bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of human respiratory syncytial virus to cells: implication of sulfated cell surface proteoglycans
Authors: Martínez Isidoro , Melero José A.
Published: August 2015

Binding of human respiratory syncytial virus (HRSV) to cultured cells was measured by flow cytometry. Using this assay and influenza virus as a control virus with a well-characterized receptor, a systematic search of cell surface molecules that might be implicated in HRSV binding was carried out. Treatment of cells with different enzymes or with other reagents suggested that heparin-like glycosaminoglycans (GAGs) were involved in attachment of HRSV, but not influenza virus, to host cells. This was further confirmed by a lack of binding of HRSV to CHO-K1 mutant cell lines deficient in glycosylation or GAGs biosynthesis and by an inhibition of binding after preincubation of virus with heparin and other GAGs. The degree of sulfation, more than the polysaccharide backbone of GAGs, seems to be critical for virus binding.
Nucleotide sequences of the F, L and G protein genes of two non-A/non-B avian pneumoviruses (APV) reveal a novel APV subgroup
Authors: Bäyon-Auboyer Marie-Hélène , Arnauld Claire , Toquin Didier , Eterradossi Nicolas
Published: August 2015

Sequence analysis was performed of all or part of the genes encoding the fusion (F), polymerase (L) and attachment (G) proteins of two French non-A/non-B avian pneumovirus (APV) isolates (Fr/85/1 and Fr/85/2). The two isolates shared at least 99·7% nt and 99·0% aa sequence identity. Comparison with the F genes from subgroup A, subgroup B or Colorado APVs revealed nt and aa identities of 70·0–80·5% and 77·6–97·2%, respectively, with the L gene sharing 76·1% nt and 85·3% aa identity with that of a subgroup A isolate. The Fr/85/1 and Fr/85/2 G genes comprised 1185 nt, encoding a protein of 389 aa. Common features with subgroup A and subgroup B G proteins included an amino-terminal membrane anchor, a high serine and threonine content, conservation of cysteine residues and a single extracellular region of highly conserved sequence proposed to be the functional domain involved in virus attachment to cellular receptors. However, the Fr/85/1 and Fr/85/2 G sequences shared at best 56·6% nt and 31·2% aa identity with subgroup A and B APVs, whereas these isolates share 38% aa identity. Phylogenetic analysis of the F, G and L genes of pneumoviruses suggested that isolates Fr/85/1 and Fr/85/2 belong to a previously unrecognized APV subgroup, tentatively named D. G-based oligonucleotide primers were defined for the specific molecular identification of subgroup D. These are the first G protein sequences of non-A/non-B APVs to be determined.
Quantitative analysis of viral RNA kinetics in coxsackievirus B3-induced murine myocarditis: biphasic pattern of clearance following acute infection, with persistence of residual viral RNA throughout and beyond the inflammatory phase of disease
Authors: Reetoo K. Nundita , Osman Shabina A. , Illavia Shirin J. , Cameron-Wilson Charlotte L. , Banatvala Jangu E. , Muir Peter
Published: August 2015

Although the association remains controversial, enteroviruses have been implicated in the aetiology of several chronic diseases of humans. To further understand the mechanism of enterovirus persistence and its relationship to organ pathology, virus infectivity and viral RNA kinetics in the heart and other target organs during acute and persistent phases of murine coxsackievirus B3 infection were investigated. These studies revealed a biphasic pattern of virus clearance. Thus, there was a rapid but incomplete clearance of viral RNA from the myocardium following the acute phase of virus replication, which paralleled the elimination of virus infectivity. The mean half-life of viral RNA between days 5 and 14 post-infection (p.i.) was 13·4 h. In contrast, a much slower rate of decline in viral RNA levels was observed during the post-infectious inflammatory phase of myocarditis. The mean half-life of viral RNA between days 14 and 90 p.i. was 14·1 days. Viral RNA persisted in the myocardium beyond the resolution of inflammation and was still detectable in a proportion of animals 90 days after infection. Clearance of viral RNA from other target organs occurred more rapidly, but the rate of clearance was largely independent of the level of viral RNA present during the acute phase of infection. Thus, while antiviral immune responses effectively eliminated infectious virus, clearance of residual viral RNA from the myocardium and other target organs was significantly delayed, despite a prolonged inflammatory response. These findings suggest that clearance of persistent enterovirus infection requires mechanisms different from those responsible for the elimination of virus infectivity.
The inhibitors of apoptosis of Epiphyas postvittana nucleopolyhedrovirus
Authors: Maguire Terry , Harrison Penelope , Hyink Otto , Kalmakoff James , Ward Vernon K.
Published: August 2015

In this study, four inhibitor of apoptosis genes (iaps) in the genome ofEpiphyas postvittananucleopolyhedrovirus (EppoMNPV) that are homologous toiap-1,iap-2,iap-3andiap-4genes of other baculoviruses have been identified. All fouriapgenes were sequenced and theiap-1andiap-2genes were shown to be functional inhibitors of apoptosis. Theiap-1,iap-2andiap-3genes contain two baculovirus apoptosis inhibitor repeat motifs and a C3HC4RING finger-like motif. The activity of theiapgenes was tested by transient expression inSpodoptera frugiperda(Sf-21) cells treated with the apoptosis-inducing agents actinomycin D, cycloheximide, anisomycin, tumour necrosis factor-α and UV light. Theiap-2gene prevented apoptosis induced by all agents tested, indicating activity towards a conserved component(s) of multiple apoptotic pathways. However, theiap-2gene was unable to function in the absence of a gene immediately upstream ofiap-2that has homology to theorf69gene ofAutographa californicaMNPV. The use of a CMV promoter rescued the apoptosis inhibition activity of theiap-2gene, indicating that the upstreamorf69homologue is associated with expression ofiap-2. Theiap-1gene was able to delay the onset of apoptosis caused by all of the induction agents tested but, unlikeiap-2, was unable to prevent the development of an apoptotic response upon prolonged exposure of cells to the apoptosis induction agents. No anti-apoptotic activity was observed for theiap-3andiap-4genes of EppoMNPV.
Unidirectional RNA polymerase I–polymerase II transcription system for the generation of influenza A virus from eight plasmids
Authors: Hoffmann Erich , Webster Robert G.
Published: August 2015

Recently, we developed a system for the generation of influenza A virus by cotransfecting only eight plasmids from which negative-sense vRNA and positive-sense mRNA are expressed (Hoffmann et al., Proceedings of the National Academy of Sciences, USA 97, 6108–6113, 2000). Here we report the establishment of a different transcription system for the expression of virus-like RNAs, allowing the intracellular synthesis of noncapped positive-sense cRNA and 5′-capped mRNA from one template. Cotransfection of eight RNA pol I–pol II tandem promoter plasmids containing the cDNA of A/WSN/33 (H1N1) resulted in the generation of infectious influenza A virus, albeit with a lower yield than the bidirectional system. Our approach of producing either vRNA and mRNA or cRNA and mRNA intracellularly from a minimum set of plasmids should be useful for the establishment or optimization of reverse genetics systems for other RNA viruses.
Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus
Authors: Evans Sharon , Cavanagh David , Britton Paul
Published: August 2015

Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3′-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3′ ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.
Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding
Authors: Patel Arvind H. , Wood Jonny , Penin Francois , Dubuisson Jean , McKeating J. A.
Published: August 2015

We compared the ability of two closely related truncated E2 glycoproteins (E2660) derived from hepatitis C virus (HCV) genotype 1a strains Glasgow (Gla) and H77c to bind a panel of conformation-dependent monoclonal antibodies (MAbs) and CD81. In contrast to H77c, Gla E2660 formed disulfide-linked high molecular mass aggregates and failed to react with conformation-dependent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla–H77c E2660 chimeric glycoproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525–660 of Gla E2660, produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this region are responsible for protein misfolding. The presence of Gla hypervariable region 1 (aa 384–406) on H77 E2660, chimera C4, had no effect on protein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384–524 or 407–524 of Gla E2660, respectively, were recognized by conformation-dependent MAbs and yet failed to bind CD81, suggesting that amino acids in region 407–524 are important in modulating CD81 interaction without affecting antigen folding. Comparison of Gla and H77c E2660 aa sequences with those of genotype 1a and divergent genotypes identified a number of variant amino acids, including two putative N-linked glycosylation sites at positions 476 and 532. However, introduction of G476N–G478S and/or D532N in Gla E2660 had no effect on antigenicity or aggregation.
Primate foamy virus Pol proteins are imported into the nucleus
Authors: Imrich Horst , Heinkelein Martin , Herchenröder Ottmar , Rethwilm Axel
Published: August 2015

Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127Pol, p85RT/RN and p40IN served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains.
Identification and functional analysis of sequence rearrangements in the long control region of human papillomavirus type 16 Af-1 variants isolated from Ugandan penile carcinomas
Authors: Tornesello Maria Lina , Buonaguro Franco M. , Buonaguro Luigi , Salatiello Immacolata , Beth-Giraldo Elke , Giraldo Gaetano
Published: August 2015

Human papillomavirus type 16 (HPV-16) is the predominant HPV isolate found in malignancies of male and female lower genital tracts. However, only a small percentage of individuals infected with high-risk HPVs develop a genital neoplasia, suggesting that additional events at both the cellular and the virus level are necessary for the progression to cancer, including genetic mutations/rearrangements of viral sequences involved in the oncogenic process. In this study, the genetic stability of the long control region (LCR) (nt 7289–114), which regulates expression levels of oncoproteins E6 and E7, was analysed in HPV-16 isolates from penile carcinoma (PC) biopsies of patients recruited from Uganda, one of the countries with the highest incidence of genital cancers in both men and women. Nucleotide changes within the LCR region typical of the African-1 (Af-1) lineage were observed in all HPV-16 isolates. Two out of five samples showed further rearrangements of the enhancer region. The functional activity of LCR with Af-1 mutations and/or rearrangements was evaluated by cloning each LCR into CAT expression vectors, followed by transfection in several epithelial and non-epithelial cell lines. CAT expression levels driven by a rearranged LCR were significantly higher than those driven by Af-1 or European prototype LCRs. Furthermore, in the NIH3T3 focus formation assay, the transforming activity of E6 and E7 genes, driven by a mutated or rearranged LCR, was 1·4- to 3·0-fold higher, respectively. These results indicate that rearrangements within the LCR of HPV-16 isolated from African PCs are frequently found (2 out of 5, 40%). It is also shown that increased HPV LCR activity is associated with an increased E6/E7-mediated in vitro transforming activity, suggesting that natural variants can play a major role in the pathogenesis of genital carcinomas.
Interaction of the herpes simplex virus type 1 packaging protein UL15 with full-length and deleted forms of the UL28 protein
Authors: Abbotts Adrian P. , Preston Valerie G. , Hughes Michelle , Patel Arvind H. , Stow Nigel D.
Published: August 2015

The UL15 and UL28 proteins of herpes simplex virus type 1 are both required for the packaging of replicated viral DNA into the viral capsid. We have expressed UL28 and a functional epitope-tagged form of UL15 in mammalian and insect cells. Immunoprecipitation experiments confirmed that the two proteins can interact. In agreement with previous results, UL15, when expressed alone, entered the nucleus but UL28 remained cytoplasmic. When co-expressed the two proteins co-localized in the nucleus. Six UL28 deletion mutants were constructed and similarly analysed. The results obtained by immunoprecipitation and immunofluorescence were consistent and demonstrate that at least two separate regions of the UL28 polypeptide chain have the ability to interact with UL15. Surprisingly, three of the mutants prevented the UL15 protein from localizing to the cell nucleus, and these were not functional in a transient DNA packaging assay. Of the three UL28 mutant proteins that entered the nucleus with UL15, one containing an internal deletion of 13 amino acids was able to complement a UL28 null mutant in both DNA packaging and virus yield assays, demonstrating that this region of the protein is not essential for function. In addition to interacting with the UL28 protein we also demonstrated that UL15 molecules can interact with each other, and that sequences within the second exon contribute to this interaction.
Co-expression of the capsid proteins of Cowpea mosaic virus in insect cells leads to the formation of virus-like particles
Authors: Shanks Michael , Lomonossoff George P.
Published: August 2015

The regions of RNA-2 of Cowpea mosaic virus (CPMV) that encode the Large (L) and Small (S) coat proteins were expressed either individually or together in Spodoptera frugiperda (sf21) cells using baculovirus vectors. Co-expression of the two coat proteins from separate promoters in the same construct resulted in the formation of virus-like particles whose morphology closely resembled that of native CPMV virions. No such particles were formed when the individual L and S proteins were expressed. Sucrose gradient centrifugation of the virus-like particles showed that they had the sedimentation characteristics of empty (protein-only) shells. The results confirm that the 60 kDa L–S fusion is not an obligate intermediate in the virion assembly pathway and indicate that expression of the coat proteins in insect cells will provide a fruitful route for the study of CPMV morphogenesis.
Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions
Authors: van Hulten Mariëlle C. W. , Tsai Meng-Feng , Schipper Christel A. , Lo Chu-Fang , Kou Guang-Hsiung , Vlak Just M.
Published: August 2015

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12·3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. Therr1andrr2genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream ofrr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within theBaculoviridaeor a member of an entirely new virus family.
Analysis of human papillomavirus type 16 E6 variants in relation to p53 codon 72 polymorphism genotypes in cervical carcinogenesis
Authors: van Duin Mark , Snijders Peter J. F. , Vossen Mireille T. M. , Klaassen Erik , Voorhorst Feja , Verheijen René H. M. , Helmerhorst Theo J. , Meijer Chris J. L. M. , Walboomers Jan M. M.
Published: August 2015

This study aimed to assess the role of specific human papillomavirus type 16 (HPV-16) variants, in combination with p53 codon 72 polymorphism genotypes, in cervical carcinogenesis. An initial sequence analysis of HPV-16 long control, E6 and E7 regions of 53 well-defined cervical samples containing HPV-16 revealed that a T to G transition at nucleotide position 350 within the E6 open reading frame was the most common variation, the frequency of which seemed to decrease with increasing severity of the lesion. Therefore, a total of 246 cervical samples of residents of The Netherlands was specifically analysed for HPV-16 350G/T variants and/or p53 codon 72 genotypes. These comprised HPV-negative normal cervical scrapes (n=40), normal cervical scrapes containing HPV-16 (n=46), scrapes containing HPV-16 from women with abnormal cervical cytology participating in a non-intervention follow-up study without (n=38) and with (n=51) a histologically proven cervical intraepithelial neoplasia (CIN) III lesion at the end of the study, and cervical squamous cell carcinomas (n=71). Neither specific HPV-16 350G/T variants nor specific p53 genotypes were associated with a higher risk of developing CIN III or cervical cancer. However, HPV-16 350T variants were significantly over-represented in p53 Arg homozygous women with cervical cancer. This suggests that, in p53 Arg/Arg women, infection with HPV-16 350T variants confers a higher risk of cervical cancer.
Genetic characterization of feline parvovirus sequences from various carnivores
Authors: Steinel A. , Munson L. , van Vuuren M. , Truyen U.
Published: August 2015

Infections with viruses of the feline parvovirus subgroup such as feline panleukopenia virus (FPV), mink enteritis virus (MEV) and canine parvovirus (CPV-2) [together with its new antigenic types (CPV-2a, CPV-2b)] have been reported from several wild carnivore species. To examine the susceptibility of different species to the various parvoviruses and their antigenic types, samples from wild carnivores with acute parvovirus infections were collected. Viral DNA was amplified, and subsequently analysed, from faeces or formalin-fixed small intestines from an orphaned bat-eared fox (Otocyon megalotis), a free-ranging honey badger (Mellivora capensis), six captive cheetahs (Acinonyx jubatus), a captive Siberian tiger (Panthera tigris altaica) and a free-ranging African wild cat (Felis lybica). Parvovirus infection in bat-eared fox and honey badger was demonstrated for the first time. FPV-sequences were detected in tissues of the African wild cat and in faeces of one cheetah and the honey badger, whereas CPV-2b sequences were found in five cheetahs and the bat-eared fox. The Siberian tiger (from a German zoo) was infected with a CPV-type 2a virus. This distribution of feline parvovirus antigenic types in captive large cats suggests an interspecies transmission from domestic dogs. CPV-2 sequences were not detected in any of the specimens and no sequences with features intermediate between FPV and CPV were found in any of the animals examined.
BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus
Authors: Devireddy Laxminarayana R. , Kumar Kotlo U. , Pater Mary M. , Pater Alan
Published: August 2015

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCVE promoter and to a lesser extent the JCVL promoter. Mutations in the NF-1 sites in the JCVE promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.
Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype
Authors: Sobotta Dirk , Sominskaya Irina , Jansons Juris , Meisel Helga , Schmitt Sigrid , Heermann Klaus-Hinrich , Kaluza Georg , Pumpens Paul , Gerlich Wolfram H.
Published: August 2015

Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3–15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38–48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.
Mitochondrial distribution and function in herpes simplex virus-infected cells
Authors: Murata Takayuki , Goshima Fumi , Daikoku Tohru , Inagaki-Ohara Kyoko , Takakuwa Hiroki , Kato Keisuke , Nishiyama Yukihiro
Published: August 2015

In this study, mitochondria migrated to a perinuclear region in the cytoplasm in herpes simplex virus (HSV)-infected cells. HSV infection did not promote the expression of cytochrome c oxidase subunit 2 but did promote that of stress-responsive HSP60, both of which are known to be components of mitochondria. The levels of cellular ATP and lactate and mitochondrial membrane potential were maintained for at least 6 h but decreased at the late stage of infection. It was also found that the UL41 and UL46 gene products, both of which are known to be tegument proteins, accumulated in the perinuclear region. The clustering of mitochondria and the accumulation of tegument proteins were completely blocked by the addition of nocodazole and vinblastine. These results suggest that mitochondria respond to the stimulation of HSV infection, migrating with tegument proteins along microtubules to a site around the nucleus, and maintain function until at least the middle stage of infection.
Infection of intestinal epithelial cells and development of systemic disease following gastric instillation of murine gammaherpesvirus-68
Authors: Peacock James W. , Bost Kenneth L.
Published: August 2015

Murine gammaherpesvirus-68 (γHV-68) induces a lymphocytosis in mice and establishes a latent infection of B lymphocytes following intranasal administration in anaesthetized animals. Because γHV-68 is a gammaherpesvirus, it has been used as a model to understand the pathogenesis of Epstein–Barr virus (EBV) and human herpesvirus-8 (HHV-8) infections. In this study, we investigated the unlikely possibility that γHV-68 could survive the harsh gastrointestinal environment to efficiently infect intestinal epithelial cells, and then disseminate from mucosal sites to cause systemic disease. Surprisingly, oral administration, or gastric instillation which by-passed the oral cavity, readily caused a systemic lymphocytosis and established a latent infection in splenic leukocytes. The finding that γHV-68 could readily infect adult mice following gastric instillation strongly suggested that intestinal epithelial cells could be productively infected. Unlike the more routinely used method of intranasal inoculation, γHV-68 given intragastrically resulted in lytic virus, viral RNA and viral DNA being present in isolated intestinal epithelial cells. Furthermore, γHV-68 RNA and DNA, but not latent virus, could be detected in epithelial cells as long as 30 days post-infection, suggesting that some of these cells might be persistently infected. Taken together, these studies demonstrate that γHV-68 can survive passage through the gastrointestinal tract and infect intestinal epithelial cells. Following infection of gut epithelial cells, γHV-68 can disseminate from mucosal sites to induce a systemic lymphocytosis which is similar to the disease induced following intranasal inoculation.
Nucleotide sequence and genome organization of Apple latent spherical virus: a new virus classified into the family Comoviridae
Authors: Li Chunjiang , Yoshikawa Nobu , Takahashi Tsuyoshi , Ito Tsutae , Yoshida Kouji , Koganezawa Hiroki
Published: August 2015

A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1·41 and 1·43 g/cm3 in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3′ poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa (‘23K’; ORF1) and 235 kDa (‘235K’; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa (‘108K’). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the Comoviridae. The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.
Cytokine profile of draining lymph node lymphocytes in mice grafted with syngeneic keratinocytes expressing human papillomavirus type 16 E7 protein
Authors: López María C. , Stanley Margaret A.
Published: August 2015

Studies on the immune response to human papillomaviruses are compromised by the extreme host and tissue specificity of these viruses. To circumvent this, a mouse model system has been used in which antigen is presented via a differentiated, syngeneic keratinocyte graft expressing human papillomavirus type 16 (HPV-16) E7 protein. Using this model, previous studies have shown that animals grafted with a high cell inoculum (1×107NEK 16 cells) exhibit a delayed-type hypersensitivity response that is E7-specific and CD4+-mediated, but those receiving a low cell inoculum (5×105NEK 16 cells) are rendered unresponsive to subsequent and repeated antigen challenge. To investigate the mechanisms underlying this phenomenon, we have analysed the early changes in the cytokine profile of the graft-draining lymph node (GDLN) after high- or low-dose grafts. At 4 days post-grafting, there was a peak secretion of IL-2 associated with a decreased secretion of IL-4 by γδ-TCR+cells in the group receiving 1×107NEK 16 cells. At 5 days post-grafting, there was a peak secretion of IL-10 by CD8+cells in both the high- and low-dose graft groups compared with controls. In contrast, low dose-grafted animals showed an increase in IL-4 production by CD8+cells at this time-point. Low antigen challenge in this model system is associated with the appearance of a CD8+population in the GDLN that secretes both IL-4 and IL-10. This population may represent a Tc2 or Ts subset that could induce further unresponsiveness.
Neuronal pathways for the propagation of herpes simplex virus type 1 from one retina to the other in a murine model
Authors: Labetoulle M. , Kucera P. , Ugolini G. , Lafay F. , Frau E. , Offret H. , Flamand Anne
Published: August 2015

Herpetic retinitis in humans is characterized by a high frequency of bilateral localization. In order to determine the possible mechanisms leading to bilateral retinitis, we studied the pathways by which herpes simplex virus type 1 (HSV-1) is propagated from one retina to the other after intravitreal injection in mice. HSV-1 strain SC16 (90 p.f.u.) was injected into the vitreous body of the left eye of BALB/c mice. Animals were sacrificed 1, 2, 3, 4 and 5 days post-inoculation (p.i.). Histological sections were studied by immunochemical staining. Primary retinitis in the inoculated eye (beginning 1 day p.i.) was followed by contralateral retinitis (in the uninoculated eye) starting at 3 days p.i. Infected neurons of central visual pathway nuclei (lateral geniculate nuclei, suprachiasmatic nuclei and pretectal areas) were detected at 4 days p.i. Iris and ciliary body infection was minimal early on, but became extensive thereafter and was accompanied by the infection of connected sympathetic and parasympathetic pathways. The pattern of virus propagation over time suggests that the onset of contralateral retinitis was mediated by local (non-synaptic) transfer in the optic chiasm from infected to uninfected axons of the optic nerves. Later, retinopetal transneuronal propagation of the virus from visual pathways may have contributed to increase the severity of contralateral retinitis.
Detection of lymphocytes productively infected with Epstein–Barr virus in non-neoplastic tonsils
Authors: Ikeda Tetsuya , Kobayashi Ryo , Horiuchi Manabu , Nagata Yoshifumi , Hasegawa Makoto , Mizuno Fumio , Hirai Kanji
Published: August 2015

Epstein–Barr virus (EBV) persists for life in the infected host. Little is known about EBV reactivation and regulation of virus persistence in healthy individuals. We examined tonsils of chronic tonsillitis patients to detect EBV transcripts, EBV genomes and lytic proteins. LMP1 transcripts were observed in 11 of 15 specimens and BZLF1 transcripts were detected in six. Multiple copies of EBV genome equivalents per cell, and ZEBRA- and viral capsid antigen-positive cells were also detected in tonsillar lymphocytes. These results indicate that EBV productively infected cells may survive in the face of immune surveillance in the tonsils. Thus, EBV replication may occur in tonsillar lymphocytes, and tonsillar lymphoid tissues may play a role in the maintenance of EBV loadin vivo.
The punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral DNA synthesis
Authors: Domi Arban , Beaud Georges
Published: August 2015

Several vaccinia virus early proteins (encoded by genes B1R, H5R and I3L) synthesized in the presence of an inhibitor of DNA synthesis localize, at least in part, to punctate inclusions that are visible by immunofluorescence in the cytoplasm of poxvirus-infected cells. It is shown that these inclusions contain DNA (visualized by DAPI staining of the infected cells) and that the number of inclusions is proportional to the amount of input virus. Their mean diameter (about 680 nm) was larger than that of purified vaccinia virus particles. When the inhibition of DNA synthesis was reversed, incorporation of BrdU into the B1R particles was demonstrated after labelling for 30 min, suggesting that these cytoplasmic focal sites correspond to viral DNA replication complexes that have initiated normally but are inhibited at the step of DNA chain elongation. These experiments suggest strongly that these inclusions are the precursors of the virosomes.
CD8+ cell noncytotoxic anti-human immunodeficiency virus response inhibits expression of viral RNA but not reverse transcription or provirus integration
Authors: Mackewicz Carl E. , Patterson Bruce K. , Lee Sandra A. , Levy Jay A.
Published: August 2015

CD8+T cells from human immunodeficiency virus (HIV)-infected individuals can suppress HIV replication in CD4+cells by a noncytotoxic mechanism that inhibits the expression of viral RNA. The present study examined whether other step(s) in the virus replicative cycle could be affected by the CD8+cells. Culturing HIV-infected CD4+T cells with antiviral CD8+T cells did not significantly reduce the amounts of (i) early HIV DNA reverse transcripts (detected by LTR-U3/R), (ii) total nuclear HIVgagDNA, or (iii) integrated proviral DNA. However, exposure to the CD8+T cells did cause a reduction in the amount of multiply splicedtatand full-lengthgagmRNA expressed by the infected CD4+T cells, confirming previous observations. The levels of glyceraldehyde-3-phosphate dehydrogenase and interleukin-2 receptor-α mRNA were not affected. The results support the conclusion that the noncytotoxic anti-HIV response of CD8+T cells, demonstrablein vitro, does not affect any of the virus replication steps leading to the integration of proviral HIV, but specifically interrupts the expression of viral RNA.
Suboptimal splice sites of equine infectious anaemia virus control Rev responsiveness
Authors: Rosin-Arbesfeld Rina , Yaniv Abraham , Gazit Arnona
Published: August 2015

The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3′ half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in
trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site-directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression.
Genome structure of Sagiyama virus and its relatedness to other alphaviruses
Authors: Shirako Yukio , Yamaguchi Yuka
Published: August 2015

Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3′ poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3′ terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.
Inverse relationship between the expression of the human papillomavirus type 16 transcription factor E2 and virus DNA copy number during the progression of cervical intraepithelial neoplasia
Authors: Stevenson Mark , Hudson Lucy C. , Burns Julie E. , Stewart Roy L. , Wells Michael , Maitland Norman J.
Published: August 2015

The human papillomavirus type 16 (HPV-16) status of 43 cervical biopsies, which had been characterized histologically as normal, various grades of cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinoma, was examined by using (i) a novel antibody against the HPV-16 E2 protein, (ii) sensitive HPV-16 DNA in situ hybridization and (iii) microdissection/PCR for the E2 ORF. The data indicate that E2 protein expression is highest in koilocytes in lower-grade CIN (I), but decreases with increasing grade, whereas the detection of HPV DNA is delayed until CIN I/II, rising to the highest levels in carcinoma cells. Co-localization of E2 with HPV-16 DNA-positive cells was most commonly observed in koilocytes in CIN II lesions. PCR analyses of microdissected epithelium from the same or serial sections indicated that E2 ORFs were retained in an intact form in a number of higher-grade CIN lesions and invasive carcinomas.
Filamentous actin is required for lepidopteran nucleopolyhedrovirus progeny production
Authors: Kasman L. M. , Volkman L. E.
Published: August 2015

Autographa californicaM nucleopolyhedrovirus (AcMNPV) is the prototypical member of theNucleopolyhedrosisgenus of theBaculoviridae, a family of large, double-stranded DNA viruses that are highly diverse. Nucleocapsid morphogenesis of AcMNPV and others in theNucleopolyhedrovirusgenus takes place within the nuclei of infected host cells. Previously, we showed that filamentous actin (F-actin) is essential for this process to occur in AcMNPV-infected cells, an unprecedented finding for a DNA virus that replicates within the nucleus. Because of the fundamental importance of this requirement to our understanding of virus–host interactions, and because of the diversity of viruses included within theNucleopolyhedrovirusgenus, we were compelled to determine whether the replication of other nucleopolyhedroviruses was also F-actin dependent. We report here that progeny virus production of six other lepidopteran nucleopolyhedroviruses, representing both phylogenetic groups I and II within the genus, is also F-actin dependent. The six viruses studied (Spodoptera frugiperdaMNPV,Bombyx moriNPV,Orgyia pseudotsugataMNPV,Lymantria disparMNPV,Anticarsia gemmatalisMNPV andHelicoverpa zeaSNPV) were unable to produce progeny in the presence of either cytochalasin D or latrunculin A, two actin-binding agents that interfere with F-actin-dependent processes but differ in their modes of action. F-actin-dependent progeny morphogenesis, therefore, appears to be a characteristic common among viruses in this genus that have lepidopteran hosts.
Rescue of synthetic salmonid rhabdovirus minigenomes
Authors: Biacchesi Stéphane , Yu Yan-Xing , Béarzotti Monique , Tafalla Carolina , Fernandez-Alonso Miriam , Brémont Michel
Published: August 2015

Synthetic T7-driven cDNA minigenomes containing the bacterial chloramphenicol acetyltransferase gene as a reporter were derived from the genome of two salmonid novirhabdoviruses, infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV). We showed that an exogenous IHNV RNA minigenome transfected into fish cells could be rescued following IHNV infection as it was replicated, encapsidated and transcribed. When cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase (vTF7-3), transfected with the plasmid carrying the IHNV minigenome (genomic- and antigenomic-sense) and superinfected with IHNV, rescue of the minigenome was more efficient. Heterologous VHSV/IHNV rescue experiments failed. Finally, when the IHNV N, P and L proteins were expressed from cDNAs in cells, the minigenome was also successfully rescued, indicating that the nucleocapsid proteins were biologically functional. These data represent the first example of rescue experiments for non-mammalian rhabdoviruses replicating at a low temperature.
Transcriptional regulation of the channel catfish virus genome direct repeat region
Authors: Stingley Robin L. , Gray Wayne L.
Published: August 2015

Channel catfish virus (CCV), a member of the herpesvirus family, causes a severe haemorrhagic disease in juvenile channel catfish. In this report, we confirm that CCV gene expression is temporally regulated into immediate-early (IE), early and late phases, similar to that of other herpesviruses. The transcriptional regulation of the 14 genes within the direct repeat region of the CCV genome was determined by Northern hybridization analysis of RNA isolated from infected cells in the presence or absence of metabolic inhibitors. Two CCV genes within the direct repeat, ORFs 1 and 3, expressed IE transcripts. Early RNAs were encoded by ORFs 2–9 and 11–14. ORFs 4, 7 and 10–13 expressed late transcripts after the onset of viral DNA replication. A time-course study conducted without metabolic inhibitors confirmed that CCV direct repeat transcription is temporally regulated. The characterization of CCV transcription during cytolytic infection in vitro will provide a foundation for the analysis of CCV gene expression in tissues of acutely and latently infected catfish.
Evidence that the first strand-transfer reaction of duck hepatitis B virus reverse transcription requires the polymerase and that strand transfer is not needed for the switch of the polymerase to the elongation mode of DNA synthesis
Authors: Gong Yunhao , Yao Ermei , Stevens Melissa , Tavis John E.
Published: August 2015

Deletion of amino acids 79–88 in the duck hepatitis B virus reverse transcriptase had minimal effects on polymerase activities prior to the minus-strand DNA transfer reaction, yet it greatly diminished strand transfer and subsequent DNA synthesis. This mutation also reduced reverse transcription on exogenous RNA templates. The reaction on exogenous RNAs employed the phosphonoformic acid (PFA)-sensitive elongation mode of DNA synthesis rather than the PFA-resistant priming mode, despite the independence of DNA synthesis in this assay from the priming and minus-strand transfer reactions. These data provide experimental evidence that the polymerase is involved directly in the minus-strand transfer reaction and that the switch of the polymerase from the early PFA-resistant mode of DNA synthesis to the later PFA-sensitive elongation mode does not require the strand-transfer reaction.
Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors
Authors: Handa Atsushi , Muramatsu Shin-ichi , Qiu Jianming , Mizukami Hiroaki , Brown Kevin E.
Published: August 2015

Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID50of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.