- Volume 84, Issue 7, 2003

Envelopes of retroviruses, including human immunodeficiency virus and simian immunodeficiency virus (SIV), contain host cell proteins that potentially represent novel targets for vaccine development. We show here that sera from rhesus macaques recognized simian major histocompatibility complex (MHC) molecules in response to infection with SIV. Antibodies from these animals did not cross-react with human MHC antigens on mitogen-activated peripheral blood mononuclear cells. The development of antibodies to MHC class I α-chain did not correlate with anti-SIV envelope antibody responses, suggesting that these antibodies did not arise through molecular mimicry. In contrast to the species-specific response in infected animals, sera from animals vaccinated with inactivated human cell-grown SIV reacted to both human and rhesus MHC class I and class II molecules.

Cultivation of retrovirus packaging cells at 32 °C represents a common procedure to achieve high titres in mouse retrovirus production. Gene expression profiling of mouse NIH 3T3 cells producing amphotropic mouse leukaemia virus 4070A revealed that 10 % of the 1176 cellular genes investigated were regulated by temperature shift (37/32 °C), while 5 % were affected by retrovirus infection. Strikingly, retrovirus production at 32 °C activated the cholesterol biosynthesis/transport pathway and caused an increase in plasma membrane cholesterol levels. Furthermore, these conditions resulted in transcriptional activation of smoothened (smo), patched (ptc) and gli-1; Smo, Ptc and Gli-1, as well as cholesterol, are components of the Sonic hedgehog (Shh) signalling pathway, which directs pattern formation, diversification and tumourigenesis in mammalian cells. These findings suggest a link between cultivation at 32 °C, production of MLV-A and the Shh signalling pathway.

Several host proteins have been shown to play key roles in the life-cycle of vesicular stomatitis virus (VSV). We have identified an additional host protein, cyclophilin A (CypA), a chaperone protein possessing peptidyl cis-trans prolyl-isomerase activity, as one of the cellular factors required for VSV replication. Inhibition of the enzymatic activity of cellular CypA by cyclosporin A (CsA) or SDZ-211-811 resulted in a drastic inhibition of gene expression by VSV New Jersey (VSV-NJ) serotype, while these drugs had a significantly reduced effect on the genome expression of VSV Indiana (VSV-IND) serotype. Overexpression of a catalytically inactive mutant of CypA resulted in the reduction of VSV-NJ replication, suggesting a requirement for functional CypA for VSV-NJ infection. It was also shown that CypA interacted with the nucleocapsid (N) protein of VSV-NJ and VSV-IND in infected cells and was incorporated into the released virions of both serotypes. VSV-NJ utilized CypA for post-entry intracellular primary transcription, since inhibition of CypA with CsA reduced primary transcription of VSV-NJ by 85–90 %, whereas reduction for VSV-IND was only 10 %. Thus, it seems that cellular CypA binds to the N protein of both serotypes of VSV. However, it performs an obligatory function on the N protein activity of VSV-NJ, while its requirement is significantly less critical for VSV-IND N protein function. The different requirements for CypA by two serologically different viruses belonging to the same family has highlighted the utilization of specific host factors during their evolutionary lineages.

Infectious salmon anaemia virus (ISAV), a pathogen in marine aquaculture, belongs to the genus Isavirus, family Orthomyxoviridae. There is limited information on how ISAV interacts with host defences. To study ISAV–antibody interactions, virus neutralization (VN) assays were performed in the cell lines CHSE-214, SHK-1 and TO using three strains of ISAV and rabbit or fish anti-ISAV sera. Homologous VN titres of >1 : 1280 in CHSE-214 cells corresponded to titres of only 1 : 80 in the macrophage-like fish cell lines SHK-1 and TO, despite using 1000 and 2000 times less virus, respectively. However, rabbit antiserum to infectious pancreatic necrosis virus (IPNV) had a VN titre of 1 : 10 260 against IPNV in both CHSE-214 and TO cells. Poor ISAV neutralization in TO cells was attributed to Fc receptors mediating virus infectivity, because (1) neutralization by rabbit antiserum to ISAV was increased 48-fold in the presence of staphylococcal Protein A and (2) when using FITC-labelled virus and spectrofluorometry, a significant increase (P=0·018) in the intensity of fluorescence of intracellular virus was observed in assays of virus–antiserum mixtures in the absence of Protein A as compared to those in the presence of Protein A. Neutralization of ISAV with fish antisera was observed only in CHSE-214 cells, as Protein A could not restore neutralization in TO cells. These findings demonstrate for the first time antibody-mediated infection of macrophage-like fish cell lines by a fish virus, ISAV, and, as ISAV in Atlantic salmon targets leukocytic and endothelial cells, this may have implications for ISA pathogenesis and vaccination.

Recently, we presented evidence that the E1 fusion protein of the alphavirus Semliki Forest virus forms ion-permeable pores in the target membrane after fusion. We proposed that the homologous fusion proteins of flaviviruses and hepatitis C virus form similar pores. To test this hypothesis for the E fusion protein of flaviviruses, the release of [3H]choline from liposomes by the flavivirus West Nile (WN) virus was determined. [3H]Choline was released at mildly acid pH. The pH threshold depended on the lipid composition. Release from certain liposomes was activated even at neutral pH. To identify the generation of individual pores, single cells were investigated with the patch-clamp technique. The formation of individual pores during low pH-induced WN virus entry at the plasma membrane occurred within seconds. These experiments were performed in parallel with Semliki Forest virus. The results indicated that, similar to alphavirus infection, infection with flaviviruses via endosomes leads to the formation of ion-permeable pores in the endosome after fusion, which allows the flow of protons from the endosome into the cytoplasm during virus entry. However, in vitro translation experiments of viral cores showed that, in contrast to alphaviruses, which probably need this proton flow for core disassembly, the genome RNA of WN virus present in the viral core is directly accessible for translation. For entry of flaviviruses, therefore, a second pathway for productive infection may exist, in which fusion of the viral membrane is activated at neutral pH by contact with a plasma membrane of appropriate lipid composition.

Enhancement of flavivirus infection in vitro in the presence of subneutralizing concentrations of homologous or heterologous antiserum has been well described. However, the importance of this phenomenon in the enhancement of flavivirus infection in vivo has not been established. In order to study antibody-mediated enhancement of flavivirus infection in vivo, we investigated the effect of passive immunization of mice with Japanese encephalitis virus (JE) antiserum on the outcome of infection with Murray Valley encephalitis virus (MVE). We show that prior treatment of mice with subneutralizing concentrations of heterologous JE antiserum resulted in an increase in viraemia titres and in mortality following challenge with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo. These findings are of potential importance for the design of JE vaccination programs in geographic areas in which MVE co-circulates. Should subneutralizing concentrations of antibody remain in the population following JE vaccination, it is possible that enhanced disease may be observed during MVE epidemics.

This study identified nine genes whose expression is upregulated in the central nervous system (CNS) of mice during Japanese encephalitis virus (JEV) infection. These include: cathepsin S, oligoadenylate synthetase (OAS), GARG49/IRG2, lymphocyte antigen-6A (Ly-6A), macrophage activation gene-2 (Mpa2), early growth response gene1 (Egr1), pyrimidine 5′-nucleotidase (P5N), apolipoprotein D (ApoD) and STAT1. Activation of all nine genes during JEV infection was confirmed by Northern blot analysis. JEV replication was inhibited in the majority of mice immunized with Biken JEV vaccine, and these mice also exhibited reduced expression of JEV-inducible CNS genes. Thus, there is a good correlation between virus load and upregulation of host CNS genes. It was also demonstrated that all the CNS genes activated by JEV are also upregulated during rabies virus infection. In addition, GARG49, STAT1, cathepsin S and ApoD are known to be upregulated in the CNS by Sindbis virus, an alphavirus, and this supports the proposal that common host cell pathways are activated in the CNS by different neurotropic viruses.

Entry of Japanese encephalitis virus (JEV) into cells was analysed by using the vertebrate cell line Vero. Vero cells were treated with chlorpromazine, nystatin or cytochalasin D, which inhibit clathrin- and caveola-dependent endocytosis, and macropinocytosis of the cells, respectively. Productive JEV infection was inhibited by pretreatment with chlorpromazine; the number of JEV antigen-positive cells was less than one-fifth of that in untreated cultures, but was not significantly decreased by pretreatment with nystatin or cytochalasin. Viral antigens were detected in the membrane fractions, but not in the endosome fractions from chlorpromazine-treated JEV-inoculated cells. When the cells were treated with chlorpromazine, clathrin heavy chain antigen and JEV antigen were not detected in cytoplasm by indirect immunofluorescence staining. These results indicate that JEV is taken up by cells through the clathrin-dependent endocytic pathway, and this process leads to infection.

This paper investigates the role of CTL and antibody responses in hepatitis C virus (HCV) dynamics and pathology. Mathematical models suggest that a strong CTL response is required for resolution of HCV infection and that a weak CTL response can result in persistent infection. According to the model, establishment of persistent infection is accompanied mainly by an ongoing antibody response, while CTLs are not maintained at high levels. In the model, this outcome correlates with absence of pathology. Persistent infection in the face of an ongoing antibody response can result in evolution of antigenic escape. According to the model, evolution towards escape from antibodies can shift the balance of immune responses so that the weak CTL levels become increasingly more dominant relative to antibodies. This shift results in onset of liver pathology as the virus evolves towards increased levels of antigenic escape. Therefore, the relative balance of the immune response can be a decisive factor that determines whether patients are asymptomatic or whether pathology is observed. Virus evolution can shift this balance towards pathology over time. Theoretical results are discussed in the context of published data.

Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is approximately 9·6 kb in length and encodes a polyprotein that is proteolytically cleaved to generate at least 10 mature viral protein products. Recently, a new protein, named F, has been described to be expressed through a ribosomal frameshift within the capsid-encoding sequence, a mechanism unique among members of the family Flaviviridae. Here, expression of the F protein was investigated in an in vitro transcription/translation assay. Its expression in mammalian cells was confirmed using specific recombinant vaccinia viruses; under these conditions, protein expression is dependent on the HCV IRES. The F protein was tagged with firefly luciferase or the Myc epitope to facilitate its identification. Ribosomal frameshifting was dependent on the presence of mutations in the capsid-encoding sequence. No frameshifting was detected in the absence of any mutation. Furthermore, analysis of the F protein in time-course experiments revealed that the protein is very unstable and that its production can be stabilized by the proteasome inhibitor MG132. Finally, indirect immunofluorescence studies have localized the F protein in the cytoplasm, with notable perinuclear detection.

Hepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5′NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5′NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5′NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5′NTRs. A two-step analysis of these 5′NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5′NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5′NTRs, while replication of subgenomic replicons with these chimeric 5′NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.

Various pathogens have been shown to infect antigen-presenting cells and affect their capacity to interact with and stimulate T-cell responses. We have used an antigenically identical pair of non-cytopathic (ncp) and cytopathic (cp) bovine viral diarrhoea virus (BVDV) isolates to determine how the two biotypes affect monocyte and dendritic cell (DC) function. We have shown that monocytes and DCs are both susceptible to infection with ncp BVDV and cp BVDV in vitro. In addition, monocytes infected with ncp BVDV were compromised in their ability to stimulate allogeneic and memory CD4+ T cell responses, but DCs were not affected. This was not due to down-regulation of a number of recognized co-stimulatory molecules including CD80, CD86 and CD40. Striking differences in the response of the two cell types to infection with cytopathic virus were seen. Dendritic cells were not susceptible to the cytopathic effect caused by cp BVDV, whereas monocytes were killed. Analysis of interferon (IFN)-α/β production showed similar levels in monocytes and DCs exposed to cp BVDV, but none was detected in cells exposed to ncp BVDV. We conclude that the prevention of cell death in DCs is not associated with enhanced production of IFN-α/β, as proposed for influenza virus, but is by a distinct mechanism.

Formaldehyde-inactivated virus samples from type 1 poliovirus live-attenuated strains were prepared in the laboratory. The effect of treatment with formaldehyde on virus infectivity and immunogenicity in mice was investigated and the results compared with those from Mahoney wild-type poliovirus strain, the common type 1 component in commercial inactivated polio vaccines (IPV). Differences in the potency and specificity between these experimental vaccines were identified in both normal mice and transgenic mice expressing the human poliovirus receptor. The possible advantages/disadvantages of using live-attenuated strains for IPV production are discussed in the context of the global polio eradication initiative. A novel transgenic mouse model to study in vivo the immune protection induced by IPV preparations is described.

Flock house virus (FHV) is a non-enveloped, positive-sense RNA virus of insect origin that belongs to the family Nodaviridae. FHV has been shown to overcome the kingdom barrier and to replicate in plants, insects, yeast and mammalian cells. Although of insect origin, FHV has not previously been shown to replicate in mosquitoes. We have tested FHV replication in vitro in C6/36 cells (derived from neonatal Aedes albopictus) and in vivo in four different genera of mosquitoes, Aedes, Culex, Anopheles and Armigeres. FHV replicated to high titres in C6/36 cells that had been subcloned to support maximum growth of FHV. When adult mosquitoes were orally fed or injected with the virus, FHV antigen was detected in various tissues and infectious virus was recovered. Vectors developed from an infectious cDNA clone of a defective-interfering RNA, derived from FHV genomic RNA2, expressed green fluorescent protein in Drosophila cells and adult mosquitoes. This demonstrates the potential of FHV-based vectors for expression of foreign genes in mosquitoes and possibly other insects.

Diadromus pulchellus is a solitary endoparasitoid wasp that parasitizes the pupae of the leek-moth, Acrolepiosis assectella (Lepidoptera). Hitherto, every individual D. pulchellus from France that has been investigated was infected by an orthoreovirus, DpRV-1, and an ascovirus, DpAV-4. Recently, a new strain of D. pulchellus, established from a French field population, was found to be able to develop on leek-moth pupae, but lacked both DpRV-1 and DpAV-4. However, all these wasps were infected with a new cypovirus, DpRV-2. This cypovirus is transmitted to the A. assectella pupae at each wasp oviposition and is replicated mainly in the gut cells of the parasitized pupae. DpRV-2, like the ascovirus DpAV-4, is able to inhibit the defence reaction of A. assectella pupae and so contributes to the parasitic success of D. pulchellus wasps.

Marine birnavirus (MABV), a member of the genus Aquabirnavirus, family Birnaviridae, is an unenveloped icosahedral virus with two genomes of double-stranded RNA. The mechanisms of MABV adsorption and penetration are still undetermined. This work examined MABV infection in susceptible and resistant fish cell lines. MABV adsorbed not only onto the cell surfaces of susceptible (CHSE-214 and RSBK-2) cells but also onto resistant (FHM and EPC) cells. Furthermore, the virus entered the cytoplasm through the endocytotic pathway in CHSE-214, RSBK-2 and FHM cells but did not penetrate EPC cells. Thus, restriction of the MABV replication cycle is different between resistant FHM and EPC cells. The virus was found to bind to an around 250 kDa protein on CHSE-214, RSBK-2, FHM and EPC cells. Thus, this 250 kDa protein may be a major MABV receptor that exists in the plasma membranes of all four cell lines examined. This result suggests further that another receptor for virus penetration may exist in CHSE-214, RSBK-2 and FHM cells but not in EPC cells.

The late expression factor-8 gene (lef-8) of Autographa californica M nucleopolyhedrovirus encodes the largest subunit of the virally encoded DNA-directed RNA polymerase specific for the transcription of late and very late viral genes. The sequence of lef-8 predicts a C-terminal motif of 13 amino acids that is conserved in other polymerases. Detailed mutagenesis throughout lef-8 was performed, including this C-terminal motif, to define sequences required for late promoter activation. It was found that the conserved C-terminal motif was critical for late gene expression. In addition, regions throughout the entire lef-8-encoding sequence were important for optimal function, suggesting complex protein–protein and protein–DNA interrelationships in the late gene-specific viral transcriptosome.

The Rachiplusia ou multiple nucleopolyhedrovirus (RoMNPV) is a variant of Autographa californica MNPV (AcMNPV) but is significantly more virulent against several major agricultural pests. The genome sequence of the R1 strain of RoMNPV was determined and compared to that of AcMNPV strain C6. The RoMNPV genome is approximately 131·5 kbp with a G+C content of 39·1 %. The homologous repeat regions (hrs) described for AcMNPV-C6 are present in RoMNPV-R1 but the hrs of RoMNPV have fewer palindromic repeats. The RoMNPV-R1 nucleotide sequence is almost completely collinear with the sequence of AcMNPV-C6 and contains homologues of 150 of the 155 ORFs described for AcMNPV-C6. Deletions, insertions and substitutions have resulted in the loss of homologues for AcMNPV ORFs ac2 (bro), ac3 (ctl), ac97, ac121 and ac140 from the RoMNPV genome. The average amino acid sequence identity between RoMNPV and AcMNPV ORFs is 96·1 % and there are differences in promoter motif composition for 23 of these ORFs. Maximum-likelihood analysis of selection pressures on AcMNPV and RoMNPV ORFs indicate that ORFs ro18/ac20-ac21 (arif-1) and ro135/ac143 (odv-e18) have undergone positive selection.