- Volume 89, Issue 3, 2008

West Nile virus (WNV) is an emerging mosquito-borne flavivirus that causes neuronal damage in the absence of treatment. In many flaviviruses, including WNV, the NS2B cofactor promotes the productive folding and the functional activity of the two-component NS3 (pro)teinase. Based on an analysis of the NS2B–NS3pro structure, we hypothesized that the G22 residue and the negatively charged patch D32DD34 of NS2B were part of an important configuration required for NS2B–NS3pro activity. Our experimental data confirmed that G22 and D32DD34 substitution for S and AAA, respectively, inactivated NS2B–NS3pro. An additional D42G mutant, which we designed as a control, had no dramatic effect on either the catalytic activity or self-proteolysis of NS2B–NS3pro. Because of the significant level of homology in flaviviral NS2B–NS3pro, our results will be useful for the development of specific allosteric inhibitors designed to interfere with the productive interactions of NS2B with NS3pro.

Plasma leakage is one of the characteristic features of dengue haemorrhagic fever. The interaction among peripheral blood mononuclear cells (PBMCs), dengue virus and endothelial cells was analysed in vitro. Human umbilical vein endothelial cells (HUVECs) were infected with dengue-2 virus (DV-2) at an m.o.i. of 0.5 p.f.u. per cell. PBMCs were added to DV-2-infected HUVECs, and transendothelial electrical resistance (TEER) and transalbumin permeability were assessed. Dengue virus infection at an m.o.i. of 0.5 p.f.u. per cell alone did not decrease the TEER, but addition of PBMCs decreased the TEER, increased the albumin permeability and induced morphological changes of HUVECs. The extent of the decrease was more profound with adherent PBMCs than with non-adherent PBMCs. The expression of vascular endothelial cadherin (VE-cadherin) was examined using real-time RT-PCR and immunofluorescence. Addition of PBMCs to DV-2-infected HUVECs decreased the levels of mRNA transcripts and cell-surface expression of VE-cadherin. The results indicate that PBMCs increased the permeability of DV-2-infected HUVECs and that the increased permeability was concomitant with morphological change and the decrease in VE-cadherin expression. The results suggest that functional impairment of the DV-2-infected HUVEC monolayer was caused by interaction with PBMCs.

The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1–6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.

A virus was isolated from aborted dromedary (Camelus dromedarius) fetuses during an abortion storm in Dubai, United Arab Emirates. Laboratory investigations showed the causative agent to be indistinguishable from equine rhinitis A virus (ERAV), a picornavirus. Two pregnant dromedaries experimentally infected with the camel virus isolate both aborted and an identical virus was reisolated from both fetuses, thus confirming the diagnosis. The extremely high prevalence of antibody (>90 %) and the high titres recorded against ERAV in the dromedary herd clearly showed that ERAV does infect dromedaries. Unlike horses, where ERAV targets the upper respiratory tract, in dromedaries the target organ appears to be the genital tract.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hooved animals that carries enormous economic consequences. CD8+ cytotoxic T lymphocytes play an important role in protection and disease outcome in viral infections but, to date, the role of the CD8+ T-cell immune response to FMDV remains unclear. This study aimed to investigate major histocompatibility complex (MHC) class I-restricted CD8+ T-cell responses to FMDV in vaccinated and in infected cattle. An in vitro assay was used to detect antigen-specific gamma interferon release by CD8+ T cells in FMDV-infected cattle of known MHC class I genotypes. A significant MHC class I-restricted CD8+ T-cell response was detected to both FMDV strain O1 BFS and a recombinant fowlpox virus expressing the structural proteins of FMDV. Antigen-specific MHC class I-restricted CD8+ T-cell responses were also detected in cattle vaccinated with inactivated FMDV. These responses were shown to be directed, at least in part, to epitopes within the structural proteins (P12A region) of the virus. By using mouse cells expressing single cattle MHC class I alleles, it was possible to identify the restriction elements in each case. Identification of these epitopes will facilitate the quantitative and qualitative analysis of FMDV-specific memory CD8+ T cells in cattle and help to ensure that potential vaccines induce a qualitatively appropriate CD8+ T-cell response.

The cytotoxicity of Semliki Forest virus (SFV) infection is caused partly by the non-structural protein nsP2, an essential component of the SFV replicase complex. Due to the presence of a nuclear localization signal (NLS), nsP2 also localizes in the nucleus of infected cells. The present study analysed recombinant SFV replicons and genomes with various deletions or substitutions in the NLS, or with a proline-to-glycine mutation at position 718 of nsP2 (P718G). Deletion of one or two arginine residues from the NLS or substitution of two of the arginines with aspartic acid resulted in a virus with a temperature-sensitive phenotype, and substitution of all three arginines was lethal. Thus, most of the introduced mutations severely affected nsP2 functioning in viral replication; in addition, they inhibited the ability of SFV to induce translational shut-off and kill infected cells. SFV replicons with a P718G mutation or replacement of the NLS residues 648RRR650 with RDD were found to be the least cytotoxic. Corresponding replicons expressed non-structural proteins at normal levels, but had severely reduced genomic RNA synthesis and were virtually unable to replicate and transcribe co-electroporated helper RNA. The non-cytotoxic phenotype was maintained in SFV full-length genomes harbouring the corresponding mutations; however, during a single cycle of cell culture, these were converted to a cytotoxic phenotype, probably due to the accumulation of compensatory mutations.

We previously demonstrated the presence of tyrosine-dependent motifs for specific sorting of two measles virus (MV) glycoproteins, H and F, to the basolateral surface in polarized epithelial cells. Targeted expression of the glycoproteins was found to be required for virus spread in epithelia via cell-to-cell fusion in vitro and in vivo. In the present study, recombinant MVs (rMVs) with substitutions of the critical tyrosines in the H and F cytoplasmic domains were used to determine whether the sorting signals also play a crucial role for MV replication and spread within lymphocytes, the main target cells of acute MV infection. Immunolocalization revealed that only standard glycoproteins are targeted specifically to the uropod of polarized lymphocytes and cluster on the surface of non-polarized lymphocytes. H and F proteins with tyrosine mutations did not accumulate in uropods, but were distributed homogeneously on the surface and did not colocalize markedly with the matrix (M) protein. Due to the defective interaction with the M protein, all mutant rMVs showed an enhanced fusion capacity, but only rMVs harbouring two mutated glycoproteins showed a marked decrease in virus release from infected lymphocytes. These results demonstrate clearly that the tyrosine-based targeting motifs in the MV glycoproteins are not only important in polarized epithelial cells, but are also active in lymphocytes, thus playing an important role in virus propagation in different key target cells during acute MV infection.

Highly pathogenic avian influenza H5N1 virus has swept west across the globe and caused serious debates on the roles of migratory birds in virus circulation since the first large-scale outbreak in migratory birds of Lake Qinghai, 2005. In May 2006, another outbreak struck Lake Qinghai and six novel strains were isolated. To elucidate these QH06 viruses, the six isolates were subjected to whole-genome sequencing. Phylogenetic analyses show that QH06 viruses are derived from the lineages of Lake Qinghai, 2005. Five of the six novel isolates are adjacent to the strain A/Cygnus olor/Croatia/1/05, and the last one is related to the strain A/duck/Novosibirsk/02/05, an isolate of the flyway. Antigenic analyses suggest that QH06 and QH05 viruses are similar to each other. These findings implicate that QH06 viruses of Lake Qinghai may travel back via migratory birds, though not ruling out the possibility of local circulation of viruses of Lake Qinghai.

Mutations in the surface glycoprotein (G) of infectious hematopoietic necrosis virus (IHNV), a rhabdovirus that causes significant losses in hatcheries raising salmonid fish, were studied. A 303 nt segment (mid-G region) of this protein from 88 Idaho isolates of IHNV was sequenced. Evidence of positive selection at individual codon sites was estimated by using a Bayesian method (MrBayes). A software algorithm (CPHmodels) was used to construct a three-dimensional (3D) representation of the IHNV protein. The software identified structural homologies between the IHNV G protein and the surface glycoprotein of vesicular stomatitis virus (VSV) and used the VSV structure as a template for predicting the IHNV structure. The amino acids predicted to be under positive selection were mapped onto the proposed IHNV 3D structure and appeared at sites on the surface of the protein where antigen–antibody interaction should be possible. The sites identified as being under positive selection on the IHNV protein corresponded to those reported by others as active sites of mutation for IHNV, and also as antigenic sites on VSV. Knowledge of the sites where genetic variation is positively selected enables a better understanding of the interaction of the virus with its host, and with the host immune system. This information could be used to develop strategies for vaccine development for IHNV, as well as for other viruses.

Feline immunodeficiency virus (FIV) interacts with dendritic cells (DC) during initiation of infection, but whether DC support or transfer FIV infection remains unclear. To address this issue, we studied the susceptibility of feline myeloid DC to FIV infection and assessed potential transfer of infection from DC to CD4+ T cells. FIV was detected in membrane-bound vesicles of DC within 2 h of inoculation, although only low concentrations of FIV DNA were found in virus-exposed isolated DC. Addition of resting CD4+ T cells increased viral DNA levels; however, addition of activated CD4+ T cells resulted in a burst of viral replication manifested by FIV p27 capsid antigen generation. To determine whether transfer of FIV infection required productively infected DC (vs virus bound to DC but not internalized), virus-exposed DC were cultured for 2 days to allow for degradation of uninternalized virus and initiation of infection in the DC, then CD4+ T blasts were added. Infection of T cells remained robust, indicating that T-cell infection is likely to be mediated by de novo viral infection of DC followed by viral transfer during normal DC/T-cell interactions. We conclude that feline DC support restricted FIV infection, which nevertheless is sufficient to efficiently transfer infection to susceptible T cells and trigger the major burst of viral replication. Feline DC/FIV/T-cell interactions (similar to those believed to occur in human immunodeficiency virus and simian immunodeficiency virus infections) highlight the means by which immunodeficiency-inducing lentiviruses exploit normal DC/T-cell interactions to transfer and amplify virus infection.

We have shown previously that a type-specific neutralization domain is located within a 39 aa sequence in the fourth variable domain of gp135 in visna/maedi virus. We now show that neutralizing antibodies detected early in infection are directed to this epitope, suggesting an immunodominant nature of this domain. Ten antigenic variants were previously analysed for mutations in this region, and all but one were found to be mutated. To assess the importance of these mutations in replication and neutralization, we reconstructed several of the mutations in an infectious molecular clone and tested the resulting viruses for neutralization phenotype and replication. Mutation of a conserved cysteine was shown to alter the neutralization epitope, whilst the replication kinetics in macrophages were unchanged. Mutations modulating potential glycosylation sites were found in seven of the ten antigenic variants. A frequently occurring mutation, removing a potential glycosylation site, had no effect on its own on the neutralization phenotype of the virus. However, adding an extra potential glycosylation site in the region resulted in antigenic escape. The results indicate that the conserved cysteine plays a role in the structure of the epitope and that glycosylation may shield the principal neutralization site.

APOBEC3G (A3G), a member of cytidine deaminase family, has potent anti-human immunodeficiency virus type 1 (HIV-1) activity. It has been demonstrated that alpha interferon (IFN-α) can significantly enhance the expression of A3G in human primary resting CD4+ T-cells, macrophages and primary hepatocytes, subsequently decreasing their viral susceptibility. Plasmacytoid dendritic cells (pDCs) are key effectors in innate host immunity, mediating adaptive immune responses and stimulating IFN-α production in reaction to various stimuli. In this report, we demonstrate that IFN-α, either exogenously added to- or endogenously secreted by pDCs, can enhance the expression of A3G and its family members such as A3A, A3C and A3F. We have also shown that IFN-α can inhibit HIV-1 expression in pDCs. This inhibitory effect could be countered by addition of an A3G-specific short interfering RNA, indicating that IFN-α-induced A3G plays a key role in mediating pDCs response to HIV-1. Given the central role played by pDCs in orchestrating the IFN-α/A3G intercellular network and intracellular signal pathway, our data indicate that pDCs themselves are also protected by an IFN-α/A3G-mediated innate immunity barrier from HIV-1 infection.

A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50–pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.

Recombinant simian varicella viruses (rSVVs) were engineered to express respiratory syncytial virus (RSV) antigens. The RSV surface glycoprotein G and second matrix protein M2 (22k) genes were cloned into the SVV genome, and recombinant viruses were characterized in vitro and in vivo. rSVVs were also engineered to express the membrane-anchored or secreted forms of the RSV-G protein as well as an RSV G lacking its chemokine mimicry motif (CX3C), which may have different effects on priming the host immune response. The RSV genes were efficiently expressed in rSVV/RSV-infected Vero cells as RSV-G and -M2 transcripts were detected by RT-PCR, and RSV antigens were detected by immunofluorescence and immunoblot assays. The rSVVs replicated efficiently in Vero cell culture. Rhesus macaques immunized with rSVV/RSV-G and rSVV/RSV-M2 vaccines produced antibody responses to SVV and RSV antigens. The results demonstrate that recombinant varicella viruses are suitable vectors for the expression of RSV antigens and may represent a novel vaccine strategy for immunization against both pathogens.

Natural killer (NK) cells are known for their ability to lyse tumour cell targets. Studies of infections by a number of viruses, including poxviruses and herpesviruses, have demonstrated that NK cells are vital for recovery from these infections. Little is known of the ability of viruses to infect and complete a productive replication cycle within NK cells. Even less is known concerning the effect of infection on NK cell biology. This study investigated the ability of ectromelia virus (ECTV) to infect NK cells in vitro and in vivo. Following ECTV infection, NK cell gamma interferon (IFN-γ) production was diminished and infected cells ceased proliferating and lost viability. ECTV infection of NK cells led to early and late virus gene expression and visualization of immature and mature virus particles, but no detectable increase in viable progeny virus. It was not unexpected that early gene expression occurred in infected NK cells, as the complete early transcription system is packaged within the virions. The detection of the secreted early virus-encoded immunomodulatory proteins IFN-γ-binding protein and ectromelia inhibitor of complement enzymes (EMICE) in NK cell culture supernatants suggests that even semi-permissive infection may permit immunomodulation of the local environment.

Porcine circovirus type 2 (PCV2) is the necessary agent for the occurrence of post-weaning multisystemic wasting syndrome (PMWS) in pigs. It has been suggested that PMWS-affected pigs are immunosuppressed and, therefore, more prone to develop co-infections. In this study, we elucidated that PCV2 downregulates in vitro the immune cell functions during recall antigen responses. We showed that PCV2, but not the non-pathogenic porcine circovirus type 1, induces interleukin (IL)-10 secretion by monocytic cells. Notably, PCV2-induced IL-10 led to effective repression of IL-12 in blood peripheral mononuclear cells (PBMCs). Besides alpha and gamma interferon synthesis by PBMCs from pseudorabies virus (PRV)-immunized animals, activated in vitro PRV also was repressed by subsequent infection by PCV2. The ability of PCV2 to hamper the development of immune responses may contribute to the Th1 suppressed responses, immune suppression and co-infections.

Open reading frame (ORF) 67 (Bm67) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that is found in all completely sequenced baculoviruses; its function is unknown. In the present study, a Bm67-knockout virus was generated for studying the role of Bm67 in the BmNPV infection cycle. Furthermore, a Bm67-repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67-knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67-knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67-repair bacmid could rescue the defect, and budded virus titres reached wild-type levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electron microscopic analysis revealed that the formation of normal-appearing nucleocapsids is reduced in Bm67-knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of ‘enveloped’ nucleocapsids at the nucleoplasm bilayer indicated that they are enveloped abnormally. These results indicated that Bm67 is required for the production of infectious budded viruses and for assembly of envelope and nucleocapsids.

Six clones of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) were plaque-purified from field isolates collected in Missouri, USA. In bioassays, four of the plaque-purified isolates killed neonate S. frugiperda larvae more rapidly than the field isolates from which they were derived, with LT50 values (mean time to kill 50 % of the test larvae) ranging from 34.4 to 49.7 h post-infection. The complete genomic sequence of one of these isolates, SfMNPV-3AP2, was determined and analysed. The SfMNPV-3AP2 genome was 131 330 bp with a G+C content of 40.2 %. A total of 144 open reading frames (ORFs) was identified and examined, including the set of 62 genes in common among lepidopteran nucleopolyhedrovirus genomes. Comparisons of ORF content, order and predicted amino acid sequences with other nucleopolyhedoviruses indicated that SfMNPV is part of a cluster of viruses within NPV group II that includes NPVs isolated from Spodoptera, Agrotis and Mamestra host species. SfMNPV-3AP2 shared a high degree of nucleotide sequence similarity with partial sequences from other SfMNPV isolates. Comparison of the SfMNPV-3AP2 genome sequence with a partial sequence from a Brazilian isolate of SfMNPV revealed that SfMNPV-3AP2 contained a deletion that removed parts of ORF sf27 and the gene encoding ecdysteroid UDP-glucosyltransferase (egt). An examination of the egt region in the other isolates revealed that the other five SfMNPV clones also contained deletions of varying length in this region. Variant genotypes with deletions extending around the egt gene have been reported previously from a Nicaraguan field isolate of SfMNPV, suggesting that the presence of such variants is a common feature of SfMNPV populations.