Molecular analysis of simian varicella virus DNA.

Clinical and pathological studies indicate that simian varicella virus (SVV) infection in primates is the counterpart of human varicella zoster virus (VZV) infection. The SVV and VZV genomes are also similar in size and structure. To extend studies of SVV DNA, we analyzed virus DNA from African green monkey kidney cells infected with the Delta-herpes-virus strain of SVV. The infectivity of SVV DNA was 88 PFU/micrograms. The buoyant density of SVV DNA, determined by isopycnic banding in CsCl gradients, was 1.700 +/- 0.002 g/ml, corresponding to a G + C molar ratio of 40.8%. The size of SVV DNA, estimated by analysis of restriction endonuclease digestion products and pulsed-field gel electrophoresis was 125.1 and 124.9 kbp, respectively. Electron microscopy of SVV DNA revealed a long region of 110.0 kbp, a unique short (Us) region of 5.1 kbp, and inverted repeat regions of 7.5 kbp flanking the Us. An EcoRI map of SVV DNA revealed two fragments not previously reported; our complete Pstl map also shows some differences. Mapping of SVV DNA with an additional restriction enzyme, measurement of full-length SVV DNA molecules, and the first use of pulsed-field electrophoresis to size SVV DNA, confirm and extend Gray's recent finding that SVV DNA has the same size and molecular configuration as VZV. We also show for the first time that the density of SVV DNA is similar to that of VZV DNA and that SVV DNA is infectious.     

The genomes of simian varicella virus and varicella zoster virus are colinear.

Simian varicella virus (SVV) causes an exanthematous disease in non-human primates which is clinically similar to varicella zoster virus (VZV) infection of humans. In this study, the genetic relatedness of SVV and VZV was confirmed and the location of SVV DNA sequences homologous to VZV restriction endonuclease (RE) fragments and viral genes was determined. VZV DNA RE fragments representing 98.3% of the VZV genome were 32P-labeled and hybridized to RE digested, immobilized SVV DNA. Homologous sequences were located throughout the viral DNAs in similar map positions, indicating a colinear relationship between the VZV and SVV genomes. 32P-labeled VZV glycoprotein (gp I, II, III, and IV) and gene 62 DNA probes also hybridized to SVV DNA in a colinear manner. The results suggest that the location of specific SVV genes may be predicted from the known map positions of homologous VZV genes. This study provides further support for SVV infection of non-human primates as a model for VZV infection of humans.     

Expression of the simian varicella virus glycoprotein E

Simian varicella virus (SVV) is closely related to human varicella-zoster virus (VZV) and induces a varicella-like disease in nonhuman primates. The SVV genome encodes a glycoprotein E (gE) which is homologous to the gE of VZV and other alphaherpesviruses. The SVV gE was expressed in Escherichia coli and rabbits were immunized with the recombinant gE fusion proteins to generate polyclonal gE antiserum. Immunofluorescence and immunoprecipitation analyses demonstrated that the SVV gE is expressed on the surface and within SVV-infected cells. The gE is also expressed on SVV virions as indicated by serum neutralization assay. The mature SVV gE is glycosylated and is similar in size ( approximately 100 kd) to the mature VZV gE. Immunohistochemical analysis detected gE within skin vesicles and lung tissue of SVV-infected monkeys. Analysis of the humoral immune response to gE in an SVV-infected monkey determined that anti-gE antibody is induced as early as day 9 postinfection and persists at high titer for longer than 4 months. The simian varicella model offers an opportunity to investigate the role of gE in viral pathogenesis and immunity and to evaluate its potential as a varicella vaccine.     

A cosmid-based system for inserting mutations and foreign genes into the simian varicella virus genome

Simian varicella is a natural varicella-like disease of nonhuman primates. The etiologic agent, simian varicella virus (SVV), is genetically related to varicella-zoster virus (VZV) and SVV infection of nonhuman primates is a useful model to investigate VZV pathogenesis and latency. In this study, we report development of a cosmid-based genetic system to generate SVV mutant viruses. SVV subgenomic DNA fragments (32–38 kb) that span the viral genome were cloned into cosmid vectors. Co-transfection of Vero cells with four overlapping cosmid clones representing the entire SVV genome resulted in recombination and generation of infectious virus. SVV mutants were produced by manipulation of one cosmid and substitution into the genetic system. This genetic approach was used to insert a site-specific mutation within the SVV open reading frame 14 which encodes the nonessential glycoprotein C gene. In a subsequent experiment, the green fluorescent protein (GFP) gene was inserted into the SVV genome within ORF 14. These SVV mutants replicate as efficiently as wild-type SVV in cell culture. This cosmid-based genetic system will be useful to investigate the effect of viral mutations on SVV pathogenesis and latency and also to develop and evaluate recombinant varicella vaccines that express foreign antigens.     

The white spot syndrome virus DNA genome sequence.

White spot syndrome virus (WSSV) is at present a major scourge to worldwide shrimp cultivation. We have determined the entire sequence of the double-stranded, circular DNA genome of WSSV, which contains 292,967 nucleotides encompassing 184 major open reading frames (ORFs). Only 6% of the WSSV ORFs have putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication, and protein modification. The remaining ORFs are mostly unassigned, except for five, which encode structural virion proteins. Unique features of WSSV are the presence of a very long ORF of 18,234 nucleotides, with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. The collective information on WSSV and the phylogenetic analysis on the viral DNA polymerase suggest that WSSV differs profoundly from all presently known viruses and that it is a representative of a new virus family.     

The simian varicella virus and varicella zoster virus genomes are similar in size and structure.

Simian varicella virus (SVV) DNA was purified from viral nucleocapsids and the molecular structure of the SVV genome was determined. SVV DNA was analyzed by agarose gel electrophoresis of BamHI, BglII, EcoRI, and PstI restriction endonuclease digests. SVV and varicella zoster virus (VZV) DNAs were demonstrated to have distinct restriction endonuclease profiles. Summation of the sizes of individual restriction endonuclease fragments indicate the size of SVV DNA is congruent to 121 kilobase pairs (kbp) or congruent to 76.8 megadaltons (Md). Electron microscopy, lambda exonuclease analysis, and Southern blot DNA hybridizations were utilized to determine the molecular structure of the SVV genome and to construct restriction endonuclease maps. The results indicate that SVV DNA consists of a long component (L, congruent to 100 kbp) covalently linked to a short component (S, congruent to 20 kbp) which is composed of a unique short sequence (Us, 5.3 +/- 0.7 kbp) bracketed by inverted repeat sequences (TRs and IRs, congruent to 7.2 kbp). The presence of 0.5 M PstI restriction endonuclease fragments indicates that the S component may invert relative to the L component and that the genome exists in two major isomeric forms. The findings demonstrate that the SVV and VZV genomes are similar in size and structure.     

Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome

Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid, and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectious SVV (rSVV-BAC). The mini-F vector sequences flanked by loxP sites were removed by co-infection of Vero cells with rSVV-BAC and adenovirus expressing Cre-recombinase. Recombinant SVV generated using the SVV-BAC genetic system has similar molecular and in vitro replication properties as wild-type SVV. To demonstrate the utility of this approach, a SVV ORF 10 deletion mutant was created using two-step Red-mediated recombination. The results indicate that SVV ORF 10, which encodes a homolog of the HSV-1 virion VP-16 transactivator protein, is not essential for in vitro replication but is required for optimal replication in cell culture.     

Prevalence and distribution of latent simian varicella virus DNA in monkey ganglia.

We used polymerase chain reaction to analyze the prevalence and distribution of latent simian varicella virus (SVV) in ganglionic and nonganglionic tissues from nine African green monkeys experimentally infected with SVV. Primers specific for three different regions of the SVV genome were used for amplification. SVV DNA sequences were detected in trigeminal ganglia from seven of nine monkeys and in thoracic ganglia from seven of nine monkeys. Analysis of DNA from nonneuronal tissues of three monkeys and from adrenal glands of nine monkeys revealed the presence of SVV-specific sequences in the adrenal gland of one monkey. The results indicate that, like human varicella, SVV becomes latent primarily in ganglia at multiple levels of the neuraxis, and more than one region of the SVV genome is present in latently infected ganglia. SVV latency in primates may be a useful model for varicella latency in humans.     

基孔肯雅病毒深圳分离株的全序列分析

目的了解基孔肯雅病毒SZ_20101028株的全基因组分子遗传特征。方法通过C6／36细胞从患者血清中分离得到CHIKV,针对病毒的基因组设计了6条特异性引物,对病毒的全基因组进行扩增,测序并通过生物信息学软件分析该病毒株的全基因组分子遗传特征。结果病毒SZ_20101028株的全基因组核苷酸序列长度为12377nt,含两个开放阅读框。编码3722个氨基酸,两个编码区之间含有68nt非编码连接区;发现该毒株与2010年引起东莞基孔肯雅热疫情暴发的病原体核苷酸的同源性达到了99％,进化树分析结果表明,SZ_20101028株与2010年引起东莞疫情暴发的病原体的亲缘性最高,并且和2004年引起印度洋地区疫情暴发流行的病原体同属于印度洋亚型。结论深圳市首例输人性基孔肯雅病毒属于印度洋亚型,与2010年引起东莞疫情暴发的病原体的亲缘性最高。     

Improved yields and assay of simian varicella virus, and a comparison of certain biological properties of simian and human varicella viruses

Studies were performed to define conditions under which propagation, assay and stabilization of the Delta herpesvirus (DHV) strain of simian varicella virus might be improved, and to compare biological properties of DHV with those of human varicella zoster virus (VZV). A mycoplasma contaminant was successfully eliminated from the DHV seed virus by treatment with a specific anti-serum. DHV was found to replicate more efficiently in the BS-C-1 line of African green monkey kidney cells than in Vero cells, and seed virus preparations in the form of virus-infected cells were produced which had infectivity titers greater than or equal to 1 X 10(6) p.f.u./ml. Greater yields of virus were produced in cultures infected as dispersed cells than as preformed monolayers. Infectious DHV could be released from host cells by sonic treatment of heavily infected cultures at 48 h post infection. Certain agents reported to enhance replication of herpes viruses (caffeine, carbaryl, the tumor promoter 12-0-tetra-decanoyl-phorbol-13-acetate, and DEAE-dextran) had no enhancing effect on replication of DHV. However, DEAE-dextran in the maintenance medium enhanced spontaneous release of DHV into culture fluids. Plaquing efficiency and plaque size of DHV were greater in BS-C-1 than in Vero cells, and plaque assays and plaque reduction neutralization tests were developed in this cell system using a solid overlay medium with neutral red vital stain. Neutralization of DHV was markedly enhanced by fresh guinea pig complement. The newly developed neutralization test demonstrated more vigorous antibody responses to DHV in active and latent VZV infections than were demonstrated with previous procedures. In addition to their preferential growth in monkey and human cells respectively, DHV and VZV were found to differ markedly in their rates of attachment to host cells, with DHV requiring over 6 h of adsorption, while VZV adsorption was essentially complete at 1 h. Also, cell-free DHV was much more resistant than cell-free VZV to repeated cycles of freezing and thawing.     

The complete genome sequence and genome structure of frangipani mosaic virus

In this study, the complete sequence of the genomic RNA of frangipani mosaic virus (FrMV) has been determined and compared to those of other known tobamoviruses. The complete genome sequence of FrMV consisted of 6,643 nucleotides. The FrMV genomic RNA encoded four open reading frames (ORFs), for proteins of M(r) 128 kDa (1,147 aa), 186 kDa (1,651 aa), 30 kDa (257 aa) and 18 kDa (175 aa) from the 5′ to the 3′ end. Overall similarities for the four ORFs of FrMV-P ranged from 26.8 to 53.0% at the amino acid level when compared to those of 24 other tobamoviruses. Phylogenetic analysis of the FrMV replicase (186 kDa) and MP revealed that FrMV is closely related to SHMV and CMMoV, while the FrMV replicase (128 kDa) is more closely related to cucurbit-infecting and malvaceous-infecting tobamoviruses, and the FrMV CP is closely related to that of CMMoV and solanaceous-infecting tobamoviruses.     

The complete genome sequence of polygonum ringspot virus

The complete genome sequence of polygonum ringspot virus (PolRSV), genus Tospovirus, family Bunyaviridae, was determined. This is the first report of the complete genome sequence for a European tospovirus isolate. The large RNA of PolRSV was 8893 nucleotides (nt) in size and contained a single open reading frame of 8628 nucleotides in the viral-complementary sense, coding for a predicted RNA-dependent RNA polymerase of 330.9 kDa. Two untranslated regions of 230 and 32 nucleotides were present at the 5′ and 3′ termini, respectively, which showed conserved terminal sequences, as commonly observed for tospovirus genomic RNAs. The medium and small (S) RNAs were 4710 and 2485 nucleotides in size, respectively, and showed 99 % homology to the corresponding genomic segment of a previously partially characterized PolRSV isolate, Plg3. Protein sequences for G_N/G_C, N and NSs were identical in length in the two PolRSV isolates, while an amino acid insertion was observed for the NSm protein of the newly characterized isolate. The noncoding intergenic region of the S RNA was very short (183 nt) and was not predicted to form a hairpin structure, confirming that this unique characteristic within tospoviruses, previously observed for Plg3, is not isolate specific.     

Completion of the Norwalk virus genome sequence

Norwalk virus (NV) is the prototype human calicivirus, and causes epidemic outbreaks of acute gastroenteritis. The sequence and predicted genome organization of NV and a NV-like virus [Southampton virus (SHV)] suggested they are similar viruses at the nucleotide and amino acid level, although SHV was reported to be antigenically distinct from NV. A recent review described the discovery of an additional 12 nucleotides at the 5 end of SHV and prompted us to investigate the possibility of additional nucleotides at the 5 end of the NV genome. The results obtained by homopolymeric tailing of NV cDNA with dCTP and dATP showed 12 additional nucleotides also are present on the NV genomic RNA. These data are important with respect to the biology of the virus, and make the genome sequence of NV complete.     

Simian varicella virus DNA shares homology with human varicella-zoster virus DNA

Delta herpesvirus (DHV) and Medical Lake Macaque (MLM) virus are cell-associated simian herpesviruses that cause varicella-like disease in nonhuman primates, and are antigenically related to human varicella-zoster virus (VZV). The results of studies designed to determine if homology exists between the DNA of DHV and MLM and the DNA of VZV are reported here. Southern blot hybridizations conducted at Tm-20 degrees did not detect DNA homology between the VZV and simian varicella virus genomes. However, under conditions of lower stringency (Tm-36 degrees and Tm-43 degrees), VZV DNA probes hybridized to specific HindIII fragments within DNA isolated from simian varicella virus-infected cells. Under similar hybridization conditions, DNA homology was not detected between VZV DNA and herpes simplex virus DNA. Further studies using cloned VZV DNA HindIII fragments as probes suggested that the homology between VZV DNA and DHV DNA is distributed across the viral genomes. These results demonstrate that the genomes of VZV and simian varicella virus share regions of conserved nucleotide sequences, and indicate a close evolutionary relationship between VZV and simian varicella viruses. In addition, the studies show that the DHV and MLM strains of simian varicella virus are more closely related to each other than to human VZV.