- Volume 82, Issue 7, 2001

We have demonstrated previously that the envelope protein of a murine retrovirus, Moloney murine leukaemia virus, has immunosuppressive properties in vivo. This property was manifested by the ability of the protein, when expressed by tumour cells normally rejected by engrafted mice, to allow the env-expressing cells to escape immune rejection and to proliferate. Here, it is shown that this property is not restricted to the envelope of a murine retrovirus, but is also shared by the envelope encoded by a primate retrovirus, Mason–Pfizer monkey virus.

In this study we compared the efficacy of live attenuated human immunodeficiency virus type 2 (HIV-2) vaccine alone versus boosting with live non-pathogenic HIV-2 following priming with ALVAC HIV-2 (recombinant canarypox virus expressing HIV-2 env, gag and pol). Six monkeys were first inoculated intravenously with live HIV-2SBL-6669 and 7 to 10 months later were challenged intrarectally with 10 MID50 of cell-free simian immunodeficiency virus (SIV) strain SIVsm. One monkey was completely protected against SIV infection and all five monkeys that became SIV-infected showed a lower virus replication and an initial lower virus load as compared with a parallel group of six control animals. In another experiment five monkeys were immunized either three times with ALVAC HIV-2 alone or twice with ALVAC HIV-2 and once with purified native HIV-2 gp125. The monkeys were then challenged with HIV-2 given intravenously and finally with pathogenic SIVsm given intrarectally. After challenge with SIVsm, three of five monkeys were completely protected against SIVsm infection whereas the remaining two macaques became SIV-infected but with limited virus replication. In conclusion, vaccination with an ALVAC HIV-2 vaccine followed by exposure to live HIV-2 could induce cross-protection against mucosal infection with SIVsm and seemed to be more efficient than immunization with a live HIV-2 vaccine only.

A method for encoding insertions and deletions (indels) has been developed and adapted to the SplitsTree program. Following phylogenetic reconstruction, the relative frequencies of indels were estimated for a large number of in vivo sequence sets corresponding to the env V1 hypervariable region of the simian immunodeficiency virus SIVmac251. The method allowed recovery of many point mutations hitherto lost due to gap stripping. Deletions were as frequent as transversions and were 4- to 8-fold more frequent than insertions, invariably duplications. The high proportion of deletions among mutation events suggests that lentivirus vectors may readily delete parts of their cargo.

The intra-host evolution of 73 human immunodeficiency virus type 1 quasispecies was analysed by split decomposition analysis. Non-synonymous and synonymous nucleotide substitutions were counted along the shortest path connecting all sequences and compared with the numbers expected under the assumption of a random model of mutation. For the majority of substitutions, drift and negative selection seemed to prevail.

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins, E1 and E2. These proteins contain a large N-terminal ectodomain, and are anchored into membranes by their C-terminal transmembrane domain (TMD). The TMDs of HCV envelope proteins are multifunctional. In addition to their role as membrane anchors, they possess a signal sequence function in their C-terminal half, and play a major role in subcellular localization and assembly of these envelope proteins. In this work, the expression of full-length E2 led to secretion of a proportion of this protein, which is likely to be due to inefficient membrane insertion of a fraction of E2 expressed alone. However, when E1 and E2 were coexpressed from the same polyprotein, E2 was not secreted and remained tightly associated with membranes, suggesting that an early interaction between the TMDs of HCV envelope proteins improves the stability of membrane insertion of E2. These results reinforce the hypothesis that the TMDs of E1 and E2 are major factors in the assembly of the HCV envelope glycoprotein complex.

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system – under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.

The N-terminal one-third of the NS3 protein of Dengue virus type 2 (DEN-2) complexes with co-factor NS2B to form an active serine proteinase which cleaves the viral polyprotein. To identify sites within NS3 that may interact with NS2B, seven regions within the NS3 proteinase outside the conserved flavivirus enzyme motifs were mutated by alanine replacement. Five sites contained clusters of charged residues and were hydrophilic. Two sites were hydrophobic and highly conserved among flaviviruses. The effects of five mutations on NS2B/3 processing were examined using a COS cell expression system. Four retained significant proteinase activity. Three of these mutations and two more were introduced into genomic-length cDNA and tested for their effects on virus replication. The five mutant viruses showed reduced plaque size and two of the five showed significantly reduced titres. All seven mutations were mapped on the X-ray crystal structure of the DEN-2 NS3 proteinase: three were located at the N terminus and two at the C terminus of the NS2B-binding cleft. Two mutations were at the C terminus of the proteinase domain and one was solvent-exposed. The study demonstrated that charged-to-alanine mutagenesis in the viral proteinase can be used to produce growth-restricted flaviviruses that may be useful in the production of attenuated vaccine strains.

To determine whether Powassan virus (POW) and deer tick virus (DTV) constitute distinct flaviviral populations transmitted by ixodid ticks in North America, we analysed diverse nucleotide sequences from 16 strains of these viruses. Two distinct genetic lineages are evident, which may be defined by geographical and host associations. The nucleotide and amino acid sequences of lineage one (comprising New York and Canadian POW isolates) are highly conserved across time and space, but those of lineage two (comprising isolates from deer ticks and a fox) are more variable. The divergence between lineages is much greater than the variation within either lineage, and lineage two appears to be more diverse genetically than is lineage one. Application of McDonald–Kreitman tests to the sequences of these strains indicates that adaptive evolution of the envelope protein separates lineage one from lineage two. The two POW lineages circulating in North America possess a pattern of genetic diversity suggesting that they comprise distinct subtypes that may perpetuate in separate enzootic cycles.

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3′ untranslated region (3′UTR). Two mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R496→H in the envelope (E) gene and C10884→T in the 3′UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R496 in the E protein produced plaques of smaller size than viruses with H496 at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T10884→C substitution within the 3′UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H496 and T10884, showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H496→R and T10884→C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I45→F) and the NS5 (T2688→A and M3385→I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.

The aim of this study was to assess whether the infection of antigen-presenting cells (APC) in vivo, evident in calves persistently infected (PI) with bovine viral diarrhoea virus (BVDV), compromised their ability to stimulate virus-specific T cell responses. Major histocompatibility complex (MHC) molecule-identical cattle were identified from the inbred family at the Institute for Animal Health. One was PI and immunotolerant to BVDV. Virus was not isolated from the remaining calves, which were classified as BVDV-immune or BVDV-naïve depending on the presence or absence of BVDV-specific antibodies in sera. Two-colour flow-cytometric analysis of PBMC from the PI calf showed that 40% of CD14+ monocytes were infected in vivo. Monocytes from the PI calf (PI monocytes) were used as naturally infected ex vivo APC with CD4+ or CD8+ T cells isolated from the BVDV-naïve or BVDV-immune animals. PI monocytes stimulated proliferative responses with CD4+ and CD8+ T cells from BVDV-immune animals, but not from BVDV-naïve calves. This provided evidence for the presence of virus-specific CD4+ and CD8+ memory T cells after acute infection and indicated that ex vivo monocytes from PI, immunotolerant calves stimulated both MHC class I- and MHC class II-restricted T cell responses to BVDV. Additionally, naturally infected ex vivo monocytes cultured in vitro for 3 days stimulated effective T cell responses to the virus with which they were infected.

A unique hepatitis E virus (HEV) strain was identified as the aetiological agent of acute hepatitis in a United States (US) patient who had recently returned from vacation in Thailand, a country in which HEV is endemic. Sequence comparison showed that this HEV strain was most similar, but not identical, to the swine and human HEV strains recovered in the US. Phylogenetic analysis revealed that this new HEV isolate was closer to genotype 3 strains than to the genotype 1 strains common in Asia. The fact that this HEV was closely related to strains recovered in countries where HEV is not endemic and was highly divergent from Asian HEV strains raises the questions of where the patient’s infection was acquired and of whether strains are geographically as localized as once thought.

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5′ and 3′ hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.

Foot-and-mouth disease virus (FMDV) is known to employ the conserved Arg–Gly–Asp (RGD) tripeptide located on the variable βG–βH loop of the VP1 capsid protein for binding to cells. Coxsackievirus A9 (CAV9) also carries an RGD sequence, but on a short C-terminal extension of its VP1 and in a different amino acid context. This apparent relationship raised the question of whether insertion of the heterologous CAV9 sequence into FMDV would influence infection by the genetically modified FMDV. Four VP1 mutants were generated by PCR mutagenesis of a full-length FMDV cDNA plasmid. After transfection of BHK-21 cells, viral protein synthesis and virus particle formation could be detected. Two of the four mutants, mV9b and mV9d, could be propagated in BHK-21 cells, but not in CV-1 cells. Both of these mutants contained 17 amino acids of the C terminus of CAV9 VP1. Infection of BHK cells could be specifically inhibited by rabbit immune serum raised against a synthetic peptide representing the amino acid sequence of the C-terminal extension of CAV9 VP1. This demonstrated the direct involvement of the inserted sequence in cell infection. In fact, genetically modified FMDV O1K was capable of employing the VP1 C-terminal RGD region of CAV9 for infection of BHK cells. In addition, these results show that, even in cell culture-adapted viruses, the RGD-containing βG–βH loop plays an important role in virus infectivity.

This work focuses on the development of a potential recombinant DNA vaccine against foot-and-mouth disease virus (FMDV). Such a vaccine would have significant advantages over the conventional inactivated virus vaccine, in particular having none of the risks associated with the high security requirements for working with live virus. The principal aim of this strategy was to stimulate an antibody response to native, neutralizing epitopes of empty FMDV capsids generated in vivo. Thus, a plasmid (pcDNA3.1/P1–2A3C3D) was constructed containing FMDV cDNA sequences encoding the viral structural protein precursor P1–2A and the non-structural proteins 3C and 3D. The 3C protein was included to ensure cleavage of the P1–2A precursor to VP0, VP1 and VP3, the components of self-assembling empty capsids. The non-structural protein 3D was also included in the construct in order to provide additional stimulation of CD4+ T cells. When swine were immunized with this plasmid, antibodies to FMDV and the 3D polymerase were synthesized. Furthermore, neutralizing antibodies were detected and, after three sequential vaccinations with DNA, some of the animals were protected against challenge with live virus. Additional experiments suggested that the antibody response to FMDV proteins was improved by the co-administration of a plasmid encoding porcine granulocyte–macrophage colony-stimulating factor. Although still not as effective as the conventional virus vaccine, the results encourage further work towards the development of a DNA vaccine against FMDV.

Equine rhinitis A virus (ERAV) is a picornavirus that has been reclassified as a member of the Aphthovirus genus because of its resemblance to foot-and-mouth disease virus at the level of nucleotide sequence and overall genomic structure. The N-terminal amino acid sequence of three of the four capsid proteins of ERAV was determined and showed that the proteolytic cleavage sites within the precursor P1 polypeptide occur exactly as those predicted for an aphthovirus-like 3C protease, which generates the capsid proteins VP1 and VP3. However, the autocatalytic cleavage site between VP4 and VP2, which is independent of 3C protease cleavage, was different from that predicted previously. ERAV.393/76 antisera from horses and rabbits showed different reactivity to the viral structural proteins in both serum neutralization assays and Western blots. High neutralizing antibody titres appeared to correlate with strong reactivity to VP1 in Western blots.

A previous report showed that insertion of a foreign gene encoding chloramphenicol acetyltransferase (CAT) between the HN and L genes of the full-length cDNA of a virulent Newcastle disease virus (NDV) yielded virus with growth retardation and attenuation. The NDV vector used in that study was pathogenic to chickens; it is therefore not suitable for use as a vaccine vector. In the present study, an avirulent NDV vector was generated and its potential to express CAT protein was evaluated. The CAT gene was under the control of NDV transcriptional start and stop signals and was inserted immediately before the open reading frame of the viral 3′-proximal nucleocapsid protein gene. A recombinant NDV expressing CAT activity at a high level was recovered. The replication and pathogenesis of the CAT-expressing recombinant NDV were not modified significantly. These results indicate the potential utility of an avirulent NDV as a vaccine vector.

The potential of RNA-based vaccines was evaluated for the generation of a protective immune response in the mouse model of influenza type A virus infection using the internal nucleoprotein (NP) as antigen. This antigen is of particular interest, since it has the potential to elicit protective cytotoxic T lymphocytes (CTL) against heterologous strains of influenza A virus. In view of the short half-life of RNA, self-replicating RNAs or replicons of the positive-stranded genomes of Semliki Forest virus (SFV) and poliovirus were engineered to synthesize the influenza A virus NP in place of their structural proteins. NP expression was demonstrated by immunoprecipitation after transfection of cells with RNA from the SFV (rSFV-NP) and poliovirus (rΔP1-E-NP) genome-derived replicons transcribed in vitro. C57BL/6 mice were injected intramuscularly with these synthetic RNAs in naked form. Both replicons, rSFV-NP and rΔP1-E-NP, induced antibodies against the influenza virus NP, but only mice immunized with the rSFV-NP replicon developed a CTL response against the immunodominant H-2Db epitope NP366. Finally, the protective potential of the CTL response induced by immunization of mice with rSFV-NP RNA was demonstrated by the reduction of virus load in the lungs after challenge infection with mouse-adapted influenza A/PR/8/34 virus and was comparable to the protective potential of the response induced by plasmid DNA immunization. These results demonstrate that naked RNA immunization with self-replicating molecules can effectively induce both humoral and cellular immune responses and constitutes an alternative strategy to DNA immunization.

Influenza A virus infection of mice has been used extensively as a model to investigate the mechanisms of antigen presentation to cytotoxic T lymphocytes (CTL) and the phenomenon of immunodominance in antiviral CTL responses. The different virus-encoded epitopes that are recognized in H-2b and H-2d mice have been characterized and their relative immunodominance has been well-studied. In H-2k mice, four different Kk-restricted influenza virus epitopes have been described, but the dominance hierarchy of these epitopes is unknown and there is also an uncharacterized Dk-restricted response against the virus. In this study, a Dk-restricted epitope derived from the influenza virus A/PR/8/34 polymerase protein PB1, corresponding to amino acid residues 349–357 (ARLGKGYMF), was identified. This peptide is the major epitope within the PB1 polymerase and is at least as dominant as any of the four Kk-restricted epitopes that are recognized in CBA mice following primary influenza virus infection. The PB1 epitope is only the fourth Dk-presented peptide to be reported and the sequence of this epitope confirms a Dk-restricted peptide motif, consisting of arginine at position two, arginine or lysine at position five and a hydrophobic residue at the carboxy terminus.

Infectious salmon anaemia virus (ISAV) is an orthomyxo-like virus that causes serious disease in Atlantic salmon (Salmo salar). Like the orthomyxoviruses, ISAV has been shown to possess haemagglutinin (HA) activity. This study presents the cloning, expression and identification of the ISAV HA gene, which was isolated from a cDNA library by immunoscreening. The HA gene contained an ISAV-specific conserved nucleotide motif in the 5′ region and a 1167 bp open reading frame encoding a protein with a predicted molecular mass of 42·4 kDa. The HA gene was expressed in a baculovirus system. A monoclonal antibody (MAb) shown previously to be directed against the ISAV HA reacted with insect cells infected with recombinant baculovirus. Salmon erythrocytes also adsorbed to these cells and adsorption was inhibited by the addition of either the ISAV-specific MAb or a polyclonal rabbit serum prepared against purified virus, confirming the virus specificity of the reaction. Immunoblot analyses indicated that ISAV HA, in contrast to influenza virus HA, is not posttranslationally cleaved. Sequence comparisons of the HA gene from five Norwegian, one Scottish and one Canadian isolate revealed a highly polymorphic region that may be useful in epidemiological studies.