- Volume 89, Issue 6, 2008

Characterization of the J virus or, in keeping with recent nomenclature recommendations, J paramyxovirus (JPV) genome revealed a unique genome structure, consisting of eight genes in the order 3′-N-P/V/C-M-F-SH-TM-G-L-5′. The small hydrophobic (SH) protein and the transmembrane (TM) protein genes are predicted to encode proteins 69 and 258 aa in size, respectively. The 4401 nt attachment (G) protein gene, much larger than most other paramyxovirus attachment protein genes sequenced to date, encodes a putative 709 aa attachment protein and contains distally a second open reading frame (ORF-X) 2115 nt long. Experiments undertaken in this study were intended to confirm the sequence-based gene allocation of JPV and to determine if proteins encoded by the SH gene, the novel TM gene and ORF-X are expressed. Northern blot analyses carried out on mRNA purified from JPV-infected cells indicated that the putative transcription initiation and termination sequences flanking the SH and TM genes are functional, consistent with their allocation as discrete genes, although a high level of read-through was observed across almost all transcriptional boundaries. Probes specific to the G protein coding region and ORF-X both identified an mRNA species corresponding to the predicted length of the G gene, confirming sequence-based predictions. While the SH and TM proteins were both detected in infected cells, no evidence was found for the expression of ORF-X. Preliminary studies indicate that the novel TM protein is a type II glycosylated integral membrane protein, orientated with its C terminus exposed at the cell surface.

The Borna disease virus (BDV) proteins X and P are translated from a bicistronic viral mRNA. Here, it was shown that the rescue of recombinant BDV from cDNA was enhanced approximately eightfold if reconstitution of the viral polymerase complex was performed with an expression vector encoding X and P rather than P alone. The results provide evidence that appropriate amounts of X reduce the previously reported high sensitivity of the BDV polymerase to imbalances between the viral proteins N and P. These data indicate that X buffers an unfavourable excess of P, thereby stimulating the assembly of functional BDV polymerase complexes.

Ten virus isolates belonging to species Getah virus (GETV) have been obtained during surveys for arboviruses in China since 1964. Seven of these isolates (YN0540, YN0542, SH05-6, SH05-15, SH05-16, SH05-17 and GS10-2) were obtained during the current study. The full-length sequences of three Chinese isolates (M1, isolated in 1964; HB0234, isolated in 2002; YN0540, isolated in 2005) were determined. The full-length sequences of these isolates were respectively 11 696, 11 686 and 11 690 nt, and showed more than 97 % intraspecies identity. Deletions were found in the capsid protein of strain M1 and non-structural protein nsP3 of strain HB0234. The E2 gene and 3′ UTR of all ten isolates were also characterized. The E2 gene of the Chinese GETV isolates showed nucleotide sequence identities of 98–100 % when compared with other GETV isolates. In the 3′ UTR of the Chinese isolates, an insertion of 10 consecutive adenine residues (nt 189–198) appeared in strain M1, and 9 or 3 consecutive adenines were found towards the 3′ end of the third RES in strains SH05-6 and SH05-15, respectively. The 3′ UTRs of the Chinese isolates showed a deletion between positions 45 and 54 and nucleotide transitions at positions 43, 64 and 148. Sequence and phylogenetic analyses showed that there was a relatively high degree of conservation among GETV isolates. The isolation of GETV from various provinces in China and also in Russia and Mongolia (including regions of the northern tundra) are an indication of changes in the world distribution of this re-emerging virus.

Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein expression and the subcellular localization of nsP1 in induced cells were similar to those in virus-infected cells. The nsP1 expressed by stable, inducible cell lines or by SFV-infected HEK293 T-REx cells was a stable protein with a half-life of approximately 5 h. In contrast to SFV infection, induction of nsP1 expression had no detectable effect on cellular transcription, translation or viability. Induction of expression of nsP1 or nsP16D interfered with multiplication of SFV, typically resulting in a 5–10-fold reduction in virus yields. This reduction was not due to a decrease in the number of infected cells, indicating that nsP1 expression does not block virus entry or initiation of replication. Expression of nsP1 interfered with virus genomic RNA synthesis and delayed accumulation of viral subgenomic RNA translation products. Expression of nsP1 with a mutation in the palmitoylation site reduced synthesis of genomic and subgenomic RNAs and their products of translation, and this effect did not resolve with time. These results are in agreement with data published previously, suggesting a role for nsP1 in genomic RNA synthesis.

Recombination can facilitate the accumulation of mutations and accelerate the emergence of resistance to current antiretroviral therapies for human immunodeficiency virus (HIV) infection. Yet, since recombination can also dissociate favourable combinations of mutations, the benefit of recombination to HIV remains in question. The confounding effects of mutation, multiple infections of cells, random genetic drift and fitness selection that underlie HIV evolution render the influence of recombination difficult to unravel. We developed computer simulations that mimic the genomic diversification of HIV within an infected individual and elucidate the influence of recombination. We find, interestingly, that when the effective population size of HIV is small, recombination increases both the diversity and the mean fitness of the viral population. When the effective population size is large, recombination increases viral fitness but decreases diversity. In effect, recombination enhances (lowers) the likelihood of the existence of multi-drug resistant strains of HIV in infected individuals prior to the onset of therapy when the effective population size is small (large). Our simulations are consistent with several recent experimental observations, including the evolution of HIV diversity and divergence in vivo. The intriguing dependencies on the effective population size appear due to the subtle interplay of drift, selection and epistasis, which we discuss in the light of modern population genetics theories. Current estimates of the effective population size of HIV have large discrepancies. Our simulations present an avenue for accurate determination of the effective population size of HIV in vivo and facilitate establishment of the benefit of recombination to HIV.

Small ruminant lentiviruses (SRLV) are widespread amongst domesticated goats and sheep worldwide, but have not been clearly identified in wild small ruminants, where they might constitute an animal health risk through contamination from local domesticates. SRLV proviruses from three ibexes from the French Alps are described and sequences from their gag gene and long terminal repeats (LTRs) were compared with sequences from local goats and goat/ibex hybrids. The ibex and hybrid proviruses formed a closely related group with <2 % nucleotide difference. Their LTRs were clearly distinct from those of local goats or reference SRLV sequences; however, their gag sequences resembled those from one local goat and reference sequences from caprine arthritis encephalitis virus rather than visna/maedi virus. One SRLV-positive ibex from a distant site shared similarities with the other ibexes studied in both its gag and LTR sequences, suggesting that a distinct SRLV population could circulate in some wild ibex populations.

The rotavirus non-structural protein NSP4 functions as the viral enterotoxin and intracellular receptor for the double-layered particles (DLP). The full-length protein cannot be expressed and/or purified to homogeneity from bacterial or insect cells. However, a bacterially expressed and purified mutant lacking the N-terminal 72 aa (ΔN72) was recently obtained from strains Hg18 and SA11 exhibiting approximately 17–20-, 150–200- and 13166–15800-fold lower DD50 (50% diarrhoea-inducing dose) values in suckling mice compared with that reported for the partially pure, full-length protein, a C-terminal M175I mutant and a synthetic peptide comprising aa 114–135, respectively, suggesting the requirement for a unique conformation for optimal functions of the purified protein. The stretch of approximately 40 aa from the C terminus of the cytoplasmic tail of the endoplasmic reticulum-anchored NSP4 is highly flexible and exhibits high sequence variation compared with the other regions, the significance of which in diarrhoea induction remain unresolved. Here, it was shown that every amino acid substitution or deletion in the flexible C terminus resulted in altered conformation, multimerization, trypsin resistance and thioflavin T (ThT) binding, and affected DLP binding and the diarrhoea-inducing ability of the highly diarrhoeagenic SA11 and Hg18 ΔN72 in suckling mice. These studies further revealed that high ThT fluorescence correlated with efficient diarrhoea induction, suggesting the importance of an optimal ThT-recognizable conformation in diarrhoea induction by purified NSP4. These results based on biological properties provide a possible conformational basis for understanding the influence of primary sequence variations on diarrhoea induction in newborn mice by purified NSP4s that cannot be explained by extensive sequence analyses.

Insect responses that are specific for virus infection have been investigated using the genetically tractable Drosophila melanogaster. Most studies focus on interactions with Drosophila C virus (DCV), which is a member of the family Dicistroviridae. DCV is a non-enveloped, T=3 icosahedral virus with a positive-sense RNA genome. It was demonstrated recently that several genes controlled by the Jak-STAT pathway are specifically upregulated upon DCV infection. To investigate the virus factors that induce these responses, we used the Jak-STAT regulated genes as reporter genes. Challenge of flies with non-infectious DCV particles or double-stranded RNA did not stimulate significant upregulation of the antiviral response genes. In addition, there was no difference in reporter gene upregulation between Drosophila challenged with three different strains of DCV. This suggests that upregulation of these Drosophila genes may require virus replication and may involve the non-structural proteins of DCV.

Bovine herpesvirus type 1 (BHV-1) is an important pathogen that can initiate bovine respiratory disease complex. Like other members of the subfamily Alphaherpesvirinae, BHV-1 establishes latency in sensory neurons. The latency-related (LR) gene expresses a family of alternatively spliced transcripts in infected sensory neurons that have the potential to encode several LR proteins. An LR mutant virus that contains three stop codons near the 5′ terminus of the first open reading frame in the LR gene does not express two LR proteins or reactivate from latency. In addition, the LR mutant virus induces higher levels of apoptosis in trigeminal ganglionic neurons and grows less efficiently in certain tissues of infected calves. In spite of the reduced pathogenesis, the LR mutant virus, wild-type BHV-1 and the LR rescued virus exhibit identical growth properties in cultured bovine cells. In this study, we demonstrated that during early phases of productive infection the LR mutant virus expressed higher levels of LR-RNA relative to the LR rescued virus or wt BHV-1. Bovine kidney cells infected with the LR mutant virus also induced higher levels of beta interferon RNA and interferon response genes. These results suggest that inappropriate expression of LR-RNA, in the absence of LR protein expression, may influence the latency-reactivation cycle and pathogenic potential of BHV-1.

To facilitate tracing of virion movement, the non-essential capsid proteins pUL35 of herpes simplex virus type 1 and pseudorabies virus (PrV) have been tagged with green fluorescent protein (GFP). However, the biological relevance of PrV pUL35 and the functionality of the fusion proteins have not yet been investigated in detail. We generated PrV mutants either lacking the 12 kDa UL35 gene product, or expressing GFP fused to the N terminus of pUL35. Remarkably, both mutants exhibited significant replication defects in rabbit kidney cells, which could be corrected in pUL35-expressing cells. After intranasal infection of mice both mutants showed delayed neuroinvasion, and survival times of the animals were extended to 3 days, compared with 2 days after wild-type infection. Thus, fusion of pUL35 with GFP resulted in a non-functional protein, which has to be considered for the use of corresponding mutants in tracing studies.

Herpesviruses are ancient pathogens that infect all vertebrates. The most conserved component of their entry machinery is glycoprotein B (gB), yet how gB functions is unclear. A striking feature of the murid herpesvirus 4 (MuHV-4) gB is its resistance to neutralization. Here, we show by direct visualization of infected cells that the MuHV-4 gB changes its conformation between extracellular virions and those in late endosomes, where capsids are released. Specifically, epitopes on its N-terminal cell-binding domain become inaccessible, whilst non-N-terminal epitopes are revealed, consistent with structural changes reported for the vesicular stomatitis virus glycoprotein G. Inhibitors of endosomal acidification blocked the gB conformation switch. They also blocked capsid release and the establishment of infection, implying that the gB switch is a key step in entry. Neutralizing antibodies could only partially inhibit the switch. Their need to engage a less vulnerable, upstream form of gB, because its fusion form is revealed only in endosomes, helps to explain why gB-directed MuHV-4 neutralization is so difficult.

Transcripts for the Epstein–Barr virus (EBV)-encoded nuclear antigens are initiated at the alternative promoters Wp, Cp and Qp. Although the host cell-dependent activity of Cp is regulated by DNA methylation, Qp is unmethylated independently of its activity. Because histone modifications affect the chromatin structure, we compared the levels of diacetylated histone H3, tetraacetylated histone H4 and histone H3 dimethylated on lysine 4 (H3K4me2) at Cp and Qp, in well characterized cell lines representing the major EBV latency types. We found an activity-dependent histone code: acetylated histones marked active Cp, whereas active Qp was selectively enriched both in acetylated histones and H3K4me2. We concluded that active (but not silent) Cp and Qp are located to ‘acetylation islands’ in latent, episomal EBV genomes, similar to the active chromatin domains of the human genome.

Myxoma virus (MYXV), a leporide-specific poxvirus, represents an attractive candidate for the generation of safe and non-replicative vaccine vectors for other species. With the aim of developing new recombinant vaccines for ruminants, we evaluated the safety and the immunogenicity of recombinant MYXV in sheep. In vitro studies indicated that ovine primary fibroblasts were not permissive for MYXV and that infection of ovine peripheral blood mononuclear cells occurred at a low rate. Although non-specific activation significantly improved the susceptibility of lymphocytes, MYXV infection remained abortive. Histological and immunohistochemical examination at the inoculation sites revealed the development of an inflammatory process and allowed the detection of sparse infected cells in the dermis. In addition, inoculated sheep developed an antibody response directed against MYXV and the product of the transgene. Overall, these results provide the first line of evidence on the potential of MYXV as a viral vector for ruminants.

Human immunodeficiency virus (HIV)-positive women have high rates of cervical squamous intraepithelial lesions (SIL) and concurrent human papillomavirus (HPV) infections with a variety of genotypes whose oncogenic risk is poorly documented. The prevalence and persistence of HPV genotypes and HPV16 variants were analysed in 112 HIV-positive and 115 HIV-negative Italian women. HIV-positive women were more likely than HIV-negative women to be infected by HPV at the initial examination (39.3 vs 13.9 %, P<0.001) and to have a higher period prevalence of HPV infection over a 3-year follow-up (43.8 % vs 17.4 %, P<0.001), regardless of CD4+ cell counts and anti-retroviral therapy. ‘High-risk’ and ‘probable high-risk’ HPVs (types 16, 18, 31, 33, 35, 45, 52, 58 and 66), among the 20 different viral genotypes identified, were predominant in HIV-positive (33.9 %) compared with HIV-negative (13.9 %) women. Among HIV-infected women, with normal cytology as well as with SIL of any grade, the most common genotypes were HPV16 followed by HPV81, -58, -72, -33 and -62. HPV16 isolates from 18 HIV-positive and eight HIV-negative women were classified into variant lineages based on sequencing analysis of E6 and E7 genes and the long control region. Whilst the HPV16 G350 European variant was prevalent in both HIV-positive (10.7 %) and -negative women (3.5 %), HPV16 African 2 variant was only detected in HIV-positive women (3.6 %), suggesting different sexual mixing behaviours. The increased prevalence of uncommon viral genotypes and HPV16 variants in HIV-positive Italian women underscores the need to target a wide range of HPV types in cervical screening of high-risk women.

Bovine papillomaviruses of types 1 and 2 (BPV-1 and -2) chiefly contribute to equine sarcoid pathogenesis. However, the mode of virus transmission and the presence of latent infections are largely unknown. This study established a PCR protocol allowing detection of ≤10 copies of the BPV-1/-2 genes E5 and L1. Subsequent screening of peripheral blood mononuclear cell (PBMC) DNA derived from horses with and without BPV-1/2-induced skin lesions demonstrated the exclusive presence of E5, but not L1, in PBMCs of BPV-1/2-infected equines. To validate this result, a blind PCR was performed from enciphered PBMC DNA derived from 66 horses, revealing E5 in the PBMCs of three individuals with confirmed sarcoids, whereas the remaining 63 sarcoid-free animals were negative for this gene. L1 could not be detected in any PBMC DNA, suggesting either deletion or interruption of this gene in PBMCs of BPV-1/-2-infected equines. These results support the hypothesis that PBMCs may serve as host cells for BPV-1/-2 DNA and contribute to virus latency.

JC virus (JCV) DNA replication occurs in the nuclei of infected cells. The level of JCV genome expression depends on nucleotide sequences in the viral regulatory region and their interaction with host-cell nuclear transcription factors. Our previous studies showed a higher level of NF-1X in JCV-permissive cells compared with the other members of the NF-1 family, NF-1A, B and C, which suggests that NF-1X plays a positive role in JCV multiplication. It remained unclear whether a reduction in the level of NF-1A, which is expressed abundantly in JCV-non-permissive cell types, leads to an increase in JCV multiplication. In this study, we show that downregulation of NF-1A expression in JCV-non-susceptible progenitor and HeLa cells results in a reversion to susceptibility for JCV multiplication. These data demonstrate that a higher level of NF-1A protein in JCV-non-permissive cell types, compared with the level of NF-1X, may be acting as a negative regulator at the JCV promoter to control JCV multiplication.

Thysanoplusia orichacea multicapsid nucleopolyhedrovirus (ThorMNPV) carrying an enhanced green fluorescent protein (EGFP) gene expression cassette (vThGFP) was used to study host-range mechanisms. Infection kinetics showed that vThGFP replication in Sf21 cells was too slow to suppress cell growth. Wide-host-range Autographa californica MNPV (AcMNPV) could speed up vThGFP infection and enhance the vThGFP infection rate in Sf21 cells. The enhancement was not due to recombination, as no recombinant virus was isolated from co-infection by plaque assay. No improvement of vThGFP infection in Sf21 was found by AcMNPV cosmid transactivation assay. However, culture medium from Sf21 cells infected with AcMNPV did enhance vThGFP replication in Sf21. Third-instar larvae of Spodoptera frugiperda, S. exigua and Helicoverpa zea were not killed by feeding with vThGFP polyhedra but were killed by intrahaemocoelic injection using budded viruses (BVs). This suggested that insufficient BVs were generated during the primary infection in the midgut. vThGFP infected haemocytes, tracheae and Malpighian tubules but not fat bodies of larvae of S. frugiperda, S. exigua and H. zea. Third-instar S. frugiperda larvae co-infected by injection with vThGFP and vAcDsRed2, an AcMNPV expressing a red fluorescent protein gene, showed EGFP expression in the fat body. This result suggests that vAcDsRed2 could help vThGFP to replicate in the fat body or trans-activate EGFP expression in the fat body. All these results suggested that slow cell infection, insufficient primary infection and inability to replicate in the fat body control the host range of ThorMNPV.

The family Polydnaviridae is a large group of immunosuppressive insect viruses that are symbiotically associated with parasitoid wasps. The polydnavirus Microplitis demolitor bracovirus (MdBV) causes several alterations that disable the cellular and humoral immune defences of host insects, including apoptosis of the primary phagocytic population of circulating immune cells (haemocytes), called granulocytes. Here, we show that MdBV infection causes granulocytes in the lepidopteran Spodoptera frugiperda to apoptose. An expression screen conducted in the S. frugiperda 21 cell line identified the MdBV gene ptp-H2 as an apoptosis inducer, as indicated by cell fragmentation, annexin V binding, mitochondrial membrane depolarization and caspase activation. PTP-H2 is a classical protein tyrosine phosphatase that has been shown previously to function as an inhibitor of phagocytosis. PTP-H2-mediated death of Sf-21 cells was blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-(O-methyl) Asp-fluoromethylketone (Z-VAD-FMK), but cells maintained in this inhibitor still exhibited a suppressed phagocytic response. Mutagenesis experiments indicated that the essential catalytic cysteine residue required for the phosphatase activity of PTP-H2 was required for apoptotic activity in Sf-21 cells. Loss of adhesion was insufficient to stimulate apoptosis of Sf-21 cells. PTP-H2 expression, however, did significantly reduce proliferation of Sf-21 cells, which could contribute to the apoptotic activity of this viral gene. Overall, our results indicate that specific genes expressed by MdBV induce apoptosis of certain insect cells and that this activity contributes to immunosuppression of hosts.