Genomic differences between the diabetogenic and nondiabetogenic variants of encephalomyocarditis virus

Plaque purification of the M variant of encephalomyocarditis (EMC-M) virus resulted in the isolation of two stable variants. One is a highly diabetogenic D variant (EMC-D) and the other is a nondiabetogenic B variant (EMC-B). The cDNA of EMC-D and EMC-B genomes were cloned and seven overlapping cDNA clones were selected to cover the entire genome except the 5'-end 310 bases which were determined by RNA-dependent DNA sequencing and enzymatic RNA sequencing. Each clone was restriction-mapped, subcloned, and sequenced. The genomes of EMC-D and EMC-B are composed of 7829 and 7825 bases, respectively. Both genomes contain a long open reading frame of 6876 nucleotides starting at position 830 on the consensus sequence, which encodes a polyprotein of 2292 amino acids. The sequences of EMC-D and EMC-B differ by two deletions, one insertion, and eight point mutations. The first deletion of 3 nucleotides is located in the 5' poly(C) tract where EMC-B has 127 nucleotides compared with 130 nucleotides in EMC-D. The second deletion in EMC-B involves 2 nucleotides at the 3'-end polyadenylation site. A single base insertion of U occurs at the 5' noncoding region of EMC-B. The eight point mutations are located in the polyprotein coding region. Two are silent and are each located in the structural gene 1B and in the nonstructural gene 2B. The remaining six mutations, one on the L gene and the other five on the 1D gene, introduce respective amino acid changes. It is concluded that the diabetogenic EMC-D viral genome (7829 bases) differs from the nondiabetogenic EMC-B viral genome (7825 bases) by 14 nucleotides out of 7829.     

Comparison of the nucleotide sequences of diabetogenic and nondiabetogenic encephalomyocarditis virus

The nucleotide sequences of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp- phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp+ phenotype) have been compared. These variants differ at eight sites. The poly(C) tract of EMC-B is three bases shorter than that of EMC-D. Of the seven sites at which nucleotide substitutions were confirmed using RNA templates, four resulted in amino acid changes in three different proteins, the leader peptide, 1B (VP2), and 1D (VP1). The biological significance of these differences can now be investigated.     

The genomic RNA of diabetogenic encephalomyocarditis virus: characterization and molecular cloning

The RNA of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp- phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp+ phenotype) which were derived from the same virus stock (M strain) have been compared. The size of both genomes is estimated at 7.7 kb. The poly(C) tract of EMC-D is estimated at 144 bases, whereas that of EMC-B is 141 bases in length. The untranslated 5' terminal 103 nucleotides are identical for B and D with preservation of a stable terminal hairpin structure. The entire open reading frame of both variants has been cloned and the restriction maps of 12 different enzymes are identical. These maps were compared to a computer-generated restriction map of another strain of EMC virus which has been cloned and sequenced by Palmenberg et al. (1984, Nucleic Acids Res. 12, 2969-2985). Approximately 50% of the restriction sites of the B and D variants had similar locations in this strain of EMC. We conclude that significant genetic variation exists between the M strain (B and D variants) and the Palmenberg strain of EMC virus. However, the B and D variants are very similar at the molecular level and comparison of their nucleotide sequences will be necessary to reveal the basis for their different biological properties.     

Genomic analysis of two porcine encephalomyocarditis virus strains isolated in China

Summary. Two strains of encephalomyocarditis virus (EMCV), designated BJC3 and HB1, were isolated from an aborted fetus and the heart tissue of a dead piglet that had pericardial fluid, respectively. The complete genomic sequences of the two viruses were determined and analyzed. The size of the genomes of BJC3 and HB1 were 7746 and 7735 nucleotides, respectively, including poly(A) tails. Comparative analysis with the genomic sequences of other EMCV strains showed that BJC3 and HB1 shared higher identity (92.5–99.6%) with BEL-2887A/91, EMCV-R and PV21, but lower identity (83.3–84.6%) with EMC-B, EMC-D and D variants, and only 81.0% with Mengo virus. Two amino acid mutations in the leader protein of the two viruses and one amino acid substitution in VP1 of BJC3 were found in comparison to other EMCV strains Phylogenetic analysis based on the amino acid sequences of the entire ORF revealed that the two Chinese isolates BJC3 and HB1 clustered together with the strains BEL-2887/91, EMCV-R and PV21, which belong to the same genetic subgroup as EMCV-30. Our results provide genomic information for EMCV isolated in China.     

Genetic mechanisms of antigenic variation in infectious bursal disease virus: Analysis of a naturally occurring variant virus

The major immunogenic protein VP2 from a pathogenic field isolate (variant A virus) of infectious bursal disease virus (IBDV) was cloned and sequenced to examine antigenic variations. The VP2 open reading frame consists of 1509 nucleotides and codes for a 503 amino acid protein. Overall, the VP2 amino acid sequence of the variant A virus shares 98.6% identity with VP2 genes from other published IBDV strains. However, within the central region of VP2 (amino acids 222–334) lies a highly divergent area that we have termed thevariable domain. Relative to five other IBDV isolates, a total of six amino acid changes occur within the variable domain of the variant A virus. At positions 284–288, a substitution of isoleucine to threonine, a decrease in the number of Chou and Fasman turns, and a switch from a hydrophilic to a hydrophobic region are found only in the variant A virus. Together these changes predict a decrease in antigenicity as determined by calculation of potential antigenic sites. This suggests that only minor changes within VP2 contributed to the emergence of a variant virus that can cause disease in immunized birds.     

Relationship of interferon-inducing particle phenotype to encephalomyocarditis virus-induced diabetes mellitus.

The B (nondiabetogenic) and D (diabetogenic) variants of encephalomyocarditis (EMC) virus were studied to further define the role of the interferon (IFN) system in murine virus-induced diabetes mellitus. The relationship between the initial multiplicity of infection with EMC-B and the IFN yield showed that cells infected with one IFN-inducing particle produce a maximum amount of IFN, whereas IFN production is suppressed in cells infected with two or more particles. The IFN yield induced by EMC-D was less than 5% of that induced by EMC-B, allowing the designation of the B and D variants as Ifp+ and Ifp-, respectively. The Ifp+ property of the virion was shown to be responsible for the greater sensitivity of EMC-B to exogenous IFN as a result of primed local IFN induction. The data indicate that different Ifp phenotypes occur in nature and are associated with the development of diabetes in mice.     

Comparison of the genomes of two sympatric iridescent viruses (types 9 and 16)

Summary A map of the sites in the genome ofCostelytra zealandica iridescent virus (CzIV), using the restriction enzymesBamHI,KpnI, andPstI, showed the genome size to be 170.2 kbp in length. It was found that the genome was cyclically permuted and that 39% of the genome of CzIV contained repetitive sequence elements. The genome was found to hybridize with the genome of another iridescent virus, type 9 (WIV), in DNA-DNA hybridization experiments. A region of the WIV DNA genome (23.4 kbp) did not hybridize with CzIV DNA and this region is similar in size to the total genomic size difference between CzIV and WIV (22.4 kbp). A unique repeat sequence from iridescent virus type 6 (CIV) was shown to be present in the genome of WIV but not that of CzIV. Finally, the positions of the major capsid protein genes, VP53 and VP52, in the restriction enzyme maps for type 16 and type 9 respectively were determined.     

Identification of putative motifs involved in the virulence of infectious pancreatic necrosis virus

Infectious pancreatic necrosis viruses (IPNVs) belonging to the family Birnaviridae display a high degree of antigenic variability, pathogenicity, and differences in outbreak mortality in salmonid species. To determine if virus isolates of Sp serotype differ in virulence, fry of Atlantic salmon (Salmo salar L.) were challenged with nine different field strains. These viruses caused either high mortality and severe pathological changes or low mortality and no lesions. To study the molecular basis for the variation in virulence of IPNV, complete nucleotide sequences of segment A of all these strains as well as segment B of three selected strains were determined. All viruses tested had a unique genome sequence. Only minor differences were noted in the genes encoding VP1, VP3, and VP4 proteins, whereas most changes were observed in the gene encoding the VP2 protein. A high level of variation was found in the small open reading frame (ORF), which encodes a 15-kDa nonstructural (NS) polypeptide also known as VP5. One of the strains lacked the initiation codon for this protein, whereas the other four could encode a truncated version of the NS protein. Additional data obtained by sequencing of the NS and VP2 genes directly from diseased fish demonstrated changes in the VP2 gene after two passages in cell culture, which could possibly be associated with attenuation. Comparison of the deduced amino acid sequences of the NS and VP2 genes reveals that the virulent strains possess a 12-kDa coding NS gene and have residues Thr, Ala, Thr/Ala, and Tyr/His at positions 217, 221, 247, and 500 of the VP2 gene, respectively-the motifs identified in this study to be involved in the virulence of IPNV.     

Identification of amino acids located in the antibody binding sites of human hepatitis A virus.

Antigenic mutants of human hepatitis A virus (human-HAV) were isolated by their resistance to neutralizing monoclonal antibodies raised to human-HAV. The nucleotide sequence determined for the capsid regions of 12 mutants identified amino acid changes that clustered in three non-overlapping sites; one in VP3 and two in VP1. All mutants had a change at amino acid residue 70 in VP3, indicating its primary importance for antibody binding. Ten mutants had two amino acid changes occurring in the VP3 site as well as one in one of the two VP1 sites. These data suggest that both sites in VP1 interact with the single VP3 site to form the immunodominant epitope of HAV. The amino acid changes found in the antigenic mutants of human-HAV selected in this study were located in the same positions as changes found in strains of HAV isolated from Old World monkeys. These simian strains of HAV are not recognized by most monoclonal antibodies raised to human-HAV, suggesting that the observed amino acid changes are part of the antibody binding site.     

五株隐孢子虫18S rRNA部分基因克隆和序列分析

设计Nested PCR引物扩增牛源微小隐孢子虫Cryptospordium parvum、羊源微小隐孢子虫C. parvum、牛源安氏隐孢子虫C. andersoni、鸡源贝氏隐孢子虫C. baileyi及猪源隐孢子虫C. suis 18S rRNA基因突变区,PCR产物经克隆测序,其片段大小分别为212、213、213、213和210 bp.将测得的序列用DNAStar软件分析并与NCBI数据库中相同与相近种株序列进行相似性比较, 进行相似性分析并用TREECON软件绘制系统发育进化树.结果表明测得的5株隐孢子虫与各自相同种相似性为98.1%～100%,与其他种相似性为90.1%～98.6%.分析显示在此突变区设置特异酶切位点能区分开C. parvum, C. andersoni, C. baileyi与C. suis,位点分别是TaqI、BstUI、MseI.本研究为我国隐孢子虫分类、分子流行病学研究提供了新的方法.     

Localization of the regions of homology between the genomes of herpes simplex virus, type 1, and pseudorabies virus

Only 8% of the sequences of the genomes of pseudorabies (PRV) and herpes simplex (type 1) (HSV) viruses are homologous. These homologous sequences have been shown previously to be distributed throughout most of the genomes of the two viruses. By means of blot hybridization of restriction fragments of HSV-1 DNA to cloned, nick-translated restriction fragments of PRV DNA, it was possible to compare the location on the genomes of these viruses of the homologous regions. The results showed that the genome of PRV is, for the most part, colinear with the IL arrangement of the genome of HSV-1. An inversion or translocation of sequences mapping on the PRV genome between 0.07 and 0.39 map units was observed on the genome of one of these viruses. A comparison of the map positions of five genes with known functions confirmed these findings. The genes coding for the major immediate-early protein, the major capsid protein, and the thymidine kinase occupy similar positions on the genome of PRV and on the genome of HSV-1 in the IL arrangement. However, the genes for DNA polymerase and for the major DNA binding protein appear to be inverted relative to one another on the genomes of the two viruses.     

甲型肝炎病毒龙甲株结构蛋白基因cDNA序列与国外毒…

将甲型肝炎病毒龙甲株（ＬＪ）结构蛋白基因（包括部分５端非编码基因ｎｔ６３０－３０４９）ｃＤＮＡ序徇与国外ＨＭ１７５、ＭＢＢ和ＬＡ三个母株相应区域相比较，结果显示，ＬＪ株结构蛋白基因与ＭＢＢ株的同源性最高，为９６．７％，与ＨＭ１７５和ＬＡ的同源率分别为９５．４％和９１．４％。推导的氨基酸变异率分别为０．９１％，０．９１％和２．９８％。ＬＪ与ＬＪ株氨基酸差异在整个结构蛋白区均有分布，而与ＨＭ１７５和Ｍ     

Molecular basis of the TaqI p49a,f polymorphism in the DYS1 locus containing DAZ genes

The previously described TaqI p49a,f polymorphism at the DYS1 locus in the non-recombinant part of the Y chromosome is widely exploited to investigate many facets of human population genetics. It has been shown that the DYS1 locus corresponds to the four DAZ genes in the AZF-c region of the Y chromosome. As the DNA sequence of the DAZ genes is known in its entirety, it is now possible to establish correspondences between the main Southern polymorphic TaqI bands (A, C, D, F and I) at the DYS1 locus and TaqI fragments deduced from the sequence, by way of comparing band sizes and sequence homologies between hybridized fragments. Transitions between polymorphic forms for each variable TaqI fragment can be explained regarding the restriction maps, by postulating a parsimonious number of TaqI site losses during human evolution. Most of the codon changes caused by TaqI site losses located in the exons of the four DAZ genes have potentially high selective values.     

传染性法氏囊病病毒HZ96VP2cDNA的结构分析及在大肠杆菌中的表达

以随机引物反转录传染性法氏囊病病毒（ＩＢＤＶ） 杭州分离株ＨＺ９６ 基因组合成第１ 链ｃＤＮＡ。以第１ 链ｃＤＮＡ为模板,ＰＣＲ 扩增ＨＺ９６ ＶＰ２ ｃＤＮＡ ;Ｓａｎｇｅｒ 双脱氧法测序ＨＺ９６ ＶＰ２ ｃＤＮＡ, 并对其基因结构氨基酸序列进行计算分析; 构建非融合表达质粒, 在大肠杆菌中表达ＶＰ２ 蛋白。结果表明, 克隆的ＨＺ９６ＶＰ２ ｃＤＮＡ 全长为１４３１ 个核苷酸对（ｂｐ） , 含起始密码子ＡＴＧ 和终止密码子ＴＡＡ, 编码ＶＰ２ 第２０ 至第４９５ 共４７６ 个氨基酸; ＨＺ９６ ＶＰ２ 在核苷酸和氨基酸水平上, 与其它Ｉ 型ＩＢＤＶ ＶＰ２ 的同性均在９０ ％ 以上; 对ＨＺ９６ 分离株与强毒株ＶＰ２ ｃＤＮＡ 编码的氨基酸序列进行二级结构和亲水性分析发现,ＨＺ９６ 分离株ＶＰ２ 第２７９ 和２８４ 位氨基酸的变异,可导致附近区域（２７４ ～２９０ 位氨基酸） 的亲水性降低和α－ 螺旋的消失。ＨＺ９６ ＶＰ２ 在大肠杆菌中的表达量为８ ％ 。     

Genetic characterization of JC virus Tokyo-1 strain, a variant oncogenic in rodents

The genome DNA of JC virus Tokyo-1 strain [JCV(Tokyo-1)], a variant oncogenic in rodents, was molecularly cloned directly from the brain of a Japanese patient with progressive multifocal leukoencephalopathy and from tissue culture, and the restriction enzyme cleavage pattern and regulatory sequences were determined. The restriction pattern of the cloned JCV(Tokyo-1) DNA was different from those of JCVs previously reported in the United States and Germany. Also, the arrangement of the regulatory sequence was unique to this strain. Thus JCV(Tokyo-1) can be classified as a new subtype. The relationship between the restriction pattern and the regulatory sequence of JCV(Tokyo-1), and its characteristic oncogenicity, is discussed.     

Genomic location of bovid herpesvirus type 2 nucleotide sequences homologous to five herpes simplex virus type 1 genes

The location of nucleotide sequences within the bovid herpesvirus 1 (BHV-2) genome homologous to herpes simplex virus 1 (HSV-1) DNA were investigated. BHV-2 DNA was digested with restriction endonucleases and blotted to nitrocellulose paper. The blots were then probed with plasmids containing HSV-1 genes for thymidine kinase (TK), the major DNA binding protein (ICP8), the major capsid protein (VP5) and genes for HSV-1 glycoproteins gB, gD, and gC. Except for HSV-1 gC, each HSV-1 gene tested hybridized to BHV-2 nucleotide sequences that were located either on both sides of a restriction endonuclease cleavage site, within a small restriction endonuclease fragment, or to an area common to two overlapping restriction fragments. Thus, we were able to localize BHV-2 nucleotide sequences homologous to the HSV-1 ICP8 gene between 0.38 and 0.41 map units (m.u.), and BHV-2 nucleotide sequences homologous to the HSV-1 VP5 gene between 0.24 and 0.27 m.u. In addition, BHV-2 nucleotide sequences homologous to HSV-1 genes for TK, gB and gD were found to lie on both sides of restriction endonuclease cleavage sites at 0.30 0.35, and 0.94 m.u., respectively.     

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.     

Genetic heterogeneity of the human papovaviruses BK and JC

We have examined the structure and infectivity of BKV and JCV genomes from prototype strains after cell culture passage and of BKV genomes from primary isolates. Genomic structures were determined by restriction endonuclease analysis of molecularly cloned DNA. Infectivity was determined by transfection of the cloned genomes into urine-derived epithelial cells and assaying for viral proteins and virus production. Prototype BKV DNA, which was cloned after 14 passages in three different cell lines, contained no alterations in restriction enzyme sites and was infectious. In contrast, prototype JCV acquired changes in the late region of the genome during passage in cell culture and the cloned DNA was not infectious. Urine-derived cells were used to isolate virus from the urine of two renal transplant patients and one asymptomatic individual. The genome of the virus isolated from the normal individual was indistinguishable from prototype BKV except for a 60-base pair deletion, which was localized between 0.62 and 0.72 map units. Two isolates from transplant patients differed from each other and from prototype BKV at a number of restriction enzyme cleavage sites located in the early region and were infectious. Genomes containing deletions from 100 to 600 base pairs were also cloned but were not infectious.     

Genetic analysis of HAV strains in Tunisia reveals two new antigenic variants

To evaluate the genetic variability of hepatitis A virus (HAV) isolates in Tunisia, serum samples were collected from 99 patients in different Tunisian areas in 2003 containing 92 cases with acute hepatitis, five with severe acute hepatitis and two with fulminant hepatitis. The entire VP1 gene was amplified and sequenced. Sequences were then aligned and a phylogenetic analysis was performed. Additionally, the amino acid (aa) sequence of the VP1 was determined. The analysis of Tunisian HAV isolates revealed that all the isolates were sub-genotype IA with 96.4%–99.8% of identity and showed the emergence of two novel antigenic variants. The Tun31-03 antigenic variant, with a 38 aa deletion containing Met156, Val171, Leu174 and Ala176 and located between 150 and 187 aa of the VP1 protein where neutralization escape mutations, was found. The second antigenic variant, Tun36-03, was isolated from a patient with fulminant hepatitis and presented a substitution of Thr by Pro at position 10 of the VP1 protein. This amino acid is located in a peptide presenting an antigenically reactive epitope of the VP1 protein. This substitution has never been described previously.     

The genome of goose hemorrhagic polyomavirus,a new member of the proposed subgenus Avipolyomavirus

The full-length genome of goose hemorrhagic polyomavirus (GHPV), the ethiologic agent of hemorrhagic nephritis and enteritis of geese, was cloned and sequenced. Transfection of the circular ds DNA with a size of 5256 bp and an organisation typical of polyomaviruses produced viral progeny in cultured goose cells. According to the splicing sites determined by RT-PCR, five open reading frames (ORFs) were found to encode putative proteins with significant similarities to large T antigen and small t antigen as well as VP1, VP2, and VP3 of other polyomaviruses. An additional ORF located in the 5' region of late mRNA, with a coding capacity for 169 amino acids, shows a low degree of homology to VP4 of avian polyomavirus (APV). The alignment of nucleotide sequences and amino acid sequences revealed a relatively close relationship between GHPV and APV. Therefore, grouping of this new polyomavirus into the proposed subgenus Avipolyomavirus is suggested.