Genetic variation and phylogenetic analysis of 22 French isolates of equine arteritis virus

Summary Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001–2004) were determined by sequencing open reading frames (ORFs) 2a–7. The ORFs 2a–7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (nucleotides 11296–11813) of 22 French isolates and 216 additional EAV strains available in GenBank clustered the global isolates of EAV into two distinct groups: North American and European. The latter could be further divided into two large subgroups: European subgroup 1 (EU-1) and European subgroup 2 (EU-2). Phylogenetic analysis based on 100 EAV ORF3 sequences yielded similar results. Of the 22 French EAV isolates, the 11 isolates obtained before January 28, 2003 clustered with either the EU-1 (9 isolates) or EU-2 (2 isolates) subgroup. In contrast, by the criteria used in this study, the 11 isolates obtained after January 30, 2003 belong to the North American group, strongly suggesting that these strains were recently introduced into France. The first two authors contributed equally to this work.     

Phylogenetic analyses of the putative M (ORF 6) and N (ORF 7) genes of porcine reproductive and respiratory syndrome virus (PRRSV): implication for the existence of two genotypes of PRRSV in the U.S.A. and Europe

Summary The putative membrane (M) protein (ORF 6) and nucleocapsid (N) protein (ORF 7) genes of five U.S. isolates of porcine reproductive and respiratory syndrome virus (PRRSV) with differing virulence were cloned and sequenced. To determine the genetic variation and the phylogenetic relationship of PRRSV, the deduced amino acid sequences of the putative M and N proteins from these isolates were aligned, to the extent known, with other PRRSV isolates, and also other members of the proposed arterivirus group including lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). There was 96–100% amino acid sequence identity in the putative M and N genes among U.S. and Canadian PRRSV isolates with differing virulence. However, their amino acid sequences varied extensively from those of European PRRSV isolates, and displayed only 57–59% and 78–81% identity, respectively. The phylogenetic trees constructed on the basis of the putative M and N genes of the proposed arterivirus group were similar and indicated that both U.S. and European PRRSV isolates were related to LDV and were distantly related to EAV. The U.S. and European PRRSV isolates fell into two distinct groups, suggesting that U.S. and European PRRSV isolates represent two distinct genotypes.     

Genetic typing of equine arteritis virus isolates from Argentina

We report the nucleotide sequence and genetic diversity of four Equine Arteritis Virus (EAV) ORF 5 and 6 from Argentina isolates, obtained from asymptomatic virus-shedding stallions. Nucleic acid recovered from the isolates were amplified by RT-PCR and sequenced. Nucleotide and deduced amino acid sequences from the Argentine isolates were compared with 17 sequences available from the GenBank. Phylogenetic analysis revealed that the Argentine isolates grouped together in a definite cluster near European strains. Despite the greater genetic variability among ORF 5 from different isolates and strains of EAV, phylogenetic trees based on ORF 5 and 6 are similar. Both trees showed that virus sequences from America and Europe segregate into distinct clades based on sequence analysis of either ORF 5 or 6. This study constitutes the first characterization of Argentine EAV isolates. M. G. Echeverría, S. Díaz, E. Nosetto Members of CONICET (Scientific Research Council)     

Development and evaluation of ELISA procedures to detect antibodies against the major envelope protein (GL) of equine arteritis virus

Enzyme-linked immunosorbant assays (ELISAs) were developed for the detection of antibodies against the major envelope glycoprotein (G_L) of equine arteritis virus (EAV). A 6-Histidine tagged recombinant protein expressing the complete G_L ectodomain (G_L-6His), a glutathione-S-transferase recombinant protein expressing amino acids 55–98 of G_L (G_L-GST) and an ovalbumin-conjugated synthetic peptide representing amino acids 81–106 of G_L (G_L-OVA) were used as diagnostic antigens. An ELISA procedure was developed and optimised for each antigen. The G_L-OVA and G_L-6His assays showed the greatest specificity while the G_L-GST assay was slightly more sensitive that the G_L-OVA and G_L-6His assays; results based on the analysis of 50 virus neutralisation positive and 50 virus neutralisation negative sera. The G_L-OVA ELISA was selected for further evaluation since it was simpler to use than ELISAs based on recombinant antigens and did not suffer from background reactivity. The final sensitivity and specificity of the G_L-OVA ELISA were 96.75 and 95.6%, respectively, results based on the analysis of 400 virus neutralisation positive and 400 virus neutralisation negative sera. It also detected EAV antibody (100% efficiency) in seropositive shedding stallions and, in ponies infected experimentally with the UK93 isolate of EAV, the appearance of virus neutralising antibodies and G_L-OVA ELISA-specific immunoglobulins coincided.     

Amino acid substitutions in the structural or nonstructural proteins of a vaccine strain of equine arteritis virus are associated with its attenuation

Comparative sequence analysis of a series of strains of equine arteritis virus (EAV) of defined virulence for horses, ranging from the horse-adapted virulent Bucyrus (VB) strain to a fully attenuated vaccine strain derived from it, identified 13 amino acid substitutions associated with attenuation. These include 4 substitutions in the replicase proteins and 9 in the structural proteins. Using reverse genetic techniques, these amino acid substitutions were introduced into a virulent infectious cDNA clone pEAVrVBS derived from the VB strain of EAV. Inoculation of horses with the recombinant viruses clearly demonstrated that changes in either the replicase (nsp1, nsp2 and nsp7) or structural proteins (GP2, GP4, GP5 and M) resulted in attenuation of the virulent VB strain. The recombinant virus with substitutions in the structural proteins was more attenuated than the recombinant virus with substitutions only in the replicase proteins.     

Evaluation of genetic divergence among Borrelia burgdorferi isolates by use of OspA, fla, HSP60, and HSP70 gene probes.

In order to assess the genetic variation of immunologically relevant structures among isolates of the Lyme disease spirochete, Borrelia burgdorferi, three chromosomal genes encoding flagellin (fla) and the heat shock proteins HSP60 and HSP70, as well as the plasmid gene encoding outer surface protein A (OspA), from 55 different European and North American strains obtained from ticks and mammal hosts have been investigated by restriction fragment length polymorphisms (RFLPs). RFLPs of fla and the HSP60 and HSP70 genes revealed two distinct banding patterns (A and B) for each of the three genes and allowed the definition of four genomic groups [AAA, BBB, BBA, and B(A/B)A] for the three chromosomal genes. On the other hand, RFLPs of the OspA gene revealed six distinct banding patterns (types I to VI) making up six independent genomic groups for the plasmid-encoded gene. Furthermore, we have sequenced the chromosomal HSP60 gene from B. burgdorferi ZS7 and the plasmid-encoded OspA gene from two strains, ZQ1 and 19857. Alignment of the deduced HSP60 amino acid sequence from B. burgdorferi ZS7 (genomic group AAA) to a previously published HSP60 sequence derived from strain ACA-1, which according to the proposed classification is in a different genomic group (BBA), revealed a sequence identity of > 99%. Similar alignments of the OspA sequence of strain ZQ1 to those of other isolates that were published previously revealed sequence identities of between 70 and 94% among strains of distinct OspA genomic groups. These data indicate the existence of a restricted number of species-specific subgroups and clearly show that genotypic variation is much more pronounced for the OspA gene than for fla and the HSP60 and HSP70 genes. A phylogenetic tree constructed on the basis of distance matrix analyses of 12 OspA sequences supports the proposed classification of genomic groups of B. burgdorferi.     

呼吸道合胞病毒分离株N蛋白编码基因特征分析

目的 阐明人呼吸道合胞病毒(human respiratory syncytial virus,HRSV)A和B亚型病毒分离株的核蛋白(nucleoprotein,N)编码基因特征.方法 采用逆转录-聚合酶链反应(revelrse transciptionpolymerase chain reaction,RT-PCR)对北京2004年分离的HRSV代表株(2株A亚型和2株B亚型)进行N基因全长序列扩增、测序,并和GenBank下载的所有HRSV病毒的N基因全长序列进行对比和分析.结果 2株HRSV A亚型分离株与A亚型Long株(标准株)的N蛋白的核苷酸和氨基酸差异分别为36～40个(3.1%～3.4%)和4个(1.0%);2株HRSV B亚型分离株与B亚型CH18537株(标准株)的N蛋白之间的核苷酸和氨基酸差异分别为17(1.4%)和1(0.3%)个.4株HRSV代表株和从GenBank下载的HRSV的N蛋白之间的核苷酸和氨基酸差异分别为3～172个(0.25%～14.63%)和0-18个(0～4.6%).结论 N基因在HRSV基因组中是较为保守的基因,A或B亚型的型内差异相对较小;但A和B亚型之间N基因序列有较大变异,变异平均分配于整个N基因中;本研究为HRSV基因快速诊断试剂的研制提供了基因信息学数据.     

Comparison of the coat protein genes of Mirafiori lettuce big-vein virus isolates from Australia with those of isolates from other continents

The complete coat protein nucleotide encoding sequences of 13 Mirafiori lettuce big-vein virus isolates from Australia were compared to those of 23 other isolates, including one from Australia. On phylogenetic analysis, sub-clade A1 contained isolates from Australia (13), Europe and Japan, A2 contained isolates from Australia (1), Europe and South America, and B1 and B2 contained only European isolates. In the amino acid sequences deduced, the N-terminus and central regions varied considerably between clades A and B. Mean Dn/Ds ratios were 0.112, 0.076, 0.187 and 0.063 for all isolates, Australian isolates, clade A isolates and clade B isolates, respectively.     

Further properties of Equine Arteritis Virus

Summary An American (Bucyrus) and a Swiss (Bibuna) strain of Equine Arteritis Virus both had a particle size lying in between 50 and 100 m, were resistant to treatment with trypsin, were quite sensitive towards heat and low pH, and were completely inactivated by molar MgCl₂. When added to properties established earlier for EAV by the author (content of RNA, sensitivity towards lipid solvents) these features do not easily classify EAV whithin one of the presently defined groups of viruses. Emphasis is put on similarities between EAV and the viruses of hog cholera, bovine virus diarrhea, and eventually human hepatitis. All four agents might be members of a group of hemoviruses.     

European and American Subgroup C Isolates of Avian Metapneumovirus belong to Different Genetic Lineages

The gene encoding the attachment glycoprotein (G) was sequenced in three French isolates of-subgroup C avian metapneumovirus (APV-C) from ducks. With 1771 nt, this gene proved as long as recently published for North-American APV-C isolates from turkeys. The nt sequences of the duck viruses shared 99% identity but proved only 75-83% identical with their North-American counterparts, viruses of both origins encoding 585 amino acid (aa)-long G proteins. Alignments revealed more homogeneity within the European and North-American groups (at least 98 and 79% aa identity, respectively) than between European and North-American viruses (at best 70% a identity), and confirmed the presence of an extracellular divergent domain (positions 302-484) in APV-C G. A phylogenetic analysis demonstrated that North-American and French isolates of APV-C belonged to significantly different genetic lineages, in agreement with the different geographical origin and host species of these viruses.     

Genetic characterization of Aleutian mink disease viruses isolated in China

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex mediated disease in minks. To understand the genetic characterization of AMDV in China, the genomic sequences of three isolates, ADV-LN1, ADV-LN2, and ADV-LN3, from different farms in the Northern China were analyzed. The results showed that the lengths of genomic sequences of three isolates were 4,543, 4,566, and 4,566 bp, respectively. They shared only 95.5-96.3 % nucleotide identity with each other. The nucleotide and amino acid homology of genome sequence between the Chinese isolates and European or American strains (ADV-G, ADV-Utah1, and ADV-SL3) were 92.4-95.0 % and 92.1-93.8 %, respectively. The amino acid substitutions randomly distributed in the genome, especially NS gene. ADV-LN1 strain had a 9-amino-acid deletion at amino acid positions 70 and 72-79 in the VP1 gene, comparing with ADV-G strain; ADV-LN2 and ADV-LN3 strains had 1-amino-acid deletion at amino acid positions 70 in the VP1. Some potential glycosylation site mutations in VP and NS genes were also observed. Phylogenetic analysis results showed that the three strains belonged to two different branches based on the complete coding sequence of VP2 gene. However, they all were in the same group together with the strains from United States based on the NS1 sequence. It indicated that Chinese AMDV isolates had genetic diversity. The origin of the ancestors of the Chinese AMDV strains might be associated with the American strains.     

Phylogenetic analysis of Taura syndrome virus isolates collected between 1993 and 2004 and virulence comparison between two isolates representing different genetic variants

Taura syndrome virus (TSV) is highly pathogenic to Litopenaeus vannamei (Pacific white shrimp) and has caused significant economic loss in the shrimp culture industry. It was first reported from Ecuador in 1992 and has since become widely distributed throughout the Americas and southeast Asia (SE Asia). To determine the genetic relationship among various geographic isolates, we amplified and sequenced a 1.3 kb fragment of the TSV capsid protein gene 2 (CP2) from each of 34 isolates collected from cultured penaeid shrimp stocks in Ecuador, Colombia, Honduras, USA, Mexico, Belize, Thailand, China, and Indonesia. An additional six CP2 sequences obtained from GenBank were included in the analysis. The results indicated low genetic variation (0--5.6% for nucleotide sequence and 0--7.0% for deduced amino acid sequence) among these 40 isolates. A phylogenetic analysis based on the deduced CP2 amino acid sequence revealed three distinct groups: Americas, Belize, and SE Asia. The Belize and SE Asia groups were separated from each other by a 4.7% difference in amino acid sequence. The Belize and Americas groups differed by 4.4%. The Americas and SE Asia groups were the closest, separated by a difference of only 3.3%. Comparison between Belize and Hawaii TSV (reference strain for Americas group) indicated that Belize TSV was more virulent than Hawaii TSV. In bioassays, the Belize isolate caused 50% mortality by 3 days, while the Hawaii isolate caused 50% mortality over 4--6 days. Based on the phylogenetic analysis and virulence comparison, the Belize TSV isolate should be considered as a new variant.     

Phylogenetic analysis of the gag region encoding the matrix protein of small ruminant lentiviruses: comparative analysis and molecular epidemiological applications

Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs.     

Analysis and comparison of the 4b core protein gene of avipoxviruses from wild birds: evidence for interspecies spatial phylogenetic variation

Summary. Avipoxviruses have been isolated from a wide variety of avian hosts, and yet little is known regarding the host-virus species variation of the genus Avipoxvirus. We have investigated the variations in the viral 4b core protein gene from six different avipoxviruses based on PCR, Southern blot and nucleotide sequence analysis to evaluate the suitability of this region for differentiation between avipoxvirus isolates. Southern blot and nucleotide sequence analysis revealed considerable interspecies variation between the different virus isolates. In the deduced amino acid sequences (of 142 residues) of the 4b core protein gene, fowlpox virus vaccine strain (FPV-VR250) was found to be similar to the three poxvirus isolates from great tit (GTV-A310, GTV-A311 and GTV-A256), sparrowpox virus (SPV-A468), and pigeonpox virus (PPV-B7) with similarities of 79.6%, 81%, 81%, 64.8% and 84.5%, respectively. Furthermore, comparative phylogenetic analysis of the aligned DNA sequences revealed divergence among the different viruses that can be consistently correlated to the host.     

Complete genomic characterization of a potato mop-top virus isolate from the United States

Potato mop-top virus (PMTV; family Virgaviridae) was reported recently in the Pacific Northwestern USA. To better understand the genetic diversity of this virus, the complete genome of an isolate from Washington State (WA), USA, was characterized. Sequence comparisons of the WA isolate with other known sequences revealed that the RNA-Rep-encoded RdRp protein and the RNA-CP-encoded coat protein displayed >99 % amino acid sequence identity to those of two Nordic (RdRp) and several European and North American isolates (CP), respectively. The RNA-TGB-encoded TGB 1 and TGB 3 protein sequences had >99 % amino acid sequence identity to the corresponding proteins of Czech and Danish isolates, whereas the TGB 2 protein is identical to those of Colombian isolates. Phylogenetic analysis of the viral genes of the WA isolate reflected the close relationship between WA and European isolates. RFLP analysis of corresponding DNA of RNA TGB and RNA CP revealed that the WA isolate has the RNA TGB-II and RNA CP-B types, which are prevalent in Europe and other parts of world. This is the first report of the complete genome characterization of PMTV from the Americas.     

Comparison of the coat protein genes of Lettuce big-vein associated virus isolates from Australia with those of isolates from other continents

The complete coat protein (CP) nucleotide sequences of seven Lettuce big-vein associated virus (LBVaV) isolates from Australia were compared to those of 22 other LBVaV and five tobacco stunt virus (TStV) isolates. On phylogenetic analysis, clade I contained only LBVaV isolates from Europe, sub-clade IIa only Australian LBVaV isolates, IIb only Japanese LBVaV isolates, and IIc only TStV isolates from Japan. In the amino acid sequences deduced, the central region of the gene was most divergent. Mean Dn/Ds ratios were 0.283 and 0.124 for clades I and II, respectively. The suggestion that TStV is a strain of LBVaV was supported.     

Further characterization of a potent immunogen and the chromosomal gene encoding it in the Lyme disease agent, Borrelia burgdorferi.

Further characterization of a previously reported 83-kDa antigen of Borrelia burgdorferi and the gene encoding it is reported. The DNA sequence of the gene and the amino acid sequence of the protein were determined. On the basis of the amino acid content, the actual size of the antigen was determined to be 79.8 kDa, rather than 83 kDa as previously reported. The expression of the antigen in several representative North American and European B. burgdorferi isolates was demonstrated. The conservation of the gene within the species was demonstrated by DNA hybridization of a labeled gene probe to several North American and European B. burgdorferi isolates. Of other Borrelia spp. assayed (B. hermsii, B. coriaceae, B. duttonii, and B. anserina), only B. anserina, a poultry pathogen, hybridized to the gene probe and expressed the 79.8-kDa antigen.     

Genetic variation analysis of apple chlorotic leaf spot virus coat protein reveals a new phylogenetic type and two recombinants in China

In this study, we generated sequences of the apple chlorotic leaf spot virus (ACLSV) coat protein (CP) gene. Genetic variation and phylogenetic analyses were carried out on these sequences along with others reported previously. ACLSV populations clustered into four types: in three of the four types, combinations of three amino acids at positions of 40, 75 and 79 were conserved. The fourth phylogenetic type, newly identified here, was characterized by co-variation of Ser⁴⁰-Tyr⁷⁵-Ser⁷⁹. Statistically significant genetic differentiation and infrequent gene flow were detected among the four types. Two natural recombinants were detected for the first time among ACLSV isolates/genotypes from China.     

Phylogenetic analysis of rabbit haemorrhagic disease virus (RHDV) strains isolated between 1988 and 2003 in eastern Hungary

Summary. To define the genetic variability of RHDV strains collected in eastern Hungary, liver samples from rabbits that had died of RHD were collected between 1988 and 2003. The phylogenetic analysis of a 528-nucleotide-long portion of the gene encoding the VP60 capsid protein assigned the strains into three genogroups. The first group contained viruses from 1988–1993, and a second group comprised isolates from 1994–2002. A third group comprised all of the tested representatives of the RHDVa subtype and a Hungarian isolate from 2003. These findings were supported by the alignments of the deduced amino acid sequences of the VP60 gene and strongly suggest the presence of the RHDVa subtype in Hungary.