Detection of substantial porcine group B rotavirus genetic diversity in the United States, resulting in a modified classification proposal for G genotypes

Rotavirus (RV) is an important cause of gastrointestinal disease in animals and humans. In this study, we developed an RT-PCR to detect RV group B (RVB) and characterized the VP7 (G) gene segment detected in porcine samples. One hundred seventy three samples were tested for RV group A (RVA), RVB, and C (RVC) by RT-PCR and examined for RV-like lesion using histopathology. A majority (86.4%) of the samples had mixed RV infections and co-infections of RVA/RVB/RVC were detected at a higher rate (24.3%) than previously reported. RVB was identified in 46.8% of the 173 samples. An adapted VP7 classification was developed using previously published (n=57) and newly sequenced (n=68) RVB strains, resulting in 20 G genotypes based on an 80% nucleotide identity cutoff value. Our results revealed a broad genetic diversity of porcine RVB strains, suggesting RVB has been the cause of common/pre-existing, yet undiagnosed, disease in pigs.     

基于化学发光肠道病毒71型IgM抗体检测试剂的研制及初步应用

目的在肠道病毒71型(EV71)重组外壳蛋白VP1的基础上建立以化学发光为基础的IgM抗体检测技术,并与普通TMB显色酶联免疫吸附法比较,评价其临床应用前景。方法采用抗IgM单克隆抗体及辣根酶标记的重组VP1抗原,在此基础上建立EV71-IgM捕获法化学发光检测技术,并对比化学发光检测技术与普通酶联检测技术在45份EV71抗体阳性和30份阴性血清中的反应情况。结果化学发光法、普通酶联免疫吸附法、市售EV71-IgM检测试剂分别能与38份、19份、26份EV71抗体阳性血清发生阳性反应,灵敏度分别为84.4%、42.2%和57.8%;化学发光法灵敏度显著高于其他两种,差异有统计学意义(P<0.05);3种检测方法与阴性血清均无阳性反应。结论在EV71-VP1重组抗原的基础上建立的EV71-IgM捕获法化学发光检测技术具有很高的灵敏度,并有很好的临床应用前景。     

传染性法氏囊病病毒B87株VP2基因克隆及序列分析

目的获得传染性法氏囊病病毒（IBDV）B87株VP2基因,并对其序列进行分析。方法采用RT-PCR方法扩增传染性法氏囊病病毒B87株VP2基因,扩增产物提纯后克隆入pMD18-T载体,通过酶切、PCR和测序验证克隆,测序结果与GenBank下载的21株参考毒株VP2基因编码区全核苷酸序列进行比较。结果克隆的VP2基因序列长度为1536bp。所测B87株与Ⅰ型参考毒株的核苷酸同源性介于95.1%～99.2%之间,与Ⅱ型毒株的同源性仅为54.1%～54.7%。结论大多数IBDV强毒株、变异株、超强毒株与B87株的亲缘关系较远;诱导产生保护性中和抗体的VP2抗原决定簇的两个亲水区氨基酸变异较大,是逃避B87疫苗株的免疫保护、导致免疫失败而发生免疫鸡群传染性法氏囊病（IBD）流行的分子基础。     

一株引起无菌性脑膜脑炎柯萨奇B组3型病毒的分离和VP1基因分析

目的对一株从无菌性脑膜脑炎患者中分离到的柯萨奇B组3型病毒(coxsackie virus B3,CVB3)分离株KMA103-09进行VP1区的基因特征分析。方法采用Hep-2、RD细胞对患者粪便标本进行病毒分离,肠道病毒组合血清进行鉴别,反转录-聚合酶链反应(RT-PCR)扩增病毒目的片段,测序后进行VP1基因分析,用Mega4.1等软件分析处理。结果肠道病毒组合血清鉴别为柯萨奇病毒B组3型(Cox.B3),从病毒性脑膜脑炎患者粪便标本中,分离到CVB3,其VP1区的核苷酸长度为849bp,未发现核苷酸插入与丢失。与山东04433/SD/CHN/2004/CB3株、山东05280/SD/CHN/2005/CB3株及山东YZ127/SD/CHN/2005/CB3株氨基酸同源,与其余国内分离株同源性高于为99.29%,与国外毒株的同源性为95.41%~96.47%。在进化树上与AM06HZ/SD/CHN/2006/CB3株显示在同一个分支上。结论分离的肠道病毒为柯萨奇病毒B组3型(Cox.B3),分离株(KMA103-09)VP1区变异较小。     

Detection of unusual mutation within the VP1 region of different re-isolates of poliovirus Sabin vaccine

In the present study, a genomic analysis of full VP1 sequence region of 15 clinical re-isolates (14 healthy vaccinees and one bone marrow tumor patient) was conducted, aiming to the identification of mutations and to the assessment of their impact on virus fitness, providing also insights relevant with the natural evolution of Sabin strains. Clinical re-isolates were analyzed by RT-PCR, sequencing and computational analysis. Some re-isolates were characterized by an unusual mutational pattern in which non-synonymous mutations outnumbered the synonymous ones. Furthermore, the majority of amino-acid substitutions were located in the capsid exterior, specifically in N-Ags, near N-Ags and in the north rim of the canyon. Also mutations, which are well-known determinants of attenuation, were identified. The results of this study propose that some re-isolates are characterized by an evolutionary pattern in which non-synonymous mutations with a direct phenotypic impact on viral fitness are fixed in viral genomes, in spite of synonymous ones with no phenotypic impact on viral fitness. Results of the present retrospective characterization of Sabin clinical re-isolates, based on the full VP1 sequence, suggest that vaccine-derived viruses may make their way through narrow breaches and may evolve into transmissible pathogens even in adequately immunized populations. For this reason increased poliovirus laboratory surveillance should be permanent and full VP1 sequence analysis should be conducted even in isolates originating from healthy vaccinees.     

Relationships among porcine and human P[6] rotaviruses: evidence that the different human P[6] lineages have originated from multiple interspecies transmission events

Porcine rotavirus strains (PoRVs) bearing human-like VP4 P[6] gene alleles were identified. Genetic characterization with either PCR genotyping or sequence analysis allowed to determine the VP7 specificity of the PoRVs as G3, G4, G5 and G9, and the VP6 as genogroup I, that is predictive of a subgroup I specificity. Sequence analysis of the VP8* trypsin-cleavage product of VP4 allowed PoRVs to be characterized further into genetic lineages within the P[6] genotype. Unexpectedly, the strains displayed significantly higher similarity (up to 94.6% and 92.5% at aa and nt level, respectively) to human M37-like P[6] strains (lineage I), serologically classifiable as P2A, or to the atypical Hungarian P[6] human strains (HRVs), designated as lineage V (up to 97.0% aa and 96.1% nt), than to the porcine P[6] strain Gottfried, lineage II (<85.1% aa and 82.2 nt), which is serologically classified as P2B. Interestingly, no P[6] PoRV resembling the original prototype porcine strain, Gottfried, was detected, while Japanase P[6] PoRV clustered with the atypical Japanase G1 human strain AU19. By analysis of the 10th and 11th genome segments, all the strains revealed a NSP4B genogroup (Wa-like) and a NSP5/6 gene of porcine origin. These findings strongly suggest interspecies transmission of rotavirus strains and/or genes, and may indicate the occurrence of at least 3 separate rotavirus transmission events between pigs and humans, providing convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

新型肠道病毒89型结构蛋白VP1的构建表达及序列分析

目的研究克隆表达新型肠道病毒89型VP1结构蛋白．并进行活性鉴定和序列分析。方法分离肠道病毒89型VP1结构蛋白全基因,克隆入原核表达载体PET-28构建重组质粒pH—VP1,在大肠埃希菌BL21中进行表达,检测表达产物的特异性及活性。将EV89VP1核酸序列与Genbank数据库中的序列进行比对并建立系统进化树。结果重组质粒在1mmol／L的IPTG37℃诱导7h后可诱导表达得到约33kD的蛋白。经WesternBlot实验检测证明表达的融合蛋白具有EV89抗原的活性。分离的EV89病毒其VP1区核酸序列与Genbank数据库中仅有的3株EV89序列同源性分别为86．4％、85．9％、85．6％。结论成功构建EV89重组蛋白．其表达产物具有良好的特异性及活性,可进-步用于VP1检测方法的研制。进化分析表明,分离的EV89VP1区与Genbank数据库中其他分离株亲源性较远。     

Could the Ebola virus matrix protein VP40 be a drug target?

Filoviruses are filamentous lipid-enveloped viruses and include Ebola (EBOV) and Marburg, which are morphologically identical but antigenically distinct. These viruses can be very deadly with outbreaks of EBOV having clinical fatality as high as 90%. In 2012 there were two separate Ebola outbreaks in the Democratic Republic of Congo and Uganda that resulted in 25 and 4 fatalities, respectively. The lack of preventive vaccines and FDA-approved therapeutics has struck fear that the EBOV could become a pandemic threat. The Ebola genome encodes only seven genes, which mediate the entry, replication, and egress of the virus from the host cell. The EBOV matrix protein is VP40, which is found localized under the lipid envelope of the virus where it bridges the viral lipid envelope and nucleocapsid. VP40 is effectively a peripheral protein that mediates the plasma membrane binding and budding of the virus prior to egress. A number of studies have demonstrated specific deletions or mutations of VP40 to abrogate viral egress but to date pharmacological inhibition of VP40 has not been demonstrated. This editorial highlights VP40, which is the most abundantly expressed protein of the virus and discusses VP40 as a potential therapeutic target.