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.     

Attachment of coxsackievirus B3 variants to various cell lines: mapping of phenotypic differences to capsid protein VP1

The coxsackievirus B3 (CVB3) strain Nancy P establishes a persistent carrier-state infection without visible cytopathic effect in primary human fibroblasts (HuFi H), whereas the derivative variant PD induces a complete lysis of the cell monolayer. To define the molecular basis of this exceptional growth property, the complete genomes of both viruses were sequenced and compared to all published sequences of CVB3. As a result, six unique amino acid substitutions in the VP1 capsid protein were observed. Via hybrid virus construction, the lytic phenotype was transferred to a nonlytic cDNA-generated CVB3. Mapping experiments indicate that the presence of amino acid residues K78, A80, A91, and I92 in VP1 is sufficient to induce "lytic" infections in HuFi H cells. Binding assays demonstrate that CVB3 Nancy P preferentially binds to the human coxsackievirus-adenovirus receptor (CAR), while PD exhibits a very weak interaction with CAR but strong binding to the decay accelerating factor (DAF). These results suggest that the mutated amino acid residues in VP1 are involved in receptor recognition/binding. Moreover, the lytic replication of CVB3 PD and the hybrid virus in various nonpermissive rodent cell lines indicates that cell surface molecules other than CAR and DAF may be involved in attachment of this variant to cell surfaces.     

Mengo virus maturation is accompanied by C-terminal modification of capsid protein VP1

We have demonstrated that the C-terminal ends of the VP1 proteins of Mengo virus undergo a postassembly trimming of three amino acid residues. A variable proportion of the VP1 molecules isolated from purified virions terminate at Glu 277, which corresponds to the initial viral protease 3C cleavage site. The remainder of the VP1 molecules terminates at Leu 274. When chymotrypsin treatment is included in the virus purification procedure, Leu 274 is the exclusive C-terminal amino acid of the VP1 molecules. The trimming process was found to influence the pH-mediated dissociability of virions in vitro: this was demonstrated by sucrose gradient analysis and electron microscopy. Since long-term incubation of purified virions did not alter the C-terminal Glu 277/Leu 274 ratio, it appears that the trimming process is not autocatalytic.     

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS–PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.     

Genomic and receptor attachment differences between mengovirus and encephalomyocarditis virus

Antigenically, encephalomyocarditis (EMC) virus and Mengovirus cannot be distinguished by hyperimmune sera. Biologically, however, these two viruses are quite different. The D clone of EMC virus produces diabetes, but not encephalitis, whereas our 2T clone of Mengovirus produces a rapidly lethal encephalitis. Moreover, by thermal elution analysis of EMC virus and Mengovirus cDNA-RNA hybrids there was an estimated 20% difference in the nucleotide sequences of these two viruses. Receptor attachment studies revealed that the rate of binding of the neurovirulent Mengovirus was 5 to 10 times greater for neuronal cell lines than was the nonneurovirulent EMC virus. Receptor saturation experiments showed that unlabeled Mengo and EMC viruses effectively blocked the binding of labeled homologous, but not heterologous, virus. It is concluded from these and other studies that Mengo and EMC viruses are related, but distinct viruses that bind to different receptors on the cell surface.     

Methylation of the polyomavirus major capsid protein VP1

Polyomavirus VP1 has been shown to be modified by phosphorylation, sulfation, acetylation and hydroxylation. The major capsid protein VP1 is now shown to be modified by methylation. Addition of cycloheximide to infected cultures followed by addition of [³H-methyl]- -methionine and subsequent immunoprecipitation, SDS-PAGE and fluorography revealed methylation occurring on VP1. Amino acid analysis of [³H]-methyl]- -methionine-labelled polyomavirus VP1 by two-dimensional paper chromatography and HPLC of the acid-hydrolyzed protein revealed the presence of ³H-labelled trimethyllysine and monomethylarginine.     

Human anti-JC virus serum reacts with native but not denatured JC virus major capsid protein VP1

The immunoreactivity of human anti-JC virus (JCV) serum against the major capsid protein VP1 of JCV was analyzed by Western blot, dot blot, and hemagglutination inhibition (HAI) assays. JCV-positive human serum reacted with native but not denatured JCV major capsid protein VP1, as demonstrated by dot blot and Western blot. Rabbit antiserum raised against native JCV capsid had immunoreactivities similar to those of human anti-JCV serum. These results indicate that the antigenecity of native and denatured JCV VP1 is different. In addition, both JCV-positive human serum and rabbit antiserum raised against native JCV capsid protein inhibited the hemagglutination activity of JCV capsid particles. In contrast, rabbit antiserum raised against denatured JCV VP1 did not inhibit hemagglutination. These findings reveal that denaturation may alter the antigenic epitopes of JCV VP1. Therefore, keeping the JCV capsid protein native appears to be essential for serological or other immunological analyses of the virus.     

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.     

CD1d-reactive T-cell activation leads to amelioration of disease caused by diabetogenic encephalomyocarditis virus

A subset of CD161 (NK1) T cells express an invariant Valpha14Jalpha281 TCR-alpha chain (Valpha(invt) T cells) and produce Th2 and Th1 cytokines rapidly in response to CD1d, but their physiological function(s) remain unclear. We have found that CD1d-reactive T cells mediate to resistance against the acute, cytopathic virus diabetogenic encephalomyocarditis virus (EMCV-D) in relatively Th1-biased, C57BL/6-based backgrounds. We show now that these results generalize to Th2-biased, hypersensitive BALB/c mice. CD1d-KO BALB/c mice were more susceptible to EMCV-D. Furthermore, alpha-galactosylceramide (alpha-GalCer), a CD1d-presented lipid antigen that specifically activates Valpha(invt) T cells, protected wild-type (WT) mice against EMCV-D-induced encephalitis, myocarditis, and diabetes. In contrast, neither CD1d-KO nor Jalpha281-KO mice were protected by alpha-GALCER: Finally, disease in Jalpha281-KO mice was comparable to WT, indicating for the first time equivalent roles for CD1d-reactive Valpha(invt) and noninvariant T cells in resistance to acute viral infection. A model for how CD1d-reactive T cells can initiate immune responses, which synthesizes current results, is presented.     

Multiplex PCR for rapid detection of serotype A foot-and-mouth disease virus variants with amino acid deletion at position 59 of the capsid protein VP3

In India, there has been co-circulation, extinction and emergence of genotypes/lineages within serotype A foot-and-mouth disease (FMD) virus. At present an antigenically heterogeneous, unique lineage within genotype VII dominates the field outbreaks. This genetic cluster has amino acid deletion at position 59 of VP3 (VP3⁵⁹-deletion group), considered to be critical antigenically. The emergence of this group warrants rapid and accurate detection to facilitate early planning and implementation of an effective control policy. A rapid multiplex PCR assay was developed for detection of the dominating VP3⁵⁹-deletion group with 100% sensitivity and specificity, even before generating sequence data and confirmatory phylogenetic analysis. This development is important for surveillance of FMD in India.     

A correlation between capsid protein VP2 and the plaque morphology of African horse sickness virus in cell culture

The attenuated live virus vaccine that is used in South Africa to protect against African horse sickness infection was developed more than 50 years ago. With the selection of the vaccine strains by cell culture passage, a correlation between the size of plaques formed in monolayer Vero cultures and attenuation of virus virulence in horses was found. The large plaque phenotype was used as an indication of cell culture adaptation and strongly correlated with attenuation of virulence in horses. There was never any investigation into the genetic causes of either the variation in plaque size, or the loss of virulence. An understanding of the underlying mechanisms of attenuation would benefit the production of a safer AHSV vaccine. To this end, the genomes of different strains of two African horse sickness isolates, producing varying plaque sizes, were compared and the differences between them identified. This comparison suggested that proteins VP2, VP3, VP5 and NS3 were most likely involved in the determination of the plaque phenotype. Comparison between genome sequences (obtained from GenBank) of low and high passage strains from two additional serotypes indicated that VP2 was the only protein with amino acid substitutions in all four serotypes. The amino acid substitutions all occurred within the same hydrophilic area, resulting in increased hydrophilicity of VP2 in the large plaque strains.     

Amino acid sequence analysis of bovine rotavirus B223 reveals a unique outer capsid protein VP4 and confirms a third bovine VP4 type.

The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.     

Identification and production of monoclonal antibodies against a discriminating protein molecule between B and D variants of encephalomyocarditis virus

The protein profiles of two EMC virus variants B and D in the infected L929 fibroblast lysates were examined using one- and two-dimensional gel electrophoresis. Both variants yielded a protein molecule of similar molecular weight but differing in its isoelectric point (pI). The B variant lysate yielded a molecule with pI congruent to 7.5 whereas the same molecule from the D lysate focused at pI congruent to 5.2. A monoclonal antibody (MCA) produced against pI congruent to 5.2 fraction of the D variant successfully detected viral antigens in the D variant infected fibroblasts with only background cross reactivity with the B variant infected cells. This MCA also detected D viral antigen(s) in organ sections obtained from D but not from B variant infected mice. This study therefore suggests a clear shift in the pI of a 23 kD protein molecule serving as a possible discriminating antigen between the B and D variants of EMC virus.     

中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析

目的 分析中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点.方法 收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构-非结构蛋白VP3-VP1-2A区序列,进行序列同源性比较并分析其基因特点.结果 42株HAV病毒株在VP1-2A连接处核苷酸和氨基酸序列同源性分别为89.1%～100%和97.3%～100%;在全长结构蛋白VP3-VP1区的核苷酸和氨基酸序列同源性分别为87.6%～100%和98.8%～100%.VP1-2A连接处序列相同的病毒株在全长结构蛋白VP3-VP1区的核苷酸同源性为98.4%～100%,0～2个氨基酸位点不同.本实验所得序列在中和抗原位点处氨基酸序列均未变异.结论 42株病毒株均属于I型,40株是IA亚型,2株IB亚型.本实验所用HAV流行株在结构蛋白VP3-VP1区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异.VP1-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VP1区核苷酸序列相同或相近,氨基酸序列保守.     

中国部分甲肝病毒流行株结构蛋白VP3-VPl区基因特点分析

目的分析中国部分甲肝病毒流行株结构蛋白VP3-VPl区基因特点。方法收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构一非结构蛋白VP3-VPl-2A区序列,进行序列同源性比较并分析其基因特点。结果42株HAV病毒株在VPl-2A连接处核苷酸和氨基酸序列同源性分别为89．1％～100％和97．3％～100％;在全长结构蛋白VP3-VPl区的核苷酸和氨基酸序列同源性分别为87．6％～100％和98．8％～100％。VPl-2A连接处序列相同的病毒株在全长结构蛋白VP3．VPl区的核苷酸同源性为98．4％～100％,0～2个氨基酸位点不同。本实验所得序列在中和抗原位点处氨基酸序列均未变异。结论42株病毒株均属于I型,40株是IA亚型,2株IB亚型。本实验所用HAV流行株在结构蛋白VP3．VPl区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异。VPl-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VPl区核苷酸序列相同或相近,氨基酸序列保守。     

中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点分析

目的 分析中国部分甲肝病毒流行株结构蛋白VP3-VP1区基因特点.方法 收集42份甲肝患者急性期血清标本,经核酸提取、逆转录及巢氏PCR,测得结构-非结构蛋白VP3-VP1-2A区序列,进行序列同源性比较并分析其基因特点.结果 42株HAV病毒株在VP1-2A连接处核苷酸和氨基酸序列同源性分别为89.1%～100%和97.3%～100%;在全长结构蛋白VP3-VP1区的核苷酸和氨基酸序列同源性分别为87.6%～100%和98.8%～100%.VP1-2A连接处序列相同的病毒株在全长结构蛋白VP3-VP1区的核苷酸同源性为98.4%～100%,0～2个氨基酸位点不同.本实验所得序列在中和抗原位点处氨基酸序列均未变异.结论 42株病毒株均属于I型,40株是IA亚型,2株IB亚型.本实验所用HAV流行株在结构蛋白VP3-VP1区的核苷酸存在差异但是氨基酸序列高度保守且没有中和抗原位点处的变异.VP1-2A结合处核苷酸序列相同的分离株在全长结构蛋白VP3-VP1区核苷酸序列相同或相近,氨基酸序列保守.