Genetic and pathogenic characterization of Akabane viruses isolated from cattle with encephalomyelitis in Korea

A large-scale outbreak of Akabane viral encephalomyelitis in cattle was reported in the southern part of Korea in 2010. Fifteen Akabane virus (AKAV) strains were isolated from the brain and spinal cord samples by using BHK-21 and/or HmLu-1 cells. To examine the genetic relationships and characteristics of the isolates, nucleotide sequences of the S, M, and L segments of the 15 isolates were determined and analyzed. Complete sequence analysis of the 15 AKAV isolates showed 99.9-100% amino acid identities, indicating that the 15 isolates originated from a single strain. The S and M RNA segments of a representative isolate (AKAV-7/SKR/2010) were also compared with the segments of representative reference sequences. This AKAV-7/SKR/2010 strain showed the highest identity with the Iriki and KM-1/Br/06 strains. Neighbor-joining phylogenetic trees of S and M RNA segments were constructed. Four representative AKAV isolates were classified into subgroup Ia, which contains the Iriki and KM-1/Br/06 strains recognized to cause encephalomyelitis in calves and adult cattle in Japan. Moreover, experimental intraperitoneal infection was performed using the AKAV-7/SKR/2010 and AKAV-17/SKR/2010 strains to assess pathogenesis in suckling mice. The 2 isolates, genetically related to the Iriki strain, were neurovirulent and caused neurological signs in suckling mice. In contrast, the 93FMX strain and the K0505 strain, related to the OBE-1 strain, were avirulent in mice. The present results indicate that these isolates most likely had originated from the Iriki strain and are closely related to the Iriki strain both genetically and pathogenically.     

Molecular epidemiological analyses of the teratogenic Aino virus based on the sequences of a small RNA segment

The sequences of a small RNA segment of Aino virus isolates were analyzed to define the molecular epidemiology and genetic relationships to other species in the genus Orthobunyavirus in the family Bunyaviridae. The nucleotide and amino acid sequences of the segment were highly conserved among strains isolated from 1964 to 2002 in Japan. These Japanese isolates were segregated into two distinct lineages, one containing the prototype strain JaNAr28 isolated in 1964 and the other containing strains isolated after 1986, by phylogenetic analysis based on the nucleocapsid gene sequences. Japanese strains isolated after 1986 were rather more closely related to Kaikalur virus isolated in India in 1971 than to strain JaNAr28. On the other hand, an Australian strain, B7974, was closely related to Peaton virus. The B7974 strain might have been generated by inter-serotype genetic reassortment between Aino and Peaton viruses in Australia during their evolution. However, recent Aino virus strains isolated in Japan appear to be genetically stable.     

The complete nucleotide sequences of L3 and S7 segments of Ibaraki virus encoding for the major inner capsid proteins, VP3 and VP7

The complete nucleotide sequences of the genes encoding two of the major inner capsid proteins of Ibaraki virus (IBAV), belonging to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) were determined. The L3 RNA segment is 2768 nucleotides in length which encodes VP3 polypeptides of 899 amino acid residues (M.W. 103 kDa). The S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and encodes 349 amino acids (M.W. 38 kDa). These RNA segments had the characteristic consensus motifs of Orbivirus RNA segments in termini, namely 5'-GUUAAA... and ...ACUUAC-3'. The comparison of the IBAV L3 and S7 sequences with those of other two EHDV-2 isolates revealed the higher homologies of 93% and 92% against EHDV-2 Australia isolate (EHDV-2AUS) and lower homologies of 80% and 81% against EHDV-2 North America isolate, respectively. The phylogenetic analysis based on L3 and S7 genes also indicated close relationships between IBAV and EHDV-2AUS. KEY WORDS: dsRNA gene, lbaraki virus, inner capsid, VP3, VP7.     

从线粒体序列分异探讨鼢鼠凸颅亚属(Eospalax)种间差异的有效性

鼢鼠亚科动物中凸颅亚属鼢鼠的物种界限一直不清楚。研究测定了甘肃境内鼢鼠类线粒体DNA D-loop和ND4基因序列,获得了23条长度为414～424 bp的D-loop和17条长度为425～438 bp的ND4基因同源序列,通过其序列分异、遗传距离和系统发育分析,探讨该类群的分类学地位。结果表明:凸颅亚属鼢鼠序列间变异类型丰富,单倍型多样度高,其种间遗传距离大于种内遗传距离;基于邻接法(NJ)、贝叶斯推论法(BI)和最大似然法(ML)构建的分子系统树显示:甘肃鼢鼠、高原鼢鼠、斯氏鼢鼠、秦岭鼢鼠均各自构成单系,并构成2个进化枝,其中甘肃鼢鼠最为原始,单独构成一枝;高原鼢鼠与斯氏鼢鼠为姐妹群,处于较进化的位置,后与秦岭鼢鼠聚在一起构成另一枝,支持4个种均为独立种的观点。     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

Genotyping of infectious pancreatic necrosis virus isolates from Mexico state

The infectious pancreatic necrosis virus (IPNV; genus Aquabirnavirus) affects salmon and trout, causing high mortality in first-feeding fry. The classification of this virus includes nine serotypes and seven genogroups. In Mexico, two different isolates were identified in 2000 and 2008, respectively. Both isolates were classified into genogroup I according to the RNA genome of this virus. As Mexico is importing rainbow trout Oncorhynchus mykiss eggs from different countries, the aim of this study was to genotype IPNV isolates obtained from four rainbow trout producer regions within the state of Mexico. We utilized a fragment of the VP2* (outer capsid protein) gene sequence of Mexican IPNV isolates as a molecular marker to determine the genogroup to which they belong. Although all Mexican IPNV isolates were grouped into genogroup I, we identified genetic diversity among these isolates, and 14 unique nucleotide sequence types were associated with the four producer regions in Mexico State.     

不同地理种群的亚洲小车蝗mtDNA COⅠ基因序列及其相互关系

亚洲小车蝗(Oedaleus asiaticus)是北方草原重要的经济害虫,应用PCR直接测序法对7个不同地理种群的亚洲小车蝗共32个个体的mtDNA COⅠ基因中一段473 bp的序列进行测序。通过比较其同源性,计算核苷酸组成,并用槌角蝗科的宽须蚁蝗(Myrmeleotettix palpalis)和斑腿蝗科的鼓翅皱膝蝗(Angaracris barabensis)作外群构建NJ分子系统树。结果表明:在获得的亚洲小车蝗473 bp的序列中,A+T约占71.7%,其中29个核苷酸位点存在变异(约占所测核苷酸的6.3%)。就每个氨基酸密码子来看,第3位点的A+T含量最高。由NJ树显示,亚洲小车蝗mtDNA COⅠ序列不同单倍型之间有一定的分歧,形成不同的簇类关系,其分枝与地理分布没有直接的对应关系,但总体上看,这种簇类关系基本上呈平行分布,没有明显的地域性差别。     

Phylogenetic relationships of Brazilian isolates of Pythium insidiosum based on ITS rDNA and cytochrome oxidase II gene sequences

Pythium insidiosum is an aquatic oomycete that is the causative agent of pythiosis. Advances in molecular methods have enabled increased accuracy in the diagnosis of pythiosis, and in studies of the phylogenetic relationships of this oomycete. To evaluate the phylogenetic relationships among isolates of P. insidiosum from different regions of Brazil, and also regarding to other American and Thai isolates, in this study a total of thirty isolates of P. insidiosum from different regions of Brazil was used and had their ITS1, 5.8S rRNA and ITS2 rDNA (ITS) region and the partial sequence of cytochrome oxidase II (COX II) gene sequenced and analyzed. The outgroup consisted of six isolates of other Pythium species and one of Lagenidium giganteum. Phylogenetic analyses of ITS and COX II genes were conducted, both individually and in combination, using four different methods: Maximum parsimony (MP); Neighbor-joining (NJ); Maximum likelihood (ML); and Bayesian analysis (BA). Our data supported P. insidiosum as monophyletic in relation to the other Pythium species, and COX II showed that P. insidiosum appears to be subdivided into three major polytomous groups, whose arrangement provides the Thai isolates as paraphyletic in relation to the Brazilian ones. The molecular analyses performed in this study suggest an evolutionary proximity among all American isolates, including the Brazilian and the Central and North America isolates, which were grouped together in a single entirely polytomous clade. The COX II network results presented signals of a recent expansion for the American isolates, probably originated from an Asian invasion source. Here, COX II showed higher levels bias, although it was the source of higher levels of phylogenetic information when compared to ITS. Nevertheless, the two markers chosen for this study proved to be entirely congruent, at least with respect to phylogenetic relationships between different isolates of P. insidiosum.     

Molecular characterisation of Victorian Newcastle disease virus isolates from 1976 to 1999

OBJECTIVE: To characterise Newcastle disease virus isolates obtained in Victoria from 1976 to 1999 and identify the diversity of FO cleavage signal. DESIGN: RT-PCR using viral RNA extracted from positive NDV allantoic fluid was performed to amplify a segment of the NDV F and HN genes. Molecular characterisation of the nucleotide and amino acid sequences within the FO cleavage site was undertaken. RESULTS: All isolates contained 'avirulent FO cleavage signal sequence of varied amino acid composition. CONCLUSIONS: Molecular characterisation of past and present NDV FO cleavage signal sequences will provide valuable epidemiological information and assist in understanding the genetic origins and relationships of outbreaks.     

从流行毒株的系统发生看中国目前狂犬病流行的主要特征

以2006～2012年本实验室狂犬病病毒分离株核蛋白基因完整核苷酸序列和GenBank收录的主要数据为基础,通过构建系统发生树和比对病毒分离株相互间的同源性,对中国狂犬病流行特征进行分析.结果显示,目前涉及中国17个狂犬病主要流行省区的62个狂犬病病毒代表流行株,均为基因Ⅰ型,但在系统发生树上可分为i、ii、iii、iv、v共5个基因群,群内同源性91.4％～99.9％,群间同源性84.5％～90.1％.其中,i、ii群占国内新分离株的绝大多数,为2个主要流行基因群.i群遍布各主要流行省区,主要为来源于犬的分离株.ii群主要分布于南方省区,迄今分离到的多个鼬獾狂犬病病毒株在系统发生上也属于ii群.iii群仅见于广西壮族自治区、云南省两地,与东南亚国家狂犬病分离株同源性高达97.7％.iv群的地区分布不规律,在中国东北、中原、东南和西部地区均有零星报道,分离株较少.v群近年来仅偶见于内蒙东北部及黑龙江省与俄接壤地区,与俄罗斯远东及韩国流行株同源性高达98.5％.综上,中国狂犬病流行以犬间传播为主,野生动物狂犬病的流行日益严重,东北和西南地区存在境外狂犬病传入.犬等动物种群免疫覆盖率低应是狂犬病持续传播的主因.     

Genogrouping of Vaccine Breakdown Strains (VBS) of Feline Calicivirus in Japan

Although prevention of feline calcivirus (FCV) infection by vaccination has been attempted, and isolation of FCV, development of the disease, and a few fatal cases in vaccinated cats have been reported. Fifteen FCV strains isolated from cats that had been vaccinated with commercially available FCV vaccines (F9, FCV-255, and FC-7) were genogrouped. Molecular analysis of viral genomes involved the construction of a phylogenetic tree of capsid genes using the NJ method. Cat anti-F9 serum and rabbit anti-FCV-255 serum were used for virus neutralization tests. Molecular phylogenetic analysis of the amino acid sequences of 15 virus isolates and those of the previously published and GenBank-deposited 9 global and 14 Japanese strains showed that 8 (53%) of the 15 virus isolates as well as the vaccine strains F9 and FCV-255 belonged to genogroup I (G_AI), and 7 (47%) belonged to genogroup II (G_AII). Of the 8 G_AI strains, 2 were isolated from cats that had been vaccinated with an F9 strain live vaccine, 5 from cats vaccinated with an FCV-255-derived vaccine, and 1 from a cat vaccinated with an FC-7-derived vaccine. Of the 7 G_AII strains, 5 were isolated from cats that had been vaccinated with the F9 strain live vaccine, 1 from a cat vaccinated with the FCV-255-derived vaccine, and 1 from a cat vaccinated with the FC-7-derived vaccine. These results indicate that more vaccine breakdown strains isolated from the cats vaccinated with the F9 strain-derived vaccine belong to G_AII than to G_AI, whereas more vaccine breakdown strains isolated from the cats vaccinated with the FCV-255 strain-derived vaccine belong to G_AI than to G_AII, and that when the FC-7 strain-derived vaccine is used, the vaccine breakdown strains belong almost equally to G_AI and G_AII. Thus, the genogroups of virus isolates varied with the vaccine strain used (p < 0.05). On the other hand, the neutralizing titres of feline anti-F9 serum and rabbit anti-FCV-255 serum against the 15 isolates were very low, showing no relationships between neutralizing antibody titres and genogroups. The DNA sequence identities between the virus isolates and the vaccine strains were low, at 70.6–82.9%, and no strains were found to have sequences derived from the vaccine strains. Alignment of amino acid sequences showed that the G_AI or G_AII virus isolates from the F9-vaccinated cats differed at position 428 of the 5’ hypervariable region (HVR) of capsid region of the F9 strain, whereas those from the FCV-255-vaccinated cats differed at positions 438, 453, and 460 of the 5’HVR of capsid region E of the F9 strain. We speculate that these differences influence genogrouping. The amino acid changes within the F9 linear epitopes common to G _A I and G _A II were noted at positions 450, 451, 457 of 5’HVR of the capsid region E in the isolates from F9-derived vaccine-treated cats, and 449, 450, and 451 of 5’HVR of capsid region E in the isolates from FCV-255-derived vaccine-treated cats, suggesting that these amino acid changes are involved in escapes. These results suggest that alternate vaccination with the F9 and FCV-255 strains or the use of a polyvalent vaccine containing G_AII strains serves to inhibit development.     

IBV安徽分离株膜蛋白基因的克隆及序列分析

利用设计的1对特异性引物,通过RT-PCR方法扩增出4株鸡传染性支气管炎病毒(IBV)安徽地方分离株膜蛋白M基因全长片段并进行了克隆测序。将各IBV安徽地方分离株与GenBank中注册的一些毒株M基因核苷酸序列及推导的氨基酸序列进行比较和系统进化关系分析,发现毒株间核苷酸序列同源性为88.5%~100%,其相应的氨基酸序列同源性为90.3%~100%;不同毒株间存着重组、缺失、插入及点突变等变异,从ATG至第140 bp区段的核苷酸序列变异频率最高;4株分离毒株属于同一个进化群的2个不同进化亚群,与我国常用疫苗毒株H120、M41和W 93不属同一个进化亚群。     

Genotyping of Infectious Pancreatic Necrosis Virus Isolates from Mexico State

Abstract

The infectious pancreatic necrosis virus (IPNV; genus Aquabirnavirus) affects salmon and trout, causing high mortality in first-feeding fry. The classification of this virus includes nine serotypes and seven genogroups. In Mexico, two different isolates were identified in 2000 and 2008, respectively. Both isolates were classified into genogroup I according to the RNA genome of this virus. As Mexico is importing rainbow trout Oncorhynchus mykiss eggs from different countries, the aim of this study was to genotype IPNV isolates obtained from four rainbow trout producer regions within the state of Mexico. We utilized a fragment of the VP2* (outer capsid protein) gene sequence of Mexican IPNV isolates as a molecular marker to determine the genogroup to which they belong. Although all Mexican IPNV isolates were grouped into genogroup I, we identified genetic diversity among these isolates, and 14 unique nucleotide sequence types were associated with the four producer regions in Mexico State.

Received December 21, 2010; accepted July 27, 2011     

Recombinant cDNA probe from bluetongue virus genome segment 10 for identification of bluetongue virus

Genome segment 10 of bluetongue virus (BTV) serotype 11 UC8 strain was cloned and subsequently hybridized to viral double-stranded RNA extracted from 90 field isolates of BTV serotypes 10, 11, 13, and 17; the prototype strains of BTV 2, 10, 11, 13, and 17; the prototype strain epizootic hemorrhagic disease virus (EHDV) serotype 1; and 4 field isolates of EHDV serotype 2. The 90 field isolates were obtained from different counties in California, Louisiana, and Idaho during the years 1979, 1980, and 1981. The cloned genetic probe hybridized with all the BTV samples tested, showing different degrees of cross-hybridization at the stringency conditions used in this study. This indicated that BTV genome segment 10 has conserved nucleotide sequences among the BTV serotypes 2, 10, 11, 13, and 17. No cross-hybridization signals were detected between the cloned genome segment 10 of BTV 11 UC8 strain and the prototype strain of EHDV serotype 1 and the field isolates of serotype 2. This probe recognized a wide variety of BTV isolates.     

IBV分离株部分S1蛋白基因的序列分析

参考GenBank上的IBVS1基因序列,自行设计合成了一对跨幅约0.6kb的引物,RT-PCR对6株IBV分离株的S1基因进行扩增。序列分析显示,这些病毒从时间、空间和组织嗜性都没有明显的规律可循。2株从传染性腺胃炎病鸡中获得病毒(ZC4-3株和DB239-1株)属于既不同于国内其他分离毒株,又不同于国际参考毒株的独立一群。通过对IBV的分析,认为现流行的IBV已发生了很大的变异,分别利用嗜肾性、嗜呼吸道性和嗜腺胃性的毒株研制疫苗,对我国IBV的防疫十分重要。     

Differential detection of infectious bursal disease virus serotypes, using cDNA probes to VP2 coding region.

Two nonoverlapping clones, pOH405 and pOH632, containing cDNA inserts in the VP2 coding region of genome segment A were selected from a cDNA library prepared from the double-stranded RNA genome of the OH strain of infectious bursal disease virus (IBDV) of serotype 2. Clone pOH405, which is located in the hypervariable segment of VP2, is 328 base pairs long, has nucleotide sequence homology of 72 to 73%, and amino acid sequence homology of 64 to 67% with IBDV strains of serotype 1. Clone pOH632, which is located in the highly conserved C-terminal part of VP2, is 230 base pairs long, has nucleotide sequence homology of 87 to 88%, and amino acid sequence homology of 100% with IBDV serotype 1. The lower detection limit of 32P-labeled probes prepared from both clones was 10 ng of OH-IBDV double-stranded RNA, using high-stringency conditions of hybridization (54 C, 50% formamide) and washing (55 C, 0.015M NaCl, 0.0015M trisodium citrate, pH 7.0, with 0.1% sodium dodecyl sulfate), and autoradiography for 24 hours. Under these conditions, the dot-blot hybridization assay for detection of serotype 2 IBDV double-stranded RNA, was 1,000 times more sensitive, using probe pOH632, but only 10 times more sensitive, using probe pOH405, compared with the assay for IBDV serotype 1, using the same probes. Thus, probe pOH632 could differentiate between the 2 IBDV serotypes by nucleic acid hybridization.     

Physical and genomic characteristics identify chicken proventricular necrosis virus (R11/3 virus) as a novel birnavirus

Chicken proventricular necrosis virus (CPNV), isolate R11/3, previously was isolated from transmissible viral proventriculitis-affected chickens and was determined to be the likely etiology of this disease. CPNV was identified as a birnavirus on the basis of virion size and morphology (icosahedral, approximately 75 nm in diameter, nonenveloped); buoyant density in cesium chloride (1.32 g/ml); a genome comprising bisegmented, double-stranded RNA (approximately 3.8 and 3.4 kilobase pairs); and nucleotide sequence analyses. Nucleotide sequencing of CPNV RNA, segment B, identified a single large open reading frame that encodes a 903-amino acid protein. The 903-amino acid protein was identified as the putative VP1, the viral RNA-dependent RNA polymerase (RdRp), on the basis of sequence homologies with other birnavirus VP1 proteins. The CPNV VP1 possessed the unique permuted RdRp sequence motif arrangement characteristic of birnaviruses; however, phylogenetic analyses based on VP1 demonstrated that CPNV is deeply divergent from other birnaviruses.     

Molecular characterization of an H1N2 swine influenza virus isolated in Miyazaki, Japan, in 2006

Swine influenza virus (SIV) was isolated from a farm in Miyazaki Prefecture in Japan in July 2006. An isolate was genetically subtyped as H1N2 and was designated A/swine/Miyazaki/1/2006 (H1N2). The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. All segments were shown to be closely related to those of Japanese SIV H1N2 isolates, which have been circulating since the 1980s. The results indicate the persistence of the SIV H1N2 subtype in the Japanese pig population for more than two decades and emphasize the importance of continuous surveillance for SIV.