Genetic diversity among Lassa virus strains

The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a "molecular clock" was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation.     

Characterization of a simian hepatitis A virus (HAV): antigenic and genetic comparison with human HAV.

PA21, a strain of hepatitis A virus (HAV) recovered from a naturally infected captive owl monkey, is indistinguishable from human HAV in polyclonal radioimmunoassays and cross-neutralization studies. However, cDNA-RNA hybridization has suggested a significant difference at the genomic level between PA21 and a reference human virus, HM175. Further characterization of this unique HAV was undertaken in an effort to determine the extent of genetic divergence from human HAV and its relation to the conserved antigenic structure of the virus. The close similarity between PA21 and HM175 antigens was confirmed with an extended panel of 18 neutralizing murine monoclonal antibodies: a reproducible difference in binding to the two viruses was detected with only one antibody (B5-B3). The nucleotide sequence of the P1 region of the PA21 genome had only 83.2% identity with HM175 virus, a difference approximately twice as great as that found between any two human strains. Most nucleotide changes were in third base positions, and the amino acid sequences of the capsid proteins were largely conserved. Amino acid replacements were clustered in the carboxy terminus of VP1 and the amino-terminal regions of VP2 and VP1. These data indicate that PA21 virus represents a unique genotype of HAV and suggest the existence of an ecologically isolated niche for HAV among feral owl monkeys.     

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.     

Genetic Relatedness among Hepatitis A Virus Strains Associated with Food-Borne Outbreaks

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2–3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.     

我国新分离ECHO30病毒VP1序列分析

测定了引起2003年苏北地区无菌性脑膜炎暴发流行的病因病毒FDJS03分离株的VP1基因序列,并与国外同型流行毒株做比较,以了解本流行株的分子生物学特点及遗传变异规律。随机选取FDJS03分离毒株中的4株,用肠道病毒、VP1序列的特异性引物008／011进行RT-PCR,扩增产物经凝胶纯化后测序。将序列输入GenBank,用BLAgr程序进行核苷酸和氨基酸序列比对;选取32株不同地区不同年代的ECHO30分离毒株,在PHYLIP3．573C和TREE-PUZZLE5．0中构建进化树,比较它们完整VP1序列(876nt)的进化关系。核苷酸和氨基酸同源性比较结果证明：4株分离病毒均为ECHO30。进化树分析显示：本次FDJS03分离株与欧美20世纪70、80年代流行株亲缘关系最近,但自成一簇,与国外毒株仍然有地区差别。ECHO30的VP1基因进化有时间效应,但存在地区差异。本次流行的病原可能是单一基因型的ECHO30病毒。     

Phylogenetic relationship of street rabies virus strains and their antigenic reactivity with antibodies induced by vaccine strains. I. Analysis of phylogenetic relationship of street rabies virus strains isolated in Poland

The aims of these studies were: genetic characteristic of street rabies virus strains isolated from different animal species in Poland and determination of phylogenetic relationships to reference laboratory strains of the street rabies viruses belonging to genotype 1 and 5. The variability of rabies isolates and their phylogenetic relationship were studied by comparing the nucleotide sequence of the virus genome fragment. The Polish strains of genotype 1 belong to four phylogenetic groups (NE, CE, NEE, EE) corresponding to four variants: fox-racoon dog (F-RD); European fox 1 (F1); European fox 2 (F2) and European fox 3 (F3). On the Polish territories there are no rabies strains representing the variant dog-wolf and typical for arctic fox variant. The similarity of nucleotide and amino acid sequences of street rabies strains belonging to genotype 1 and laboratory strain CVS is very high. It is about 91% similarity at nucleotide level and 95% at amino acid level. Rabies strain CVS is similar to genotype 5 bat strains (EBL 1) only in about 69% and 74% at nucleotide and amino acid level, respectively. The genetic divergence of rabies strains circulating in Poland raised the need of permanent epidemiological and virological surveillance. The genotype and variant of isolated strains should be determined (using PCR and RLFP methods).     

Antigenic structure of the influenza B virus hemagglutinin: nucleotide sequence analysis of antigenic variants selected with monoclonal antibodies.

We report here the complete nucleotide sequence of the hemagglutinin (HA) gene of influenza B virus B/Oregon/5/80 and, through comparative sequence analysis, identify amino acid substitutions in the HA1 polypeptide responsible for the antigenic alterations in laboratory-selected antigenic variants of this virus. The complete nucleotide sequence of the B/Oregon/5/80 HA gene was established by a combination of chemical sequencing of a full-length cDNA clone and dideoxy sequencing of the virion RNA. The nucleotide sequence is very similar to previously reported influenza B virus HA gene sequences and differs at only nine nucleotide positions from the B/Singapore/222/79 HA gene (Verhoeyen et al., Nucleic Acids Res. 11:4703-4712, 1983). The nucleotide sequences of the HA1 portions of the HA genes of 18 laboratory-selected antigenic variants were determined by the dideoxy method. Comparison of the deduced amino acid sequences of the parental and variant HA1 polypeptides revealed 16 different amino acid substitutions at nine positions. All amino acid substitutions resulted from single-point mutations, and no double mutants were detected, demonstrating that as in the influenza A viruses, single amino acid substitutions are sufficient to alter the antigenicity of the HA molecule. Many of the amino acid substitutions in the variants occurred at positions also observed to change in natural drift strains. The substitutions appear to identify at least two immunodominant regions which correspond to proposed antigenic sites A and B on the influenza A virus H3 HA.     

重组CHO细胞C_(28)株S基因序列的测定及分析

目的测定重组CHO细胞C28株S基因序列,研究其遗传稳定性,并与已全基因序列测定的乙型肝炎病毒的S基因序列进行比较分析,预测和揭示现有疫苗株对当前疾病流行株的防病效果。方法从C28株中选取第22代、24代2、5代2、7代、28代2、9代3、0代、31代3、2代3、3代和34代细胞,根据GenBank中C基因型adr亚型乙型肝炎病毒的全基因序列设计引物。采用酚-氯仿法抽提CHO细胞基因组DNA,用PCR法扩增各代次细胞的S基因,回收700 bp左右的目的片段,克隆至pMD18-T载体上进行序列测定。利用生物学软件MEGA4.1和BioEdit进行S基因序列同源性分析,绘制系统进化树,分析与其他HBV病毒株S基因的同源性。应用实验动物测定C28株生产的重组乙型肝炎疫苗的效价。结果 C28株十一个代次之间S基因序列核苷酸和氨基酸同源性均为100%;C28株十一个代次S基因与其他病毒株S基因比较,与C基因型乙型肝炎病毒同源性最高,与其他基因型乙型肝炎病毒的核苷酸同源性达91.4%～95.1%,氨基酸同源性达84.5%～93.3%。免疫NIH小鼠结果显示5批重组乙型肝炎疫苗的效价均符合标准。结论 C28株S基因在传代及保存过程中具有较高的稳定性,对当前疾病流行株有较好的防病效果。     

Frequent reassortments may explain the genetic heterogeneity of rotaviruses: analysis of Finnish rotavirus strains

The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylogenetic analysis. The virus isolates were either P[8]G1 or P[8]G4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P[8] lineages (VP4-P[8]-1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.     

Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests.     

New recombinant variant of human immunodeficiency virus of type 1, subtype envB/envA, isolated in Novosibirsk

The nucleotide sequence of the variant of human immunodeficiency virus of type 1 (HIV-1), mostly widespread on the territory of the Novosibirsk region, was determined. The analysis of the nucleotide sequence confirmed that this variant belonged to HIV-1 of subtype A. The HIV-1 recombinant variant of subtype envB/envA with the recombination area within the second conservative region C2 of gene env, so far unknown, was detected and characterized. In HIV-1 the area at the beginning of gene env (5'-env) was found to belong to subtype B and the sequence at the end of gene env (3'-env), to subtype A. The analysis of the amino acid sequence of the third variable region of gene env demonstrated that the viruses under study belonged to macrophagotropic "slow/low" variants, characterized by low replication speed. The analysis of nucleotide sequences of the isolated variants of HIV-1 revealed their close genetic relationship with HIV-1 isolates circulating on the territory of Ukraine.     

Hepatitis A Virus Capsid Protein VP1 Has a Heterogeneous C Terminus

Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.     

引起一起暴发性急性胃肠炎疫情的埃可病毒18型VP1基因特征分析

埃可病毒18型(Echovirus 18,E18)属于B组肠道病毒(Enterovirus B,EV-B),是引起病毒性脑膜炎的重要病原体。对2019年4月广东省深圳市龙岗区一起急性胃肠炎疫情进行病原学鉴定及病原基因特征分析。此次疫情共有26名患者,均以发热、呕吐、腹泻、腹痛为主要症状。从其中18名病例采集了18份肛拭子标本,采用实时荧光RT-PCR、病毒分离(RD细胞)和半巢式聚合酶链反应法以鉴定病原,扩增病毒全长VP1区,测序并进行系统进化分析。结果显示,18份标本中11份肠道病毒核酸阳性,其中8份为E18。病毒分离得到2株病毒,从肛拭子标本直接扩增得到的7条E18全长VP1核苷酸序列,核苷酸相似性为100%,与GenBank中E18参考株的核苷酸及氨基酸同源性分别为79.2%~96.4%和93.3%~99.3%;进化树显示,深圳龙岗E18分离株(GDSZ1902)归属由中国分离株组成的C2a进化分支,在其VP1蛋白中的底部环处发现一处特异的氨基酸变异(D200N)。从流行病学和病原学结果分析,E18是引起本次深圳市龙岗区急性胃肠炎疫情的主要病原,属于中国E18主要流行株的进化分支,这是我国首次发现E18引起急性胃肠炎疫情的报道。     

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     

Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Fuyang,China, during 2008

Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.     

2018–2020年人轮状病毒锦州地方株VP4及VP7基因特征

【背景】人A组轮状病毒(Rotavirus Group A,RVA)是婴幼儿胃肠炎的主要病原体及发展中国家婴幼儿死亡的重要原因,目前无特效药物治疗,疫苗预防是唯一可行的预防感染方法。外衣壳蛋白VP7和VP4是疫苗设计的主要靶点,针对该基因加强RVA地方株分子流行病学监测十分必要。【目的】对锦州地方流行RVA株VP7和VP4基因进行型别鉴定和序列特征分析。【方法】收集锦州地区2018-2020年RVA感染腹泻患儿的粪便标本,提取病毒RNA,通过RT-PCR扩增VP7、VP4基因片段并测序,得到7株RVA VP7和VP4序列。使用在线基因分型工具Rota C V2.0对测序结果进行分型分析。应用BLAST、DNAStar、MEGA X、Bio Edit等生物软件与临床流行株及疫苗株进行系统发育分析及氨基酸序列比对分析。【结果】分型结果表明7株锦州地方株均为G9P[8]型,系统发育分析证实其VP7和VP4基因分别属于G9-Ⅵ和P[8]-3谱系,核苷酸序列相似性分别为99.32%-100%与99.41%-100%。JZ株VP7与疫苗株Rotavac和Rotasiil相比,在抗原表位区7-1a、7-1b、7-2中分别存在4个和3个氨基酸替换。JZ株VP4与疫苗株Rotarix和Rota Teq VP4氨基酸序列相比,发现7个和4个氨基酸替换,位于抗原表位区8-1和8-3。【结论】2018-2020年在辽宁锦州地区检测到7株G9P[8]型RVA株,VP7和VP4序列相似性高于99%,G9P[8]型可能是辽宁省锦州地区2018-2020年婴幼儿轮状病毒腹泻的主要流行基因型之一。与同基因型疫苗株比较,位于JZ株VP7和VP4抗原表位区的氨基酸位点差异对于野毒株免疫逃逸机制的研究具有意义。     

Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission

The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.     

Phylogenetic relationships of aquatic birnaviruses based on deduced amino acid sequences of genome segment A cDNA.

Aquatic birnaviruses, such as infectious pancreatic necrosis virus (IPNV), cause serious diseases in a variety of fish species used worldwide in aquaculture and have also been isolated from a variety of healthy fish and shellfish species. These viruses exhibit a high degree of antigenic heterogeneity and variation in biological properties such as pathogenicity, host range, and temperature of replication. To better understand genetic and biological diversity among these viruses, the nucleotide and deduced amino acid sequences were determined from cDNA of the large open reading frame (ORF) of genome segment A of the 9 type strains of Serogroup A and 4 other representative strains of Serotype A1, the predominant serotype in the United States. In addition, nucleotide and deduced amino acid sequences were determined for the VP2 coding region of a variety of isolates representing 5 of the 9 serotypes. VP2 is the major outer capsid protein of aquatic birnaviruses. RT-PCR was used to amplify a 2904 bp cDNA fragment including all but a few bp of the large ORF of genome segment A or a 1611 bp fragment representing the entire VP2 coding region. Nucleotide and deduced amino acid sequences were determined from the PCR products. Pairwise comparisons were made among our data and 2 other aquatic birnavirus sequences previously published. Several hypervariable regions were identified within the large ORF. The most divergent pair of viruses exhibited a similarity of 80.1% in the deduced amino acid sequence encoded by the large ORF. Genomic relationships revealed in a phylogenetic tree constructed from comparison of the deduced amino acid sequences of the large ORF demonstrated that these viruses were clustered into several genogroups. Phylogenetic comparison of the deduced amino acid sequences of the VP2 coding region of 28 aquatic birnavirus isolates, including the type strains of all 9 serotypes, demonstrated 6 genogroups, some of which were comprised of several genotypes. The most divergent pair of viruses exhibited a similarity of 81.2% in the deduced amino acid sequence from the VP2 coding region. In contrast to previous studies of much shorter genomic sequences within the C-terminus-pVP2/NS junction coding region, these genogroups based on the entire large ORF or the VP2 coding region generally correlated with geographical origin and serological classification. Isolates from the major Canadian serotypes were more closely related to the European isolates than to isolates from the United States.