strains by 4% –6.8% at the nucleotide sequence level. Due to variations of amino acids over the full genome (including the HN and N genes), this isolate exhibited significant variations in the antigenic sites. This report is the first to describe the full-length genome of a genotype F strain and provide an overview of the diversity of genetic characteristics of a circulating mumps virus.

Genome Sequencing and Phylogenetic Analysis of Three Avian Influenza H9N2 Subtypes in Guangxi*

Zhi-xun XIE, Jian-bao DONG, Xiao-fei TANG, Jia-bo LIU, Yao-shan PANG, Xian-wen DENG, Zhi-qin XIE, Li-ji XIE, Mazhar I Khan

2009, 24(1): 37 doi: 10.1007/s12250-009-2985-8

Received: 04 August 2008 Accepted: 08 December 2008
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Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.

Detection of Antibody to Hepatitis Delta Virus in Human Serum by Double Antigen Sandwich ELISA

Li XIE, De-zhuang HUANG, Li-xiang HE, Zhao-xia LUO, Yu-sen ZHOU, Xiao-dong WU

2009, 24(1): 45 doi: 10.1007/s12250-009-2981-2

Received: 07 July 2008 Accepted: 28 November 2008
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A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein’s purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.

Genetic Analysis and Rescue of a Triple-reassortant H3N2 Influenza A Virus Isolated From Swine in Eastern China *

Xian QI, Yong-jun JIAO, Hao PAN, Lun-biao CUI, Wei-xing FAN, Bao-xu HUANG, Zhi-yang SHI, Hua WANG

2009, 24(1): 52 doi: 10.1007/s12250-009-3006-7

Received: 13 October 2008 Accepted: 18 December 2008
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One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like H1N1, NS from classical swine H1N1, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.

Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

Wei-feng SHI, Zhong ZHANG, Ai-she DUN, Yan-zhou ZHANG, Guang-fu YU, Dong-ming ZHUANG, Chao-dong ZHU

2009, 24(1): 59 doi: 10.1007/s12250-009-2976-9

Received: 01 June 2008 Accepted: 10 October 2008
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Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.

Selection Pressure on Haemagglutinin Genes of H9N2 Influenza Viruses from Different Hosts

Wei-feng SHI, Ai-she DUN, Zhong ZHANG, Yan-zhou ZHANG, Guang-fu YU, Dong-ming ZHUANG, Chao-dong ZHU

2009, 24(1): 65 doi: 10.1007/s12250-009-2988-5

Received: 08 August 2008 Accepted: 22 October 2008
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Positive selection and differential selective pressure analyses were carried out to study Haemagglutinin (HA) genes of H9N2 influenza viruses from different hosts in this paper. Results showed that, although most positions in HAs were under neutral or purifying evolution, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection and some of them were even positively selected at the population level. In addition, there were always some positions differentially selected for viruses from different hosts. Both selection pressure working on HA codons and positions differentially selected might account for the extension of the host range and adaptations to different hosts of H9N2 influenza viruses.

Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus *

Li-juan LI, Hua-jun ZHANG, Cong ZHANG, Zheng-li SHI

2009, 24(1): 71 doi: 10.1007/s12250-009-3013-8

Received: 05 December 2008 Accepted: 12 December 2008
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The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

The HBV E Genotype Discover in Dai Nationality in Xishuangbanna, Yunnan Province *

Hai-ping ZHAO, Yuan-ying SHEN, Ru SHEN, Yuan-yi WANG, Mei-ya FU

2009, 24(1): 77 doi: 10.1007/s12250-009-2992-r

Received: 15 August 2008 Accepted: 05 December 2008
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To investigate the distribution of Hepatitis B virus (HBV) genotypes among the population of Dai nationality in Xishuangbanna, Yunnan Province HBV genotypes of the Serum samples were tested by PCR-RFLP. This is the first time to discover the B+E genotypes in China. This finding provides new information for understanding the distribution of HBV genotype in China and a provides a basis for establishing a Chinese gene bank.