bluuebunny/crossref_metadata_2025 huggingface dataset:
HERV-F, a new group of human endogenous retrovirus sequences
Authors: Kjellman Christian , Sjögren Hans-Olov , Widegren Bengt
Published: August 2015

Using primers from a conserved region of the XA34 human endogenous retrovirus (HERV) family, fourpolfragments originating from new members of the family were amplified from human genomic DNA. Southern blot analysis demonstrated similar hybridization patterns in human, chimpanzee and orangutan and distinct hybridization to macaque DNA. The probes also exhibited weaker hybridization to squirrel monkey DNA. Using large genomic clones, two full-length XA34-related HERVs have been identified. One of the HERVs is located downstream of a human Krüppel-related zinc finger protein gene,ZNF195. Both of the newly identified long terminal repeats have potential TATA boxes, poly(A) signals and transcription factor-binding sites but they differ to a high degree, especially in the U3region. The primer-binding sites were found to be homologous to tRNAPhe(TTC), and therefore these new HERVs have been given the name HERV-F. The closest relatives to the HERV-Fs are the RTVLH-RGH family. Phylogenetic analyses of the Gag, Pol and Env regions are discussed. Both of the newly identified HERV-Fs were shown to containprotease,reverse transcriptase,integraseandenvregions and had characteristic deletions in theintegraseandenvregions. In addition, thecapsid proteingene ofgagand two conserved zinc-binding motifs that are characteristic of a potential nucleic acid-binding protein were also identified. Apart from an ORF spanning theproteaseof one HERV-F, no other longer ORFs were found.
Endothelial cell infection in vivo by equine infectious anaemia virus
Authors: Oaks J. Lindsay , Ulibarri Catherine , Crawford Timothy B.
Published: August 2015

Equine infectious anaemia virus (EIAV) infection of horses is characterized clinically by recurrent episodes of fever, thrombocytopenia and anaemia.In vivo, the only site of virus replication that has been previously demonstrated for EIAV is the tissue macrophage. In this study,in situhybridization for EIAV was combined with immunohistochemistry for cell-type-specific markers to identify infected endothelial cells. EIAV-infected endothelial cells and macrophages were detected in horses infected with either virulent wild-type or with weakly virulent tissue culture-adapted strains of EIAV. The role of endothelial cell infection in the pathogenesis of EIAV remains undefined, but could contribute to the development of thrombocytopenia. However, endothelial cell infection does not appear to be a determinant of virulence for EIAV.
Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus
Authors: Rangel Aymara A. C. , Rockemann Daniel D. , Hetrick Frank M. , Samal Siba K.
Published: August 2015

Three aquareovirus strains isolated from grass carp (Ctenopharyngodon idellus), geoduck clams (Panope abrupta) and herring (Clupea harengus) in North America and Asia were examined by RNA–RNA blot hybridization to determine their genogroup. The isolates from clams and herring were identified as members of genogroup A, but the isolate from grass carp did not hybridize to any of the known genogroups, suggesting that this virus probably represents a new, seventh genogroup.
Two-hybrid analysis of the interaction between the UL52 and UL8 subunits of the herpes simplex virus type 1 helicase–primase
Authors: Constantin Nicoleta , Dodson Mark S.
Published: August 2015

Herpes simplex virus type 1 expresses a heterotrimeric helicase–primase, the subunits of which are encoded by the viralUL5,UL8andUL52genes. The interactions of the UL52 protein with the UL8 and UL5 proteins were analysed by using the yeast two-hybrid system. The UL52–UL5 interaction gave a specific but weak signal in the two-hybrid system. In contrast, the UL52–UL8 interaction gave a strong signal in the two-hybrid system. Deletion analysis showed that a 548 amino acid fragment of UL52 (amino acids 366–914) retains the ability to interact with UL8 and that the N-terminal 349 amino acids are dispensable for the interaction. A fragment library screen and co-immunoprecipitation experiments confirmed the deletion analysis results.
Identification and transcriptional analysis of the homologues of the herpes simplex virus type 1 UL30 to UL40 genes in the genome of nononcogenic Marek’s disease virus serotype 2
Authors: Izumiya Yoshihiro , Jang Hyung-Kwan , Sugawara Mie , Ikeda Yasuhiro , Miura Ryuichi , Nishimura Yorihiro , Nakamura Kazuya , Miyazawa Takayuki , Kai Chieko , Mikami Takeshi
Published: August 2015

Studies on Marek’s disease virus serotype 2 (MDV2) are important for understanding the natural nononcogenic phenotype of MDV. This study reports a 27535 bp nucleotide sequence of part of the MDV2 genome located in the central unique long (UL) region. The analysis revealed 11 complete ORFs with high amino acid sequence identities to the products of other alphaherpesviruses. The MDV2 ORFs were arranged collinearly with the prototype sequence of herpes simplex virus type 1, ranging from the UL30 to UL40 genes. Sequences that were particularly well conserved among alphaherpesviruses were the putative functional domain of the DNA polymerase (UL30) and the ribonucleotide reductase large and small subunits (UL39 and UL40). On the other hand, in contrast to oncogenic MDV1, MDV2 did not contain the conserved proline-repeat region in the UL36 homologue. All the genes identified were confirmed to be transcribed as 3′-coterminal mRNAs and/or unique transcripts in virus-infected cells.
Identification and characterization of the UL14 gene product of herpes simplex virus type 2
Authors: Wada K. , Goshima F. , Takakuwa H. , Yamada H. , Daikoku T. , Nishiyama Y.
Published: August 2015

The UL14 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 219 amino acid protein with a molecular mass of 23 kDa. In this study, the HSV-2 UL14 gene product has been identified by using a rabbit polyclonal antiserum raised against a recombinant 6×His–UL14 fusion protein expressed in E. coli. The antiserum reacted specifically with 34, 33 and 28 kDa proteins in HSV-2-infected cell lysates and also with a 34 kDa protein produced by in vitro transcription and translation reactions, suggesting that the 34 kDa protein is the primary translation product of the UL14 gene. The protein was synthesized at late times post-infection (p.i.) and was not detectable in the presence of the viral DNA synthesis inhibitor acycloguanosine. Indirect immunofluorescence studies localized the UL14 protein both to the nucleus and to perinuclear regions of the cytoplasm, and the nuclear UL14 protein was found to co-localize with the scaffolding protein ICP35 at 9 h p.i. However, the protein accumulated in a perinuclear region of the cytoplasm at 12 h p.i., while most of the ICP35 protein localized within assemblons in the nucleus. Although no detectable UL14 protein was associated with intracellular capsids isolated in the presence of 0·5 M NaCl, it was detected in purified virions. Furthermore, the UL14 protein expressed alone was detected both in the nucleus and in the cytoplasm at 24 h after transfection, but was mainly localized to the cytoplasm at later times.
A recombinant human papillomavirus (HPV) type 16 L1–vaccinia virus murine challenge model demonstrates cell-mediated immunity against HPV virus-like particles
Authors: Marais Dianne , Passmore Jo-Ann , Maclean James , Rose Robert , Williamson Anna-Lise
Published: August 2015

Human papillomavirus (HPV) virus-like particles (VLP) are emerging as the immunogen of choice for prophylactic vaccines. The inability to infect animals with HPV has prevented the testing of potential vaccines such as these in animal systems. This study describes the development of a recombinant vaccinia virus (VV)–HPV type 16 (HPV-16) VLP challenge model to evaluate the efficacy of the cell-mediated immune response following HPV-16 VLP immunization in mice. Inoculation of BALB/c and C57 BL/6 mice with HPV-16 VLP resulted in HPV VLP-specific T cell proliferative responses characterized by the production of both Th1and Th2cytokines, and afforded protection against virus challenge from recombinant VV expressing HPV-16 L1 (VVL1R-16). Protection was demonstrated by a 4·6 log10reduction in ovarian titres of VVL1R-16 in vaccinated BALB/c mice and a 2·3 log10reduction in vaccinated C57 BL/6 mice, compared with unvaccinated mice.
Highly regulated expression of adeno-associated virus large Rep proteins in stable 293 cell lines using the Cre/loxP switching system
Authors: Ogasawara Yoji , Mizukami Hiroaki , Urabe Masashi , Kume Akihiro , Kanegae Yumi , Saito Izumu , Monahan John , Ozawa Keiya
Published: August 2015

Since the Rep proteins of adeno-associated virus (AAV) are harmful to cells, it is difficult to obtain stable cell lines that express them constitutively. In this study, stable 293 cell lines were obtained in which large Rep expression was inducible by using the Cre/loxP switching system. To determine the function of the induced Rep proteins, the packaging capacity was examined after supplementation with a plasmid expressing small Rep and Cap proteins. A significant amount of recombinant AAV (5·5×108 vector particles per 10 cm dish) was produced by transfection with a vector plasmid and infection with Cre-expressing recombinant adenovirus, indicating that the large Rep proteins retained the function required for packaging. These findings indicate that large Rep protein expression can be strictly regulated by the Cre/loxP system and will also serve as a basis for the development of an efficient AAV-packaging cell line.
TT viruses (TTV) of non-human primates and their relationship to the human TTV genotypes
Authors: Verschoor Ernst J. , Langenhuijzen Susan , Heeney Jonathan L.
Published: August 2015

Sera from eight different non-human primate species, in total 216 samples, were analysed for the presence of TT virus (TTV) sequences. A very high incidence of TTV infection was found in sera from both common chimpanzees and pygmy chimpanzees, 48·8% and 66·7%, respectively. Sequence analysis of PCR fragments from two pygmy chimpanzees and seven common chimpanzees resulted in a total of 14 different TTV sequences. Phylogenetic analysis, including human TTV of all known genotypes, revealed that: (i) TTV from pygmy chimpanzees are closely related to viruses from human genotypes 2 and 3; (ii) TTV sequences obtained from common chimpanzees cluster together with human TTV genotypes 5 and 6, the latter only at the protein level; (iii) TTV from the common chimpanzee subspecies Pan troglodytes verus and Pan troglodytes schweinfurthii cluster together, suggesting an ancient host–pathogen relationship before subspeciation 1·6 million years ago; and (iv) TTV of common and pygmy chimpanzees may have been acquired by these animals in different zoonotic events not longer than 2·5 million years ago.
Brome mosaic virus defective RNAs generated during infection of barley plants
Authors: Damayanti Tri Asmira , Nagano Hideaki , Mise Kazuyuki , Furusawa Iwao , Okuno Tetsuro
Published: August 2015

Brome mosaic virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By usingin vitrotranscripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.
PrP gene polymorphism and natural scrapie in Icelandic sheep
Authors: Thorgeirsdottir Stefania , Sigurdarson Sigurdur , Thorisson Hjalti Mar , Georgsson Gudmundur , Palsdottir Astridur
Published: August 2015

The association between scrapie and polymorphism of the prion protein (PrP) gene was studied in the Icelandic sheep breed. Polymorphism of the three codons, 136, 154 and 171, that are important for scrapie susceptibility was determined. A BspHI restriction analysis was used to study the alleles of codons 136 and 154, while density gradient gel electrophoresis (DGGE) was used to analyse codon 171 and detect new polymorphisms. The PrP allelic variant, VRQ (amino acids at codons 136, 154 and 171), was found to be highly statistically associated with scrapie, whereas the allelic variant, AHQ, was never found in scrapie-affected animals, a finding that is statistically significant. Iceland has a few scrapie-free regions, which are a part of a quarantine network. Homozygotes for the VRQ variant were found there at a low frequency, indicating that genetic susceptibility is not enough for scrapie to develop and further evidence for the infectious nature of the disease. A comparison of PrP genotypes between sheep outside and within the scrapie-free zones revealed an increase in the AHQ allelic variant in the latter. No polymorphism was found at codon 171 in a total of 932 sheep studied, all individuals having the glutamine allele. Two novel, rare PrP alleles were found using DGGE at codons 138 and 151, i.e. S138N and R151C. Their relevance to scrapie is still unclear, but the former was found in scrapie-affected sheep as well as healthy sheep, whereas the latter was only found in healthy sheep.
The 2A proteins of three diverse picornaviruses are related to each other and to the H-rev107 family of proteins involved in the control of cell proliferation
Authors: Hughes Pamela J. , Stanway Glyn
Published: August 2015

The 2A protein appears to be diverse among picornaviruses, in contrast to the other non-structural proteins, which have homologous structures and functions. In enteroviruses and rhinoviruses, 2A is a trypsin-like protease involved in protein processing and in shut-off of host-cell macromolecular synthesis. The aphthovirus and cardiovirus 2A is associated with an unusual processing event at the 2A/2B junction. It is shown here that the 2A protein of several diverse picornaviruses, the human parechoviruses, Aichi virus and avian encephalomyelitis virus, possess previously unrecognized conserved motifs and are likely to have a common function. Moreover, these motifs, a conserved histidine and flanking amino acids, an asparagine–cysteine dipeptide and a putative transmembrane domain, are characteristic of a family of cellular proteins, at least two of which are involved in the control of cell growth. These observations have important implications for an understanding of picornavirus genome structure and evolution, as well as pointing to possible functions of 2A in these viruses.
The complete nucleotide sequence of apple mosaic virus (ApMV) RNA 1 and RNA 2: ApMV is more closely related to alfalfa mosaic virus than to other ilarviruses
Authors: Shiel P. J. , Berger P. H.
Published: August 2015

The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus.
Functional analysis of proteins encoded by banana bunchy top virus DNA-4 to -6
Authors: Wanitchakorn Raktham , Hafner Gregory J. , Harding Robert M. , Dale James L.
Published: August 2015

Green fluorescent protein (GFP)-tagging was used to determine the intracellular localization pattern of the proteins encoded by banana bunchy top virus (BBTV) DNA-3, -4 and -6. The protein encoded by BBTV DNA-4, which possesses a hydrophobic N terminus, was found to localize exclusively to the cell periphery while the proteins encoded by BBTV DNA-3 and -6 were found in both the nucleus and the cytoplasm. Co-expression of the DNA-4 protein and the proteins encoded by BBTV DNA-3 and -6 revealed that the DNA-4 protein was able to re-locate the DNA-6 protein, but not the DNA-3 protein, to the cell periphery. The 29 amino acid N-terminal hydrophobic region of the DNA-4 gene product appeared to be essential for specific localization of this protein since deletion of this region abolished its ability to localize to the cell periphery. These results indicate that BBTV may utilize a system analogous to that of the begomoviruses with the BBTV DNA-6 protein acting as a nuclear shuttle protein (NSP) while the DNA-4 protein transports the NSP–DNA complexes to the cell periphery for intercellular transport. The protein encoded by BBTV DNA-5 was found to contain an LXCXE motif and yeast two-hybrid analysis revealed that the DNA-5 protein has retinoblastoma (Rb)-binding activity. This activity was dependent on an intact LXCXE motif since specific mutations to either the C or E residue completely abolished Rb-binding activity. These results indicate that the gene product of BBTV DNA-5 is an Rb-binding-like protein and may play an important role in host-cell cycle manipulation.
The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles
Authors: Sanderson Christopher M. , Hollinshead Michael , Smith Geoffrey L.
Published: August 2015

The vaccinia virus (VV) A27L gene encodes a 14 kDa protein that is required for the formation of intracellular enveloped virus (IEV) and, consequently, normal sized plaques. Data presented here show that A27L plays an additional role in VV assembly. When cells were infected with the VV WR32-7/Ind 14K, under conditions that repress A27L expression, transport of intracellular mature virus (IMV) from virus factories was inhibited and some IMV was found in aberrant association with virus crescents. In contrast, other VV mutants (vΔB5R and vΔF13L) that are defective in IEV formation produce IMV particles that are transported out of virus factories. This indicated a specific role for A27L in IMV transport. Induction of A27L expression at 10 h post-infection promoted the dispersal of clustered IMV particles, but only when microtubules were intact. Formation of IEV particles was also impaired when cells were infected with WR32-7/14K, a VV strain expressing a mutated form of the A27L protein; however, this mutation did not inhibit intracellular transport of IMV particles. Collectively, these data define two novel aspects of VV morphogenesis. Firstly, A27L is required for both IMV transport and the process of envelopment that leads to IEV formation. Secondly, movement of IMV particles between the virus factory and the site of IEV formation is microtubule-dependent.
Long-term mutation rates in the hepatitis B virus genome
Authors: Hannoun Charles , Horal Peter , Lindh Magnus
Published: August 2015

Mutations in the hepatitis B virus (HBV) genome have so far been investigated in cross-sectional or short-term longitudinal studies. Information about long-term changes is lacking due to the difficulty of sampling over long observation periods. In this study, a retrospective approach was used that allowed the analysis of changes in the viral genome from transmission to late stages of infection without the requirement for sampling early during this period. The entire viral genome was sequenced from serum samples of three mothers and their 10 adult children, who presumably had been infected vertically. The emergence of mutations between birth and sampling (mean 26·5 years) was assessed by comparing the individual sequences with the sequence of the strain assumed to have been transmitted. The mean differences from this sequence were 0·02 and 0·28% in seven asymptomatic and one symptomatic hepatitis B e antigen (HBeAg)-positive carriers, respectively, and 0·62 % in five HBeAg-negative carriers. Mutations occurred throughout the genome and 88% of the mutations caused amino acid substitutions spread over all genes. In HBeAg-negative carriers, the number of nucleotide and amino acid changes was independent of the severity of liver disease and, except the 1762AGG1764→TGA changes, no specific mutation was associated with liver disease. In conclusion, by using a novel method it was found that the entire HBV genome is extremely stable over long periods of time during the HBeAg-positive phase if the immune response (inflammation) is weak, whereas an average of 20 mutations emerged after development of hepatitis and/or loss of HBeAg without association with clinical outcome.
Transient IFN-γ synthesis in the lymph node draining a dermal site loaded with UV-irradiated herpes simplex virus type 1: an NK- and CD3-dependent process regulated by IL-12 but not by IFN-α/β
Authors: Riffault S. , Carrat C. , Milon G. , Charley B. , Colle J. H.
Published: August 2015

Our previous studies have shown that UV-inactivated, non-replicating herpes simplex virus type 1 (UV-HSV-1) triggers early and transient synthesis of IFN-α/β in the mouse regional lymph node when delivered upstream (i.e. in the ear dermis). In this study, it is demonstrated, by use of a quantitative RT–PCR readout assay, that IFN-γ mRNA expression was rapidly and transiently upregulated in draining lymph nodes when UV-HSV-1 was delivered in the ear dermis of C57Bl/6 mice. An increased number of IFN-γ-producing cells was also detected in the lymph node by flow cytometric analysis. Two different subsets of cells, namely DX5+NK cells and CD3ϵ+T cells, accounted for this early IFN-γ synthesis. Prompt upregulation of IFN-α and IL-12p40 mRNA was also recorded. We took advantage of IFN-α/β-receptor knockout and wild-type 129 mice to study a potential role of IFN-α/β in the signalling pathway leading to IFN-γ transcription/translation. IFN-γ mRNA upregulation still occurred in IFN-α/β-receptor−/−mice, showing that IFN-α/β was dispensable. The use of IL-12-neutralizing antibodies, prior to UV-HSV-1 delivery, confirmed the major role played by IL-12 in the early/transient IFN-γ burst.
The effects of antiviral therapy on the distribution of herpes simplex virus type 1 to ganglionic neurons and its consequences during, immediately following and several months after treatment
Authors: Thackray Alana M. , Field Hugh J.
Published: August 2015

Both famciclovir (FCV) and valaciclovir (VACV) are potent inhibitors of herpes simplex virus type 1 (HSV-1) in a murine cutaneous infection model. The object of the present study was to determine whether either drug had an effect on the anatomical distribution of infected neurons in the peripheral nervous system and to assess the consequences for infected cells during, immediately following and several months after a 9 day period of continuous treatment. Mice were inoculated via the neck with a recombinant strain of HSV-1 expressing the lacZ reporter gene under the immediate-early gene promoter. Sensory ganglia were sampled daily up to day 11 post-inoculation (p.i.) and infected cells were detected by means of the reporter gene product. Ganglia were also removed at 1·5 and 10 months p.i. and latency was assessed by explant co-cultivation and by using in situ hybridization to detect LAT-expressing neurons. While both drugs reduced the severity of acute infection markedly, neither compound completely prevented the relentless distribution of infection among peripheral nervous tissue. Furthermore, there was a difference between the compounds regarding the expression of the reporter gene during and after termination of treatment and in the number of residual LAT-positive neurons.
Cloning and identification of regulatory gene UL76 of human cytomegalovirus
Authors: Wang Shang-Kwei , Duh Chang-Yih , Chang Tai-Tsung
Published: August 2015

The major immediate-early promoter/enhancer (MIEP, −1139 to +52) of human cytomegalovirus (HCMV) is regulated by cell type-specific transcriptional factors, its own MIE proteins (IE2p40, IE1p55, IE1p72 and IE2p86) as well as viral proteins pUL69, pUL82 and pUL84. To investigate the hypothesis that the regulation of HCMV MIEP is modulated by additional viral genes, HCMV (AD169) genomic sublibraries were constructed and in vitro transient co-transfection assays were performed to assess the ability of these sublibraries to modulate MIEP expression. In this study, enhancement of MIEP expression was exhibited by a number of sublibraries, from one of which a genomic clone was selected for augmentation of expression. Subcloning the insert fragment led to the identification of the responsible locus, UL76. To generate a UL76-specific antibody for immunodetection, the UL76 ORF was constructed as a histidine-tagged fusion protein that was produced in prokaryotic cells. A polyclonal antibody raised against the UL76 fusion protein immunoreacts with a protein of 38 kDa (pUL76) in UL76 ORF-transfected cells. Additionally, pUL76 is present in HCMV-infected cells at the immediate-early to late stages of the reproductive cycle. Characterized by its highly basic composition (predicted pI 11·6), a free form of pUL76 tagged with green fluorescent protein was found to localize exclusively to the nucleus. In this report, pUL76 is defined as a novel regulatory protein that modulates both activation and repression of gene expression, depending on the promoter context and the ratio of transfected effector DNA.
Molecular characterization of measles viruses isolated in Victoria, Australia, between 1973 and 1998
Authors: Chibo Doris , Birch Christopher J. , Rota Paul A. , Catton Michael G.
Published: August 2015

Molecular epidemiology studies have made significant contributions to the control of measles virus infection through the identification of source and transmission pathways of the virus. These studies allow observation of changes in measles virus genotypes over time in a particular geographical location, clarification of epidemiological links during measles outbreaks, separation of indigenous strains from newly imported strains and distinction between vaccine- and wild-type virus-associated illness. A total of 35 wild-type measles viruses identified in Victoria, Australia, between 1973 and 1998 were characterized by nucleic acid sequence analysis of the nucleoprotein gene and, in some cases, the haemagglutinin gene. Relatedness between the viruses was studied and genotypes were assigned using a classification scheme recently proposed by the World Health Organization. Five recognized genotypes (C2, D1, D4, D5 and H) and one previously undescribed genotype, which we propose to be D7, were identified. Successive replacement of measles virus genetic lineages occurred in Victoria, with no evidence of temporal overlap, during this 25 year period. This pattern of circulation is likely to represent serial importation of wild-type measles virus strains from overseas foci of measles virus infections.
DNA encoding the attachment (G) or fusion (F) protein of respiratory syncytial virus induces protection in the absence of pulmonary inflammation
Authors: Bembridge Gary P. , Rodriguez Nuria , Garcia-Beato Regina , Nicolson Carolyn , Melero Jose A. , Taylor Geraldine
Published: August 2015

Significant protection against respiratory syncytial virus (RSV) infection was induced in mice vaccinated intramuscularly (i.m.) with DNA encoding the F or G protein of RSV. The amounts of IgG1 of IgG2a antibodies in mice immunized with DNA-G alone were similar. However, the antibody response in mice co-immunized with DNA-G and DNA encoding IL-4 (DNA-IL-4) was strongly biased towards IgG1. In contrast, the antibody response in mice co-immunized with DNA-G and DNA-IL-2, -IL-12 or-IFN-γ was biased towards IgG2a. Mice vaccinated with DNA-F either alone or in combination with DNA encoding cytokines developed a predominant RSV-specific IgG2a response, which was most pronounced in mice co-immunized with DNA-F and DNA-IL-12 or -IFN-γ. Vaccinated mice developed only a slightly enhanced pulmonary inflammatory response following RSV challenge. More significantly, and in contrast to mice scarified with recombinant vaccinia virus expressing the G protein, mice vaccinated i.m. with DNA-G did not develop pulmonary eosinophilia, even when the immune response was biased towards a Th2 response by co-administration of DNA-IL-4.
Virus morphogenesis of Helicoverpa armigera nucleopolyhedrovirus in Helicoverpa zea serum-free suspension culture
Authors: Lua Linda H. L. , Reid Steven
Published: August 2015

Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) replication in Helicoverpa zea serum-free suspension culture was studied in detail and the sequence of virus morphogenesis was determined by transmission electron microscopy. By 16 h post-infection (p.i.), virus replication was observed in the virogenic stroma by the appearance of nucleocapsids. Polyhedron formation was detected by 24 h p.i. and the polyhedron envelope (PE) was completely formed by 72 h p.i. PE morphogenesis of HaSNPV is significantly different compared to the extensively studied Autograph californica (Ac)MNPV. In AcMNPV-infected cells, fibrillar structures are found in both cytoplasm and nuclei, and the fibrillar structures in nuclei are in close association with maturing polyhedra during PE formation. Fibrillar structures that resemble the AcMNPV fibrillar structures were detected only in the cytoplasm of HaSNPV-infected cells and appeared to interact with calyx precursors there, but their role in PE formation is unclear. However, prominent calyx precursor structures of various shapes and sizes were observed in the nuclei of HaSNPV-infected cells as well, and they appeared to interact with polyhedra during PE formation. Both the calyx precursor structure and the cytoplasmic fibrillar structure were detected only after HaSNPV virion occlusion had started, indicating that they might have a role in formation of PE. Similar calyx precursor structures and cytoplasmic fibrillar structures were observed in both serum-supplemented and serum-free suspension cultures, as well as in HaSNPV-infected larval tissues, indicating that the structures observed are not cell culture artefacts.
Strain-specific propagation of PrPSc properties into baculovirus-expressed hamster PrPC
Authors: Iniguez Volga , McKenzie Debbie , Mirwald Jean , Aiken Judd
Published: August 2015

The conversion of the cellular isoform of the prion protein (PrPC) to the abnormal disease-associated isoform (PrPSc) has been simulated in cell-free conversion reactions in which PrPSc-enriched preparations induce the conformational transition of PrPC into protease-resistant PrP (PrP-res). We explored the utility of recombinant hamster (Ha)PrPC purified from baculovirus-infected insect cells (bacHaPrPC) as a replacement for mammalian-derived HaPrPC in the conversion reactions. Protease-resistant recombinant HaPrP was generated after incubation of 35S-bacHaPrPC with PrPSc-enriched preparations. Moreover strain-specific PrP-res was also reproduced using insect-cell derived HaPrPC and PrPSc from two different strains of hamster-adapted transmissible mink encephalopathy, designated hyper (HY) and drowsy (DY). Two strain-mediated properties were tested: (i) molecular mass of the protease-digested products and (ii) relative resistance to proteinase K (PK) digestion. Similar to in vivo generation of PrPHY and PrPDY, the converted products selectively reproduced both characteristics, with the DY conversion product being smaller in size and less resistant to PK digestion than the HY product. These data demonstrate that non-mammalian sources of recombinant HaPrP can be converted into PK-resistant form and that strain-mediated properties can be transmitted into the newly formed PrP-res.
Characterization of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus)
Authors: Mäkinen Kristiina , Mäkeläinen Katri , Arshava Natalya , Tamm Tiina , Merits Andres , Truve Erkki , Zavriev Sergei , Saarma Mart
Published: August 2015

The polyprotein ofCocksfoot mottle virus(CfMV; genusSobemovirus) is translated from two overlapping open reading frames (ORFs) 2a and 2b by a −1 ribosomal frameshifting mechanism. In this study, a 12 kDa protein was purified from viral RNA-derived samples that appears to correspond to the CfMV genome-linked protein (VPg). According to the determined N-terminal amino acid sequence, the VPg domain is located between the serine proteinase and replicase motifs and the N terminus of VPg is cleaved from the polyprotein between glutamic acid and asparagine residues. Western blot analysis of infected plant material showed that the polyprotein is processed at several additional sites. An antiserum against the ORF 2a product recognized six distinct proteins, whereas, of these, the VPg antiserum clearly recognized only a 24 kDa protein. This indicates that the fully processed 12 kDa VPg detected in viral RNA-derived samples is a minor product in infected plants. An antiserum against the ORF 2b product recognized a 58 kDa protein, which indicates that the fully processed replicase is entirely or almost entirely encoded by ORF 2b. The origin of the detected cleavage products and a proposed polyprotein processing model are discussed.
Molecular analysis of a serotype 8 human astrovirus genome
Authors: Méndez-Toss Martha , Romero-Guido Pedro , Munguía Maria Elena , Méndez Ernesto , Arias Carlos F.
Published: August 2015

Human astroviruses are an important cause of gastroenteritis. As part of a molecular epidemiological study carried out in Mexico a human astrovirus isolate, Yuc-8, was adapted to grow in CaCo-2 cells, and its entire genome was sequenced. A 15 amino acid deletion in ORF1a, which has been associated with adaptation of astroviruses to grow in cells other than CaCo-2, was present in Yuc-8. Comparative sequence analysis of the Yuc-8 ORF2 with reported human astrovirus sequences revealed that this isolate belongs to genotype (serotype) 8. Two distinct domains in ORF2 were observed: an amino-terminal domain (residues 1 to 415), with identities higher than 81% among the strains analysed, and a carboxy-terminal domain (residues 416 to 782) with identities between 36 and 60%. Two non-superimposable phylogenetic trees were generated by separate analysis of these two domains, suggesting that a differential selective pressure is exerted along the structural polyprotein.
B-cell epitopes of the envelope glycoprotein of caprine arthritis–encephalitis virus and antibody response in infected goats
Authors: Bertoni Giuseppe , Hertig Christian , Zahno Marie-Luise , Vogt Hans-Rudolf , Dufour Sophie , Cordano Pablo , Peterhans Ernst , Cheevers William P. , Sonigo Pierre , Pancino Gianfranco
Published: August 2015

Goats infected with caprine arthritis–encephalitis virus (CAEV) develop high titres of antibodies to Env. Not only is no consistent neutralizing response found but anti-Env antibodies have even been associated with disease in infected goats. To identify the continuous antigenic determinants involved in this atypical anti-Env response, we mapped CAEV-CO Env by screening an epitope expression library with infected goat sera. In addition to the four previously described epitopes, seven novel antigenic sites were identified, of which five were located on the surface (SU) and two in the transmembrane (TM) subunits of Env. The SU antibody-binding domains located in the variable regions of the C-terminal part of the molecule (SU3 to SU5) showed the strongest reactivity and induced a rapid seroconversion in six experimentally infected goats. However, the response to these immunodominant epitopes did not appear to be associated with any neutralizing activity. The pattern of serum reactivity of naturally infected goats with these epitopes was restricted, suggesting a type-specific reaction. Interestingly, the reactivity of peptides representing SU5 sequences derived from CAEV field isolates varied with the geographical and/or breeding origin of the animals. This suggests that peptides corresponding to the immunodominant SU epitopes may well be useful in the serotyping of CAEV isolates. Furthermore, the identification of the CAEV Env epitopes will permit us to functionally dissect the antibody response and to address the role of anti-Env antibodies either in the protection from or in the pathogenesis of CAEV infection.
Herpes simplex virus type 2 induces secretion of IL-12 by macrophages through a mechanism involving NF-κB
Authors: Malmgaard Lene , Paludan Søren R. , Mogensen Søren C. , Ellermann-Eriksen Svend
Published: August 2015

Interleukin (IL)-12 is an important proinflammatory and immunoregulatory cytokine expressed primarily by macrophages. Although IL-12 appears to be essential for clearance of many bacterial and parasitic infections, only little is known about the production and regulation of this cytokine during viral infections. In this study we have shown that infection of mouse macrophages with herpes simplex virus type 2 (HSV-2) induces secretion of the p40 subunit of IL-12, and this induction was synergistically enhanced by interferon (IFN)-γ. The production of IL-12 p40 was accompanied by production of bioactive IL-12 p70, since HSV-2-induced IFN-γ secretion was blocked by neutralizing antibodies against IL-12. The IL-12-inducing effect of HSV-2 was abrogated when virus infectivity was destroyed by heat or UV irradiation, indicating that a functional viral genome is required and that interaction of viral glycoproteins with cellular receptors is not sufficient. Production of IL-12 p40 was transcriptionally regulated and requiredde novoprotein synthesis. Although IFN-α, IL-1β and tumour necrosis factor-α marginally influenced IL-12 production, they did not seem to constitute the endogenous factor(s) responsible for the effect of the virus infection. HSV-2 infection induced nuclear-binding activity to the κB halfsite of the IL-12 p40 promoter, and inhibitors of nuclear factor (NF)-κB activation significantly reduced IL-12 p40 production in infected cells. Collectively our data show that HSV-2 infection of murine macrophages induces production of IL-12 through a mechanism requiring intermediary synthesis of viral or host proteins and involving activation of NF-κB.
Identification of a Kd-restricted antigenic peptide encoded by murine cytomegalovirus early gene M84
Authors: Holtappels Rafaela , Thomas Doris , Reddehase Matthias J.
Published: August 2015

The two sister cytomegaloviruses (CMVs), human and murine CMV, have both evolved immune evasion functions that interfere with the major histocompatibility complex class I (MHC-I) pathway of antigen processing and presentation and are effectual in the early (E) phase of virus gene expression. However, studies on murine CMV have shown that E-phase immune evasion is leaky. An E-phase protein involved in immune evasion, namelym04-gp34, was found to simultaneously account for an antigenic peptide presented by the MHC-I molecule Dd. Recent work has demonstrated the induction of protective immunity specific for the E-phase proteinM84-p65, one of two murine CMV homologues of the human CMV matrix proteinUL83-pp65. In this study, the identification of the MHC-I Kd-restricted M84 peptide297AYAGLFTPL305is documented. This peptide is the third antigenic peptide described for murine CMV and the second that escapes immunosubversive mechanisms.
Characterization of repetitive DNA regions and methylated DNA in ascovirus genomes
Authors: Bigot Yves , Stasiak Karine , Rouleux-Bonnin Florence , Federici Brian A.
Published: August 2015

The accompanying phylogenetic study of large double-stranded DNA viruses based on their δ DNA polymerase genes suggests that ascoviruses (family Ascoviridae) and iridoviruses (family Iridoviridae) are closely related and may share a common ancestor. This relationship was unexpected because of marked differences between these viruses. Iridoviruses produce icosahedral virions and occur broadly among vertebrates and invertebrates, whereas ascoviruses typically produce reniform or bacilliform virions and are restricted to insect hosts, primarily lepidopterans. Detailed comparisons of these two virus types are not possible because fundamental information on the properties of the virions and their genomes is lacking, especially for ascoviruses. To facilitate further investigation of the putative evolutionary relationship between ascoviruses and iridoviruses, the genomes of representative viruses from each family were compared with respect to physical configuration, presence of DNA repeats and degree of DNA methylation. Genomes from Spodoptera frugiperda (SfAV1), Heliothis virescens (HvAV3) and Diadromus pulchellus (DpAV4) ascoviruses were all found to be circular and partially superhelical and to contain large interspersed repeats of 1–3 kbp. Mosquito (IV type 3), lepidopteran (IV type 6) and isopod (IV type 31) iridovirus genomes were all linear and lacked large regions of repetitive DNA. Ascovirus and iridovirus genomes were methylated and one, DpAV4, had the highest degree of methylation of any reported animal DNA virus. The major differences in the physical and biochemical characteristics of ascoviruses and iridoviruses reported here provide a foundation for further studies of their relatedness while making their possible close relationship and divergence during evolution of even greater interest.
A new ascovirus from Spodoptera exigua and its relatedness to the isolate from Spodoptera frugiperda
Authors: Cheng Xiao-Wen , Carner Gerald R. , Arif Basil M.
Published: August 2015

A new ascovirus was isolated from Spodoptera exigua in Indonesia and was tentatively assigned as a new species, Spodoptera exigua ascovirus 5a (SeAV-5a) according to the present ICTV ascovirus naming scheme based on DNA restriction fragment length polymorphism (RFLP), hybridization, formation of occlusion body, tissue tropism and host spectrum. SeAV-5a replicated primarily in the fat body of susceptible hosts. SeAV-5a could be transmitted to S. frugiperda, Pseudoplusia includens and Trichoplusia ni, but not to Heliothis virescens. Infection with SeAV-5a arrested growth of the hosts, but prolonged their survival, which continued up to 33 days. Clusters of virions were seen inside the characteristic vesicles. Occasionally, virions were contained within vacuoles (one to five per vacuole) and some virions were embedded in occlusion bodies. The size of the SeAV-5a virion was 347×134 nm; however, aberrant long secondary viral products were also seen. The presence of occlusion body and Southern hybridization and Western immunoblot analyses suggest that SeAV-5a is more closely related to S. frugiperda ascovirus 1a (SfAV-1a) than to Trichoplusia ni ascovirus
2 (TnAV-2). Certain regions of the 182 kb genome of SeAV-5a showed hybridization to that of SfAV-1a. Two fragments in each of the SfAV-1a EcoRI and HindIII digests hybridized to the SeAV-5a genomic DNA probe. Five to eight HindIII and EcoRI fragments in SeAV-5a DNA hybridized to the SfAV-1a genomic probe.
Agnoprotein-1a of avian polyomavirus budgerigar fledgling disease virus: identification of phosphorylation sites and functional importance in the virus life-cycle
Authors: Liu Qiang , Hobom Gerd
Published: August 2015

The avian polyomavirus budgerigar fledgling disease virus (BFDV) encodes an unusual set of four agnoproteins in its late upstream region. Of the two pairs of these proteins, which overlap each other in two different reading frames, the pL1-promoted agnoprotein-1a (agno-1a) is the dominant species and is able to support virus propagation in the absence of the other three polypeptides. Viral BFDV agno-1a, and also agno-1a expressed via an influenza virus vector, consists of a complex series of electrophoretically separable subspecies that can be reduced by phosphatase action down to a primary unphosphorylated protein with an apparent molecular mass of 31 kDa. Through peptide mass spectrometry and site-directed mutagenesis, the positions of four serine and three threonine residues have been determined as phosphate-accepting groups, which are partially modified by the combined action of three different cellular kinases. Since extensively phosphorylated agno-1a is required for its intracellular function, control over VP protein expression, and unphosphorylated agno-1a is observed as an additional component in the BFDV virion, both extreme subspecies appear to be drawn from that complex mixture, which also includes the intermediate stages of phosphorylation.
Insertion sites for recombinant vaccinia virus construction: effects on expression of a foreign protein
Authors: Coupar Barbara E. H. , Oke Pamela G. , Andrew Marion E.
Published: August 2015

The expression of antigens or other molecules from recombinant vaccinia viruses requires the insertion of coding sequence at specific sites in the viral genome. Here we investigate the influence of two different sites on the level of protein expressed during a viral infection. The level of immune response in mice to vaccinia virus-expressed murine interleukin 2 (IL-2) or IL-4 varied depending on whether the coding sequence was inserted into the vaccinia virus thymidine kinase (tk) gene or into the HindIII F fragment of the viral genome where herpes simplex virus (HSV) tk was used as a selectable marker. In each case the intensity of the response was greater when the relevant gene was expressed from the HindIII F insertion site. In order to quantify these differences a series of recombinant viruses expressing luciferase was constructed. Luciferase activity from coding sequence inserted into the HindIII F fragment was significantly higher than that from the tk gene insertion, provided HSV tk+ constructs were compared. Insertion of a marker gene (HSV tk) into the HindIII F site with disruption of the F7L open reading frame led to a reduced level of luciferase expressed from the tk insert, despite more than 45 kb of intervening sequence. In mice, luciferase expression was higher from the HindIII F inserted gene than from the tk insert in both lungs and ovaries.
Recombinant measles virus requiring an exogenous protease for activation of infectivity
Authors: Maisner Andrea , Mrkic Branka , Herrler Georg , Moll Markus , Billeter Martin A. , Cattaneo Roberto , Klenk Hans-Dieter
Published: August 2015

Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnartm-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.