- Volume 88, Issue 2, 2007

Chimpanzees are susceptible to human immunodeficiency virus type-1 (HIV-1) and develop persistent infection but generally do not progress to full-blown AIDS. Several host and immunological factors have been implicated in mediating resistance to disease progression. Chimpanzees have a higher prevalence of circulating natural killer (NK) cells than humans; however, their role in mediating resistance to disease progression is not well understood. Furthermore, NK cell survival and activity have been shown to be dependent on interleukin-15 (IL-15). Accordingly, the influence of IL-15 on NK cell activity and gamma interferon (IFN-γ) production was evaluated in naive and HIV-1-infected chimpanzees. In vitro stimulation of whole-blood cultures with recombinant gp120 (rgp120) resulted in enhanced IFN-γ production predominantly by the CD3− CD8+ subset of NK cells, and addition of anti-IL-15 to the system decreased IFN-γ production. Moreover, in vitro stimulation with recombinant IL-15 (rIL-15) augmented IFN-γ production from this subset of NK cells and increased NK cell cytotoxic activity. Stimulation with rgp120 also resulted in a 2- to 7-fold increase in IL-15 production. These findings suggest that chimpanzee CD3− CD8+ NK cells play a vital role in controlling HIV-1 infection by producing high levels of IFN-γ, and that IL-15 elicits IFN-γ production in this subpopulation of NK cells in HIV-1-infected chimpanzees.

The X4-tropic simian/human immunodeficiency virus (SHIV) 89.6P (or 89.6PD) causes rapid CD4+ T-cell depletion leading to an acute crash of the host immune system, whereas pathogenic R5-tropic simian immunodeficiency virus (SIV) infection, like HIV-1 infection in humans, results in chronic disease progression in macaques. Recent pre-clinical vaccine trials inducing cytotoxic T lymphocyte (CTL) responses have succeeded in controlling replication of the former but shown difficulty in control of the latter. Analysis of the immune responses involved in consistent control of SHIV would contribute to elucidation of the mechanism for consistent control of SIV replication. This study followed up rhesus macaques that showed vaccine-based control of primary SHIV89.6PD replication and found that all of these controllers maintained viraemia control for more than 2 years. SHIV89.6PD control was observed in vaccinees of diverse major histocompatibility complex (MHC) haplotypes and was maintained without rapid selection of CTL escape mutations, a sign of particular CTL pressure. Despite the vaccine regimen not targeting Env, all of the SHIV controllers showed efficient elicitation of de novo neutralizing antibodies by 6 weeks post-challenge. These results contrast with our previous observation of particular MHC-associated control of SIV replication without involvement of neutralizing antibodies and suggest that vaccine-based control of SHIV89.6PD replication can be stably maintained in the presence of multiple functional immune effectors.

Fusion of the viral and cellular membranes is a critical step in the infection of cells by the human T-cell leukemia virus type 1 (HTLV-1) and this process is catalysed by the viral envelope glycoproteins. During fusion, the transmembrane glycoprotein (TM) is thought to undergo a transition from a rod-like pre-hairpin conformation that is stabilized by a trimeric coiled coil to a more compact six-helix-bundle or trimer-of-hairpins structure. Importantly, synthetic peptides that interfere with the conformational changes of TM are potent inhibitors of membrane fusion and HTLV-1 entry, suggesting that the pre-hairpin motif is a valid target for antiviral therapy. Here, a stable, trimeric TM derivative that mimics the coiled-coil structure of fusion-active TM has been used to develop a plate-based assay to identify reagents that interfere with the formation of the six-helix bundle. The assay discriminates effectively between strong, weak and inactive peptide inhibitors of membrane fusion and has been used to identify a monoclonal antibody (mAb) that disrupts six-helix-bundle formation efficiently in vitro. The mAb is reactive with the C-helical region of TM, indicating that this region of TM is immunogenic. However, the mAb failed to neutralize HTLV-1 envelope-mediated membrane fusion, suggesting that, on native viral envelope, the epitope recognized by the mAb is obscured during fusion. This novel mAb will be of value in the immunological characterization of fusion-active structures of HTLV-1 TM. Moreover, the assay developed here will aid the search for therapeutic antibodies, peptides and small-molecule inhibitors targeting envelope and the HTLV-1 entry process.

The main routes of transmission of Visna/maedi virus (VMV), an ovine lentivirus, are thought to be through ingestion of infected colostrum and/or milk or through inhalation of respiratory secretions. Whereas oral transmission appears to be mediated via epithelial cells within the small intestine, the mechanism of virus uptake in the respiratory tract is unknown. In addition, it is not known whether infection is mediated by cell-associated or cell-free VMV, previous studies having not addressed this question. Intratracheal (i.t.) injection of VMV is known to be a highly efficient method of experimental infection, requiring as little as 101 TCID50 VMV for successful infection. However, using a tracheal organ culture system, we show here that ovine tracheal mucosa is relatively resistant to VMV, with detectable infection only seen after incubation with high titres of virus (⩾105 TCID50 ml−1). We also demonstrate that i.t. injection results in exposure of both trachea and the lower lung and that the time taken for viraemia and seroconversion to occur after lower lung instillation of VMV was significantly shorter than that observed for tracheal instillation of an identical titre of virus (P=0.030). This indicates that lower lung and not the trachea is a highly efficient site for VMV entry in vivo. Furthermore, cell-free virus was identified within the lung-lining fluid of naturally infected sheep for the first time. Together, these results suggest that respiratory transmission of VMV is mediated by inhalation of aerosols containing free VMV, with subsequent virus uptake in the lower lung.

Circularization of the herpes simplex virus type 1 (HSV-1) genome is thought to be an important early event during the lytic cycle. Previous studies from another laboratory using a cell line, tsBN2, that carries a temperature-sensitive mutation in the gene encoding the regulator of chromatin condensation 1 (RCC1) indicated that functional RCC1 was required for HSV-1 genome circularization and subsequent viral DNA synthesis. Here, HSV-1 infection of tsBN2 cells has been re-examined by utilizing both wild-type HSV-1 and a derivative that enables a direct demonstration of circularization. At the non-permissive temperature, when RCC1 was absent, both circularization and viral DNA synthesis were reduced, but not abolished. However, no infectious progeny virus was detected under these conditions. An impairment in the cleavage of concatemeric DNA and the failure to express at least one capsid protein indicated that HSV-1 replication is also blocked at a late stage in the absence of RCC1. This conclusion was supported by a temperature-upshift experiment, which demonstrated a role for RCC1 at times later than 6 h post-infection. Finally, a virus constitutively expressing β-galactosidase produced the protein in a reduced number of cells when RCC1 was inactivated, suggesting that genome delivery to the nucleus or the initial stages of gene expression may also be affected.

The R1 subunit (ICP10) of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR), which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 aa, protects cells against apoptosis. As the NH2 domain on its own is not antiapoptotic, it has been postulated that both domains of R1 or part(s) of them could be necessary for this function. Here, N- and C-terminal deletions were introduced in HSV-2 R1 to map the domain(s) involved in its antiapoptotic potential. The results showed that, whereas most of the NH2 domain including part of the recently described putative α-crystallin domain is dispensable for antiapoptotic activity, it is the integrity of the structured RR domain that is required for protection. As the α-crystallin domain appears to play an important role in protein folding and oligomerization, the N-terminal boundary of the antiapoptotic domain could not be defined precisely. In addition, this study provided evidence that overexpression of HSV-2 R2 at levels up to 30-fold more than HSV-2 R1 did not decrease protection from tumour necrosis factor alpha, indicating that the R1 surface where R2 binds is not involved in antiapoptotic activity. Importantly, this result suggests that the co-expression of both RR subunits during the lytic cycle should not affect protection from this cytokine.

The human cytomegalovirus-encoded protein kinase pUL97 is a determinant of efficient virus replication and fulfils several regulatory functions. In particular, pUL97 interacts with and phosphorylates viral and cellular proteins. Substrate phosphorylation has regulatory consequences on viral replicative stages such as DNA synthesis, transcription and nuclear capsid egress. pUL97, in accordance with related herpesviral protein kinases, possesses strong autophosphorylation activity. Here, we demonstrate that pUL97 shows a pronounced potential to self-interact. Self-interaction of pUL97 is not dependent on its kinase activity, as seen with a catalytically inactive point mutant. The property of self-interaction maps to the amino acid region 231–280 which is separable from the postulated kinase domain. The detection of high-molecular-mass complexes of pUL97 suggests the formation of dimers and oligomers. Importantly, the analysis of pUL97 mutants by in vitro kinase assays demonstrated a correlation between self-interaction and protein kinase activity, i.e. all mutants lacking the ability to self-interact were negative or reduced in their kinase activity. Thus, our findings provide novel insights into the pUL97 structure–activity relationship suggesting an importance of self-interaction for pUL97 functionality.

The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that has counterparts in all herpesviruses. Some of these proteins have been shown to function primarily at the post-transcriptional level in promoting nuclear export of viral transcripts. Consistently, this group has reported recently that pUL69 is an RNA-binding, nucleocytoplasmic shuttling protein that facilitates the cytoplasmic accumulation of unspliced mRNA via its interaction with the cellular mRNA export factor UAP56. Evidence has been presented to suggest that some of the pUL69 homologues self-interact and function in vivo as multimers. Herein, the possibility of pUL69 self-association was examined and it has been demonstrated that pUL69 can interact with itself in vitro and in vivo in order to form high-molecular-mass complexes. The self-interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses, suggesting that multimerization is a conserved feature of this protein family.

This study examined nuclear and cytoplasmic shuttling of the African swine fever virus (ASFV) A238L protein, which is an inhibitor of NF-κB and of calcineurin phosphatase. The results showed that the protein was present in both the nucleus and the cytoplasm in ASFV-infected cells and that the higher molecular mass 32 kDa form of the A238L protein was the predominant nuclear form, which accumulated later in infection. In contrast, both the 28 and 32 kDa forms of the A238L protein were present in the cytoplasm. The A238L protein was actively imported into the nucleus and exported by a CRM1-mediated pathway, although a pool of the protein remained in the cytoplasm and did not enter the nucleus. By using a recombinant ASFV from which the A238L gene had been deleted, it was shown that expression of A238L did not inhibit nuclear import of the NF-κB p50 or p65 subunit and did not inhibit nuclear export of p65 by a CRM1-mediated pathway. The results were consistent with a model in which A238L functions within both the nucleus and the cytoplasm.

The pathogenicity of two recent German field isolates of Porcine parvovirus (PPV-27a and PPV-143a) and two vaccine viruses [PPV-NADL-2 and PPV-IDT (MSV)], which are used for the production of inactivated vaccines, was investigated by inoculation of pregnant sows at day 40 of gestation. Post-infection sera of these sows as well as antisera prepared in rabbits by immunization with the four above-mentioned PPV isolates and with the virulent strain PPV-Challenge (Engl.) were tested for their homologous and heterologous neutralization activities. All antisera had high neutralization activity against the vaccine viruses, the PPV-Challenge (Engl.) virus and PPV-143a, but much lower activity against PPV-27a. These results suggest that PPV-27a represents a new antigenic variant or type of PPV and vaccines based on the established vaccine viruses may not be fully protective against this field isolate. PPV-27a has been characterized based on the amino acid sequences of the capsid protein as a member of a new and distinct PPV cluster ( Zimmermann et al., 2006 ). Interestingly, the homologous neutralizing antibody titres of the sera of all three pigs and both rabbits inoculated or immunized with PPV-27a were 100- to 1000-fold lower than the heterologous titres against any of the other viruses. The low homologous neutralizing antibody titres suggest a possible, yet undefined, immune escape mechanism of this PPV isolate.

Three genotypes (1–3) of human parvovirus B19 have been identified. Analysis of 13 nearly full-length genotype 3 sequences from Ghana, Europe and Brazil identified two genetically distinct clusters. The classification of genotype 3 strains into two subtypes (B19/3a and B19/3b) is proposed. The rate of evolutionary change of B19 genotype 3 strains (2×10−4 nucleotide substitutions per site per year) was similar to those of B19 genotype 1 and carnivore parvoviruses, supporting the hypothesis that high mutation rates are characteristic of members of the family Parvoviridae. The estimated divergence time between B19/3a and B19/3b is 525 years. In Ghana, subtype B19/3a is predominant.

G to A hypermutation of Hepatitis B virus (HBV) and retroviruses appears as a result of deamination activities of host APOBEC proteins and is thought to play a role in innate antiviral immunity. Alpha and gamma interferons (IFN-α and -γ) have been reported to upregulate the transcription of APOBEC3G, which is known to reduce the replication of HBV. We investigated the number of hypermutated genomes under various conditions by developing a quantitative measurement. The level of hypermutated HBV in a HepG2 cell line, which is semi-permissive for retrovirus, was 2.3 in 104 HBV genomes, but only 0.5 in 104 in permissive Huh7 cells. The level of APOBEC3G mRNA was about ten times greater in HepG2 cells than in Huh7 cells. Treatment of HepG2 cells with either IFN-α or -γ increased the transcription of APOBEC3G and hypermutation of HBV. These mRNAs and hypermutation of HBV genomes were induced more prominently by IFN-γ than by IFN-α. Both IFNs decreased the number of replicative intermediate of HBV. Overexpression of APOBEC3G reduced the number of replicative intermediate of HBV and increased hypermutated genomes 334 times, reaching 968 in 104 genomes. Deamination-inactive APOBEC3G did not induce hypermutation, but reduced the virus equally. Our results suggest that APOBEC3G, upregulated by IFNs, has a dual effect on HBV: induction of hypermutation and reduction of virus synthesis. The effect of hypermutation on infectivity should be investigated further.

The F protein is the major glycoprotein present in the envelopes of budded virus (BV) of members of the family Baculoviridae. The F protein mediates low-pH-activated fusion with insect cell membranes. Baculovirus F proteins are synthesized as a precursor (F0) and cleaved post-translationally into two disulfide-bonded subunits, F1 (C-terminal, large subunit) and F2 (N-terminal, small subunit). Recently, N-linked glycosylation of the F1 and F2 subunits of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was demonstrated [ Long, G., Westenberg, M., Wang, H., Vlak, J. M. & Hu, Z. (2006) . J Gen Virol 87, 839–846]. Sequence analysis frequently predicts that one or more N-linked glycosylation sites are present in the F2 subunit of baculovirus F proteins. N-glycans on envelope fusion proteins are usually required for proper conformational integrity and biological function, such as infectivity. This study examined the importance of N-linked glycosylation of the F2 subunit of HearNPV by site-directed mutagenesis. The only putative N-linked glycosylation site in F2 was eliminated by mutating asparagine (N104) to glutamine (Q), resulting in the mutant HearNPV fN104Q . When inserted into an f-null HearNPV and a gp64-null bacmid of Autographa californica multiple nucleopolyhedrovirus, infectious BV could be retrieved that contained unglycosylated F2. The virulence of HearNPV fN104Q was enhanced, as BV was produced earlier after infection and yielded larger plaques than f-null HearNPV repaired with the wild-type f gene. HearNPV fN104Q BV also induced much more efficient low-pH-activated syncytium formation. These results indicate that N-linked glycosylation of the HearNPV baculovirus F2 subunit is not essential for viral infectivity and suggest that it is involved in BV production and fusogenicity.

Polydnaviruses are only found in symbiotic association with parasitic wasps within the families Ichneumonidae and Braconidae (ichnoviruses and bracoviruses). They have a segmented genome consisting of circular double-stranded DNA. In the proviral linear form they are integrated in the wasp's genome; in two bracoviruses, segments were found to be clustered. Proviral segments have direct terminal repeats. Segment excision has been proposed to occur through juxtaposition of these repeats by formation of a loop and recombination; one copy of the repeat then ends up in the circular segment and one in the rejoined DNA. Here we analysed the excision/circularization site of four segments of the Chelonus inanitus bracovirus (CiV) and found that they are similar to the two already known sites; on the basis of the combined data an extended excision site motif was found. Analyses of segment flanking sequences led to the first identification of one complete and several partial spacers between proviral segments in a polydnavirus. The spacer between the proviral segments CiV14 and CiV22.5 has a length of 2065 bp; the terminal repeats of CiV14 and CiV22.5 were seen to have an opposite orientation and from this a model on the spacial organization of the loops of the proviral cluster is proposed. Through various approaches it was shown that spacers are not excised or injected into the host. Measurement of relative abundances of various segments in proviral and excised form indicates for the first time that abundant segments are present in multiple copies in the proviral form.

Because of the confounding effects of long incubation duration and flock management, accurate epidemiological studies of scrapie outbreaks are difficult to carry out. In this study, 641 Manech red-faced sheep from six scrapie-affected field flocks in Pyrénées Atlantiques, France, were monitored for clinical scrapie over a 6–9 year period. Over this period, 170 scrapie clinical cases were recorded and half of the culled animals were submitted for post-mortem transmissible spongiform encephalopathy diagnosis to assess their infectious status. Collected data were analysed using a ‘mixture cure model’ approach, which allowed for the discriminating effect of PrP genotype and flock origin on incidence and incubation period. Simulations were performed to evaluate the applicability of such a statistical model to the collected data. As expected, ARR heterozygote sheep were less at risk of becoming infected than ARQ/ARQ individuals and had a greater age at clinical onset. Conversely, when compared with ARQ/ARQ, the VRQ haplotype was associated with an increased infection risk, but not a shorter incubation period. Considering the flock effect, we observed that a high incidence rate was not associated with shorter incubation periods and that the incubation period could be significantly different in flocks harbouring similar infection risks. These results strongly support the conclusion that other parameters, such as the nature of the agent or flock management, could interfere with epidemiological dynamics of the infection in scrapie-affected flocks.

Despite circumstantial evidence that prions can be found extracellularly or at the surface of infected cells, little is known about how these infectious agents spread from cell to cell. In order to gain better insight into this critical issue, this study used two different cell lines (neuroglial MovS and epithelial Rov cells) that have previously been shown to be permissive for ovine prion multiplication. Co-culture of infected cells and uninfected target cells at a ratio of 1 : 9 resulted in total infection of MovS cells within 10 days but not of Rov cell cultures, suggesting that the efficiency of prion dissemination may vary greatly depending on the type of permissive cell. Analysis of the spatial distribution of the newly infected cells revealed that, although long-range spread could also occur, cells proximal to the infected donor cells consistently accumulated more abnormal PrP, consistent with preferential infection of nearby cells. This experimental approach, focused on dissemination among living cells, could help in the analysis of mechanisms involved in the cell-to-cell spread of prion infections.

Titration studies of the infectivity of experimental bovine spongiform encephalopathy (BSE) in sheep are necessary to assess the risk for human health posed by the ovine infection relative to the original cattle disease. Here, a comparative titration was performed of sheep-passaged BSE infectivity in Romney sheep and RIII mice, by the intracerebral (i.c.) and i.c. plus intraperitoneal (i.p.) routes, respectively. The sheep-to-mouse species barrier was lower than anticipated, as similar titres were obtained for both sheep [1×105.4 (i.c.) ID50 g−1)] and mice [1×105.0 (i.c.+i.p.) ID50 g−1]. Moreover, sheep of the ARR/ARR PrP genotype all succumbed to i.c. challenge with a 10−3 dilution of 0.5 g of a brainstem pool from BSE-affected sheep, indicating that resistance to natural infection in sheep of this genotype must reside in some mechanism of peripheral pathogenesis.