Archives of Virology

Erles, K.; Brownlie, J.

doi: 10.1007/s00705-005-0533-xpmid: 15841339

Summary.Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.

Wang, D.; An, S.-H.; Guo, Z.-J.; Xu, H.-J.; Zhang, C.-X.

doi: 10.1007/s00705-005-0534-9pmid: 15834653

Homologues of Helicoverpa armigera nucleopolyhedrovirus (HearSNPV) orf33 are found in all 22 completely sequenced members of the lepidopteran nucleopolyhedroviruses and granuloviruses, but so far their functions are unknown. In this report, we describe the characterization of HearSNPV orf33 ( ha33 ). Northern blot analysis showed a single transcript of ha33 of approximately 0.7 kb was transcribed beginning at 3 h post-infection in infected Helicoverpa zea cells (HzAM1) and the gene product could be detected as early as 6 h post-infection by western blot analysis using a rabbit derived polyclonal antibody, suggesting it was an early gene. Western blot analysis also demonstrated the ha33 protein in infected cells was a 31 kDa protein, larger than the theoretical size of 28.4 kDa, and located in the envelope fraction of budded virions (BVs). The results suggested that HearSNPV ha33 gene is a functional gene that encodes a novel structural protein of baculovirus BVs, BV-e31.

Takahashi, M.; Ido, E.; Uesaka, H.; Fukushima, T.; Ibuki, K.; Miura, T.; Hayami, M.; Takahashi, H.

doi: 10.1007/s00705-005-0536-7pmid: 15841338

CD4-bearing T cells are the primary targets for human immunodeficiency virus type 1(HIV-1)/simian immunodeficiency virus (SIV) infection. However, it is unclear whether the susceptibility of CD4-bearing T cells including CD4 single positive and CD4/8 double positive T cells to HIV/SIV infection is the same or not. In this study, we compared the susceptibility to SIV infection between CD4 + and CD4 + 8 + T cells, using Herpesvirus saimiri (HVS)-transformed CD4 + and CD4 + 8 + T cells established from peripheral blood mononuclear cells (PBMC) of rhesus macaques. Although there was little difference between the two CD4-bearing T cell population in the expression level of CD4 molecules and chemokine receptors such as CXCR4 and CCR5, SIV replicated more efficiently in CD4 + 8 + T cells than in CD4 + T cells. Moreover, we found that reverse transcription initiated more efficiently in CD4 + 8 + T cells than in CD4 + T cells and that the cell lysates from CD4 + T cells impaired the RT activity more strongly than that from CD4 + 8 + T cells. These findings suggest that intracellular environment in CD4 + 8 + T cells is better for reverse transcription and that the infection of those CD4 + 8 + T cells might play critical and different roles in HIV-1/SIV infection and dissemination.

St-Louis, M.-C.; Abed, Y.; Archambault, D.

doi: 10.1007/s00705-005-0522-0pmid: 15821973

Previous studies have shown that BIV may encode two types of Tat proteins of 103 and 108 amino acids, respectively. Here, we report the characterization of a new BIV Tat protein (Tat236) derived from a tat/rev cDNA. The tat/rev cDNA was obtained by reverse transcription-PCR from RNA extracted from cells infected with BIV. BIV was rescued by cell co-cultivation from the spleen of rabbits exposed for 3 years to the R29 isolate of BIV. Sequence analysis indicated that BIV Tat236 contains the first 98 amino acids of Tat103 and the 3′ end 138 amino acids of Rev. Reporter gene assays indicated that transactivation of BIV long terminal repeat (LTR) by Tat236 is higher than by the original BIV Tat proteins in several cell types. By using overlapping deletion mutants, evidence was given that the predicted basic domain of Rev within Tat236 plays a major role in the observed enhanced transactivation activity of the protein. However, the intact functional domain of the original BIV Tat is required for efficient transactivation. This is the first report of a hybrid Tat protein from BIV or any lentiviruses that shows higher transactivation than the original transactivator Tat proteins.

Carpes, M. Padilha; de Castro, M. E. Batista; Soares, E. Filgueiras; Villela, A. Guimarães; Pinedo, F. J. Rivera; Ribeiro, B. Morais

doi: 10.1007/s00705-005-0529-6pmid: 15834654

Programmed cell death or apoptosis is one of the defense mechanisms used by insect cells in response to baculovirus infection. Baculoviruses harbour antiapoptotic genes to prevent apoptosis and to maintain the normal course of infection. In this work, we showed that, like other baculoviruses, Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) has a functional inhibitor of apoptosis gene ( iap-3 ). The iap-3 gene was cloned, sequenced and its transcription confirmed by RT-PCR. The putative iap-3 gene of the baculovirus AgMNPV has 864 nucleotides and codes an ORF of 287 amino acids. We have found two BIR motifs (baculoviral iap repeats) at the amino-terminal region and a carboxi-terminal RING finger motif. The IAP-3 protein of AgMNPV is closely related to IAP-3 proteins of baculoviruses and lepidopteran IAPs, with most amino acid identity (75%) with the IAP-3 protein of C fDef NPV ( Choristoneura fumiferana DEF nucleopolyhedrovirus). Transcriptional analysis of the AgMNPV iap-3 gene showed that iap-3 -specific transcripts could be detected early and late in the infection. The iap-3 gene of AgMNPV was shown to encode a functional IAP since insect cells transfected with increasing amounts of a plasmid containing the iap-3 of AgMNPV showed increased resistance to apoptosis induced by a AgMNPV mutant virus.

Crouch, E. A.; Passarelli, A. L.

doi: 10.1007/s00705-005-0527-8pmid: 15868097

Baculovirus infection of permissive cells proceeds in a cascade fashion with the transcription of early, late and very late genes. The structure of a number of baculovirus early gene promoters has been dissected in detail and the viral factors necessary to stimulate their expression have been identified. Early baculovirus gene promoters in general have a resemblance to host promoters while late and very late gene promoters are different from early baculovirus promoters and are more defined. In this study we investigated whether two key Autographa californica M nucleopolyhedrovirus (Ac M NPV) transactivators have the ability to regulate the commonly used cellular promoter from the Drosophila heat shock 70 protein gene, during transient gene expression assays in two insect cell lines permissive for Ac M NPV infection, SF-21 and TN-368, or during viral infection. The Ac M NPV ie-1 transactivator gene stimulated gene expression of this cellular promoter in both cell lines when the promoter was cis -linked to an enhancer element, but stimulation in the absence of enhancer elements was either undetected or lower than in the presence of enhancer elements in SF-21 and TN-368 cells, respectively. The transactivator ie-2 stimulated gene expression in the presence of cis -linked enhancer elements and ie-1 in SF-21 cells. During viral infection, the heat shock 70 promoter was maximally activated at 12 hours post infection. We discuss how these results affect the interpretation of transient gene expression assays performed in the presence of viral transcription factors.

Zhang, S. M.; Sun, D. C.; Lou, S.; Bo, X. C.; Lu, Z.; Qian, X. H.; Wang, S. Q.

doi: 10.1007/s00705-005-0521-1pmid: 15789261

HBx, a transcriptional transactivating protein of hepatitis B virus (HBV), is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the molecular mechanism for its influence on cell remains largely unknown. It was proved that HBx need the help of host cell proteins to exert its function by binding to them. During purifying of GSTX (fusion protein of GST and HBx) expressed in E. coli , we found that it can bind specifically with GrpE (HSP60) and DnaK (HSP70) of E. coli while GST cannot. Using GST pull-down, two-dimensional gel electrophoresis and mass spectrum, we found that GSTX can also bind to human mitochondrial HSP60 and HSP70, which are homologues of GrpE and DnaK. These interactions between HBx and mitochondrial HSP60 and HSP70 are supported by the result of co-immunoprecipitation experiment. It means that HBx can form complex with E. coli and human HSP60 and HSP70. The implication of HBx, HSP60 and HSP70 complex in molecular mechanism of virus infection is discussed.

Zhou, H.; Wang, H.; Huang, L. F.; Naylor, M.; Clifford, P.

doi: 10.1007/s00705-005-0510-4pmid: 15834656

When conventional phylogenetic trees were built using 14 genome sequences of 9 sobemoviruses, two main lineages were apparent: monocot-infecting viruses and dicot-infecting viruses. To investigate whether members of the genus Sobemovirus originated from monocot hosts or from dicot hosts, we constructed relationship trees based on Relative Synonymous Codon Usage (RSCU) of the viruses. The RSCU relationship trees grouped the monocot-infecting and dicot-infecting viruses even better than the genome phylogenetic trees. The RSCU approach also enabled direct comparisons among viral and host species. When host species were added into the RSCU tree, the viral species clustered with the monocot hosts, indicating codon usage homologies to monocots. The stability of the RSCU tree was improved when RSCU values were calculated for individual viral open reading frames (ORFs). Most interestingly, the codon usages of the viral ORF-2 that encodes the replicase showed affinity to that of the plants whereas codon usages of the other viral ORFs were not relevant to the host species. All ORF-2s from 3 monocot viruses and 4 out of 6 dicot viruses had greater RSCU affinities to sequences of ORFs in monocot than to dicot hosts, possibly indicating that ORF-2, and therefore the replicase module of sobemovirus has a monocot origin.

Fonseca, F.; Neto, J. D.; Martins, V.; Nolasco, G.

doi: 10.1007/s00705-005-0507-zpmid: 15789267

Summary.Twelve new sequences of the coat protein gene of Prune dwarf virus (PDV) variants, obtained from almond trees, are presented. Comparison with previously reported sequences of the same region, obtained from other hosts (plum, cherry and peach) revealed not only the existence of a wider range of variants of PDV than formerly predicted, but also the frequent presence of a mixture of variants in each sample. In spite of the heterogeneity found in almond, the amino acid composition of the domain at the N terminus of the coat protein maintained the potential to form an amphipathic helix, and hence the capacity to serve the previously suggested function of binding the viral RNA during particle formation.Except for synonymous substitutions, measures of nucleotide diversity calculated for the two groups, respectively 13 sequences from almond and 14 sequences from other hosts, were found to be significantly different, with the almond group showing a much higher variability. Analysis of the dendrogram constructed based in all 27 PDV CP sequences did not reveal host specificity, in agreement with previous findings. However, a clear divergence between almond and other hosts sequences could be found. It is discussed that the observed differences between almond and other hosts variants may derive from differences in agricultural practices.

Mikalsen, A. B.; Sindre, H.; Mjaaland, S.; Rimstad, E.

doi: 10.1007/s00705-005-0502-4pmid: 15824888

Infectious salmon anemia (ISA) virus belongs to the proposed genus Isavirus of Orthomyxoviridae and is an emerging pathogen in Atlantic salmon ( Salmo salar ) farming. The hemagglutinin-esterase (HE) of ISA virus share several characteristics with the influenza virus hemagglutinin. This study reports recombinant expression of different ISA virus HE mutants in fish cell lines. Some introduced mutations, representing minimal differences in single amino acid residues, resulted in remarkable effects on expression efficiency in general and on surface expression specifically. Receptor binding assays showed that amino acid residues in the N-terminal half part are important in receptor binding, either being directly involved in the binding, or by influencing the structure of the binding site. Deletion of the putative N-glycosylation sites of the ISA virus HE, located near the transmembrane region, did not influence expression, receptor binding properties or staining by either a neutralising MAb, or salmon convalescent sera. The humoral immune response of Atlantic salmon after ISA virus infection showed weak neutralising activity and the results indicated that it was directed against HE.