Molecular characterisation of Australasian isolates of aquatic birnaviruses

An aquatic birnavirus, first isolated in Australia from farmed Atlantic salmon in Tasmania in 1998, has continued to be re-isolated on an infrequent but regular basis. Due to its low pathogenicity, there has been little urgency to undertake a comprehensive characterisation of this aquatic birnavirus. However, faced with possible incursions of any new aquatic birnaviruses, specific identification and differentiation of this virus from other, pathogenic, aquatic birnaviruses such as infectious pancreatic necrosis virus (IPNV) are becoming increasingly important. The present study determined the nucleic acid sequence of the aquatic birnavirus originally isolated in 1998, as well as a subsequent isolate from 2002. The sequences of the VP2 and VP5 genes were compared to that of other aquatic birnaviruses, including non-pathogenic aquatic birnavirus isolates from New Zealand and pathogenic infectious pancreatic necrosis virus isolates from North America and Europe. The deduced amino acid (aa) sequences indicate that the Australian and New Zealand isolates fall within Genogroup 5 together with IPNV strains Sp, DPL, Fr10 and N1. Thus, Genogroup 5 appears to contain aquatic birnavirus isolates from quite diverse host and geographical ranges. Using the sequence information derived from this study, a simple diagnostic test has been developed that differentiates the current Australian isolates from all other aquatic birnaviruses, including the closely related isolates from New Zealand.     

Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA

Viruses were isolated in cell culture from tissue homogenates of flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Neutralization and immunofluorescence tests with aquabirnavirus (West Buxton strain)-specific polyclonal antisera indicated that both viruses were aquabirnaviruses belonging to Serogroup A, the most common aquabirnavirus serogroup in the United States. This was confirmed by RT-PCR, with primers targeting the VP3 and VP2 gene of aquabirnaviruses. The VP2-specific RT-PCR cDNA amplification product was sequenced and deduced amino-acid sequences were compared with known sequences of the type strains of the 9 serotypes of aquabirnavirus Serogroup A. This demonstrated that the viruses from both flounder and mummichog belong to aquabirnavirus Genogroup 1. The flounder isolate exhibited deduced amino acid sequence similarities of 98.1% with the Jasper strain of serotype A9, and 97.7% with the West Buxton strain of serotype A1. The isolate from mummichog exhibited deduced amino acid sequence similarities of 99.1% with the West Buxton strain of Serotype A1 and 94.8% with the Jasper isolate of Serotype A9. Similarities of deduced amino acid sequences ranged from 79.9 to 86.9%, with representatives of the other 7 serotypes. This is the first report of an aquabirnavirus from mummichog F. heteroclitus and only the fifth report of an aquabirnavirus from a flounder species.     

传染性法氏囊病病毒中国强毒株A节段cDNA基因的克隆和序列分析

以来自哈尔滨传染性法氏囊病病毒（ＩＢＤＶ） 强毒株（Ｈａｒｂｉｎ 毒株,Ｈ） 的基因组ＲＮＡ为模板,用反转录聚合酶链反应（ＲＴ－ ＰＣＲ） 的方法得到了其Ａ 节段的全长ｃＤＮＡ 片段,分５＇端（１ ６５９ｂｐ） 和３＇端（１ ４４４ｂｐ） 上下两段分别克隆到ｐＧＥＭＢ○Ｒ － Ｔ 载体上,测定了其核苷酸顺序,在长为３ １０１ ｂｐ 中含有两个阅读框ＯＲＦＡ１ 和ＯＲＦＡ２ ,分别编码１ ０１２ 个氨基酸的前体蛋白（ＶＰ２ － ４ －３） 和１４５ 个氨基酸的ＶＰ５,ＯＲＦＡ１ 和ＯＲＦＡ２ 有部分的重叠。将核苷酸序列及推测出的氨基酸序列与已报道的ＩＢＤＶ 血清Ⅰ型和Ⅱ型毒株的相应序列进行了比较,结果表明：Ｈ 毒株与其它血清Ⅰ型毒株之间,在核苷酸水平上存在２５ｂｐ － ２６７ｂｐ 的差异;在氨基酸水平上存在１７ ～４０ 个氨基酸的差异。在ＶＰ２ － ４ － ３ 内比较显示,Ｈ 毒株与Ｐ２ 、Ｃｕ－ １ 之间氨基酸的差异最小为１ ．７％ ,Ｈ 毒株与ＵＫ６６１ 之间氨基酸的差异最大为３ ．９ ％ 。变异主要发生在ＶＰ２ 的可变区（２０６ － ３５０ 位氨基酸） ,在Ｈ 毒株所特有的１２ 个氨基酸当中,该区就占５ 个,代表１ ．７６ ％ 的变异。ＶＰ４、ＶＰ３ 和ＶＰ５区各有     

Isolation of different types of birnavirus from ayu Plecoglossus altivelis and amago salmon Oncorhynchus rhodurus cultured in the same geographic area

A birnavirus was recently isolated from cultured ayu Plecoglossus altivelis on Shikoku island, Japan. The diseased fish displayed vertebral or vertical curvature and mild haemorrhage around the brain. Cytopathic effects (CPE) of the virus, including cell roundness, filamentous change and cell lysis, were observed in CHSE-214, RTG-2 and RSBK-2 cells. The virus isolated from ayu, designated the AY-98 strain, was found to be antigenically related to the marine birnavirus (MABV) Y-6 strain that originated from yellowtail Seriola quinqueradiata. AY-98 had a bi-segmented RNA genome and the same nucleotide sequence in the 310 bp VP2/NS junction as MABV Y-6. At the same time that the ayu epizootics occurred, another birnavirus (AM-98) was isolated from amago salmon Oncorhynchus rhodurus which were cultured 66 km away from the ayu farm. AM-98 showed a similar CPE and had the same host cell ranges as AY-98. However, AM-98 was serologically similar to the VR-299 strain of infectious pancreatic necrosis virus (IPNV) and their nucleotide sequences in the VP2/NS junction region showed 98% homology without changes at the amino acid level. In this study, the ayu strain AY-98 was grouped into MABV, whereas the amago salmon strain AM-98 was grouped into IPNV. This indicates that the 2 birnaviruses originated from different sources in spite of the fact that the places where they were isolated are close to one another. The results in this paper show a new aspect of the traditional consensus that the same serogroup of birnavirus distribute in close geographic areas.     

Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40.

The nucleotide sequence of the late region of the polyoma genome has been determined. It consists of 2366 bp and encodes the virion capsid proteins VP1, VP2 and VP3. Extensive open reading frames identify the possible coding sequences of VP2 and VP3 toward the 5′ end of the late region, and of the major capsid protein VP1 toward the 3′ end of the late region. The 5′ end of the sequence encoding VP1 overlaps the 3′ VP2/VP3 region by 29 nucleotides and is in a different reading frame. The predicted amino acid sequences for all three known capsid proteins show extensive homology with the analogous capsid proteins of SV40 throughout most of their length. The VP2/VP3 amino acid homology between the two viruses is 34%, while the major capsid protein VP1 is much more highly conserved, showing 54% homology. These homologies together with the extent of open reading frames help to define the extent of the coding sequences. The VP2 initiator begins at position 269 and the coding region extends to the first termination codon beginning at 1226. The predicted size of VP2 is 35,007 daltons. A probable VP3 initiator is within the VP2 coding sequence at position 614 and is in the same frame as VP2. This coding sequence can also utilize the terminator at position 1226, and the predicted size of the VP3 translation product is 22,979 daltons. The VP1 coding region begins at position 1197 and continues in a frame different from that of VP2/ VP3 to a termination point at 2349. The molecular weight of VP1 is predicted to be 42,834 daltons. The 5′ untranslated region contains sequences that resemble a potential ribosomal binding site and a possible mRNA capping sequence similar to those found in other eucaryotic systems. There is also a sequence (5′-TCAAGTAAGTGA-3′) almost identical to one found in two regions containing potential splice sites in the early region of polyoma. The 5′ untranslated region does not show the extensive repeated sequences found in the similar region of SV40. The 3′ untranslated region contains the sequence 5′-AATAAA-3′, thought to represent a polyadenylation signal. As in the early region of polyoma, the extensive nucleotide and deduced amino acid homology with SV40 indicate a close evolutionary relationship between the two viruses, and help to identify regions of common and important structure-function relationships.     

人A组轮状病毒北京地方株VP4血清型特异片段编码基因的序列分析

轮状病毒是引起婴幼儿严重腹泻的重要病原,其第四基因编码主要中和抗原VP4,而VP4可裂解为VP8和VP5两个片段。VP8为抗原型特异性片段。克隆并测定了具有代表性的三个轮状病毒北京株VP4编码基因5′端（VPS＋VPS一部分）887个核苷酸序列并据此推导出其氨基酸序列。结果表明,相同血清型的地方株和标准株之间具有高度同源性（92％～966％）,不同血清型间则变异较大（70．5％～71％）。氨基酸最大变异处位于aa84～172,并对胰酶作用位点在致病性中的可能性进行了讨论。     

Nucleotide sequence analysis of VP7 gene of Indian isolates of bluetongue virus vis-à-vis other serotypes from different parts of the world.

Bluetongue virus (BTV), a member of genus Orbivirus, a family Reoviridae, is a non-enveloped with double shelled structure and ten segmented double stranded (ds) RNA genome. The RNA segment S7 encodes an inner capsid serogroup specific viral protein VP7. To amplify coding region of VP7 gene of BTV, new primers, forward primer (18-38 bp) and reverse primer (1156-1136 bp), were designed using VP7 gene sequences available in GenBank. This primer pair successfully amplified cell culture adapted Indian isolates of BTV belonging to two different serotypes 1 and 18. The coding sequences of two Indian isolates of BTV (BTV-1H and BTV-18B) were cloned into pPCR Script-Amp SK (+) plasmid vector and transformed into XL10-Gold Kan ultracompetent E. coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. The sequence analysis revealed that there was 93-97% nucleotide sequence identity in VP7 gene of three different Indian serotypes of BTV. The VP7 gene sequences of Indian isolates have comparatively less sequence homology (< 80%) with American (US), and French isolates compared to South African (SA), Australian (AUS) and Chinese (PRC) isolates. In silico restriction enzyme profile analysis of VP7 gene sequences revealed that Indian isolates of BTV-1 can be differentiated from other BTV-1 isolates reported from SA, AUS and PRC using TaqI. Similarly the Indian isolates of BTV belonging to three different serotypes can be differentiated using EcoRI, Hae III and TaqI restriction enzymes.     

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense"

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.     

Blotched snakehead virus is a new aquatic birnavirus that is slightly more related to avibirnavirus than to aquabirnavirus

By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala downward arrow Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.     

Primary structure of the gene for VP1 protein of the foot-and-mouth disease virus of Asia 1 serotype

The nucleotide sequence of the cDNA for the viral RNA region coding for the main antigenic protein of the epidemic stomatitis virus of Asia 1 serotype has been identified. The amino acid sequences in the regions of VP1 protein antigenic determinants of the serotype Asia 1 virus and other serotypes viruses have been compared.     

Rotavirus YM gene 4: analysis of its deduced amino acid sequence and prediction of the secondary structure of the VP4 protein.

We have determined the complete nucleotide sequence of the VP4 gene of porcine rotavirus YM. It is 2,362 nucleotides long, with a single open reading frame coding for a protein of 776 amino acids. A phylogenetic tree was derived from the deduced YM VP4 amino acid sequence and 18 other available VP4 sequences of rotavirus strains belonging to different serotypes and isolated from different animal species. In this tree, VP4 proteins were grouped by the hosts that the corresponding viruses infect rather than by the serotypes they belong to, suggesting that this protein is involved in the host specificity of the viruses. In an attempt to predict the secondary structure of the VP4 protein, we selected the more divergent VP4 sequences and made a secondary structure analysis of each protein. In spite of variations within the individual structures predicted, there was a general structural pattern which suggested the existence of at least two different domains. One, comprising the amino-terminal 63% of the protein, is predicted to be a possible globular domain rich in beta-strands alternated with turns and coils. The second domain, represented by the remaining, carboxy-terminal part of VP4, is rich in long stretches of alpha-helix, one of which, 63 amino acids long, has heptad repeats resembling those found in proteins known to form alpha-helical coiled-coils. The predicted secondary structure correlates well with the available data on the protein accessibility delineated by immunological and biochemical findings and with the spike structure of the protein, which has been determined by cryoelectron microscopy.     

中华蜜蜂囊状幼虫病毒北京分离株VP1蛋白基因序列特征及原核表达

【目的】研究中华蜜蜂囊状幼虫病毒（Chinese sacbrood virus, CSBV）VP1蛋白的分子进化特征及遗传多样性。【方法】利用RT-PCR方法,克隆了8株CSBV北京分离株VP1蛋白的基因编码区。【结果】序列分析表明,VP1蛋白基因编码区开放阅读框长945 bp,编码315个氨基酸,推测编码蛋白的相对分子量和等电点分别为35.42 kDa和9.23,具有亲水性和免疫原性。序列同源性分析表明,不同年份CSBV北京分离株VP1蛋白氨基酸序列间差异较小,仅个别氨基酸存在差异。北京分离株与辽宁分离株及越南分离株VP1核苷酸序列一致性达93%,与印度及韩国分离株VP1核苷酸序列一致性达92%,与英国分离株VP1核苷酸序列一致性最低,为88%。序列分析同时表明,CSBV北京分离株VP1蛋白序列存在特有的序列特征,同其他地区分离株比较,北京分离株VP1蛋白序列中存在着氨基酸的插入突变。序列替换率分析表明,亚洲型分离株间序列替换率低于亚洲分离株与欧洲分离株间的替换率。构建原核表达载体pEASY-E1-VP1,经IPTG诱导,CSBV VP1蛋白在大肠杆菌Escherichia coli BL21（DE3）pLysS菌株中表达。【结论】本研究提示CSBV不同分离株基因序列存在变异,结果为进一步研究CSBV致病性分化的分子机理奠定了基础。     

Identification and characterization of the herpes simplex virus type 2 gene encoding the essential capsid protein ICP32/VP19c.

We describe the characterization of the herpes simplex virus type 2 (HSV-2) gene encoding infected cell protein 32 (ICP32) and virion protein 19c (VP19c). We also demonstrate that the HSV-1 UL38/ORF.553 open reading frame (ORF), which has been shown to specify a viral protein essential for capsid formation (B. Pertuiset, M. Boccara, J. Cebrian, N. Berthelot, S. Chousterman, F. Puvian-Dutilleul, J. Sisman, and P. Sheldrick, J. Virol. 63: 2169-2179, 1989), must encode the cognate HSV type 1 (HSV-1) ICP32/VP19c protein. The region of the HSV-2 genome deduced to contain the gene specifying ICP32/VP19c was isolated and subcloned, and the nucleotide sequence of 2,158 base pairs of HSV-2 DNA mapping immediately upstream of the gene encoding the large subunit of the viral ribonucleotide reductase was determined. This region of the HSV-2 genome contains a large ORF capable of encoding two related 50,538- and 49,472-molecular-weight polypeptides. Direct evidence that this ORF encodes HSV-2 ICP32/VP19c was provided by immunoblotting experiments that utilized antisera directed against synthetic oligopeptides corresponding to internal portions of the predicted polypeptides encoded by the HSV-2 ORF or antisera directed against a TrpE/HSV-2 ORF fusion protein. The type-common immunoreactivity of the two antisera and comparison of the primary amino acid sequences of the predicted products of the HSV-2 ORF and the equivalent genomic region of HSV-1 provided evidence that the HSV-1 UL38 ORF encodes the HSV-1 ICP32/VP19c. Analysis of the expression of the HSV-1 and HSV-2 ICP32/VP19c cognate proteins indicated that there may be differences in their modes of synthesis. Comparison of the predicted structure of the HSV-2 ICP32/VP19c protein with the structures of related proteins encoded by other herpes viruses suggested that the internal capsid architecture of the herpes family of viruses varies substantially.     

Nucleotide sequence of VP4 and VP7 genes of human rotaviruses with subgroup I specificity and long RNA pattern: implication for new G serotype specificity.

We sequenced the genes coding for the two neutralization proteins, VP4 and VP7, of human rotavirus strains L26 and L27 with subgroup I specificity but the long RNA pattern. The deduced VP7 amino acid sequence of strains L26 and L27 showed a low homology (73.6 to 81.9%) to those of rotavirus strains of the established serotypes. This finding, together with the previous serological characterizations, suggests that the VP7 (G) serotype of the L26 and L27 strains is distinct from those of strains of the previously established serotypes. In contrast, the VP4 sequences of the L26 and L27 strains were quite similar to those of virulent serotype 2 strains (DS-1, S2, and RV-5).     

VP2 gene based phylogenetic relationship of Indian isolates of Bluetongue virus serotype 1 and other serotypes from different parts of the world.

Bluetongue virus (BTV), a member of genus Orbivirus, family Reoviridae, is non-enveloped with double shelled structure and 10 segmented double stranded RNA genome. The RNA segment L2 encodes an outer capsid serotype specific viral protein VP2. BTV serotype 1 (BTV-1) specific novel primer pair, forward primer (1240-1271 bp) and reverse primer (1844-1813 bp), was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1. The 604 bp PCR product of VP2 gene of BTV-1 Avikanagar (A), Chennai (C) and Sirsa 3 (S3) Indian isolates were cloned in pPCR-Script Amp SK (+) vector and transformed into XL10-Gold Kan ultracompetent Epicurian coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. BTV-1A, C and S3 isolates revealed 99% nucleotide sequence identity within 1304-1844 bp region of VP2 gene. The partial VP2 gene sequences (1240-1844 bp region) revealed that BTV-1 Indian isolates were 89% identical with Australian (AUS) BTV-1 isolates while the identity with South African (SA) BTV-1 isolate was 75%. Phylogenetically, three BTV-1 Indian isolates formed one group which is closely related to BTV-1AUS isolates followed by BTV-1SA, BTV-2, 9, 23, 13, 17, 10 and 11 isolates from different parts of world. Based on partial VP2 gene sequences, it is concluded that Indian isolates of BTV-1 are closely related to BTV-1AUS isolates than BTV-1SA and other serotypes.     

Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants

The complete nucleotide sequences of the genomes of the type 2 ( P712 , Ch, 2ab ) and type 3 (Leon 12a1b ) poliovirus vaccine strains were determined. Comparison of the sequences with the previously established genome sequence of type 1 (LS-c, 2ab ) poliovirus vaccine strain revealed that 71% of the nucleotides in the genome RNAs were common, that the 5' and 3' termini of the genomes were highly homologous, and that more than 80% of the nucleotide differences in the coding region occurred in the third letter position of in-phase codons, resulting in a low frequency of amino acid difference. These results strongly suggested that the serotypes of poliovirus derived from a common prototype. A comparison of the amino acid sequences predicted from the genome sequences showed highest variation in the capsid protein region, whereas non-structural proteins are highly conserved. Initiation of polyprotein synthesis occurs in all three strains more than 740 nucleotides downstream from the 5' end. An analysis of the non-coding region suggests that small peptides that could potentially originate from this region are conserved. The amino acid sequences immediately surrounding the cleavage signals, however, show a higher than average degree of variation. The analysis of the amino acid sequences of the capsid protein VP1 of all serotypes has led to the prediction of potential antigenic sites on the virion involved in neutralization.     

Genetic Analysis of the Capsid Region of Human Astrovirus Serotype 3 Isolated in Japan

The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.     

Genomic structure of the large RNA segment of infectious bursal disease virus.

The larger RNA segment of infectious bursal disease virus (IBDV: Australian strain 002-73) has been characterized by cDNA cloning and nucleotide sequence analysis. We believe IBDV is the first birnavirus to be sequenced and so have confirmed the coding region by N-terminal amino acid sequence analysis of intact viral proteins and several tryptic peptide fragments. The large RNA segment encodes in order the 37-kDa, 28-kDa and 32-kDa proteins within a continuous open reading frame and the primary translation product appears to be subsequently processed into the mature viral proteins. The large protein precursor is still processed into the 32-kDa host protective immunogen when expressed as a fusion protein in E. coli. These results are in marked contrast to the predictions from in vitro translation data that birnavirus genomes are expressed as polycistronic templates. We can now propose that birnaviruses, in particular IBDV, possess monocistronic segments and that the precursor is proteolytically processed in vivo. The sequence data presented for the 32-kDa host protective immunogen may provide the basic information needed for the production of an effective subunit vaccine against this commercially important virus.