- Volume 81, Issue 6, 2000

A recombinant vaccine, produced by using a highly attenuated smallpox vaccine (LC16mO) as a vector and which expresses the rinderpest virus (RPV) haemagglutinin protein, has been developed. The properties of this vaccine, including its heat stability, efficacy in short-term trials, safety and genetic stability, have been confirmed in an earlier report. In the present study, the duration of the protective immunity generated by the vaccine in cattle was examined for up to 3 years following the administration of a single vaccination dose of 108 p.f.u. The vaccinated cattle were kept for 2 (group I) or 3 years (group II) and then challenged with a highly virulent strain of RPV. Four of five vaccinated cattle in group I and all six cattle in group II survived the challenge, some showing solid immunity without any clinical signs of rinderpest. Neutralizing antibodies were maintained at a significant level for up to 3 years and they increased rapidly following challenge. Lymphocyte proliferative responses to RPV were examined in group II cattle and were observed in four of the six vaccinated cattle in this group. The long-lasting protective immunity, in addition to the other properties confirmed previously, indicate the practical usefulness of this vaccine for field use.

Three influenza C virus strains (C/Yamagata/1/92, C/Yamagata/1/93 and C/Miyagi/5/93) isolated in Yamagata and Sendai Cities, Japan, between June 1992 and May 1993 were found to possess haemagglutinin–esterase glycoproteins that were antigenically indistinguishable from one another but were clearly different from any previous Japanese isolates. To investigate the origin of the 1992/1993 strains, their antigenic and genetic properties were compared with those of eight strains isolated outside Japan between 1967 and 1982. The results showed that the 1992/1993 isolates were closely related to a virus isolated in Brazil in 1982 (C/SaoPaulo/378/82) and that these viruses (including C/SaoPaulo/378/82) are reassortants that had obtained PB1 and NP genes from a C/Yamagata/26/81-like parent and the other genes from another as yet unidentified parent.

Puumala hantavirus (PUUV) causes nephropathia epidemica (NE), a form of haemorrhagic fever with renal syndrome that occurs in northern and central Europe. The immunoglobulin A (IgA) response in NE patients was studied. The levels of total serum IgA in acute-phase samples from NE patients were found to be significantly elevated when compared with the levels in healthy controls. ELISAs for detection of the IgA1 and IgA2 responses against each PUUV structural protein (N, G1 and G2) were developed and evaluated. Sequential sera from NE patients (acute, convalescent, 2-year) and 10–20 year NE-convalescent sera were examined. Most patients developed detectable levels of IgA1 against N and G2, while the G1 responses were low or undetectable. Seven of nine 10–20 year sera contained virus-specific IgA1, which may indicate the prolonged presence of viral antigens after the initial infection. PEPSCAN analysis revealed several IgA-reactive antigenic regions in the N protein. Serum IgA and IgG was purified by affinity chromatography and examined by a virus-neutralization assay. Three of five sera from acute-phase NE patients contained neutralizing IgA1. The diagnostic potential of the PUUV-specific IgA1 response was evaluated. The N and G2 assays showed specificities of 100% with sensitivities of 91 and 84%, respectively, compared with an IgM μ-capture ELISA. Several NE patients, clinically diagnosed for acute PUUV infection, with borderline or undetectable levels of PUUV-specific IgM, were found to be highly positive for the presence of PUUV N-specific serum IgA1, proving the diagnostic value of IgA analysis as a complement to detection of IgM.

Experimental infection of cattle with bovine immunodeficiency virus (BIV) is characterized by persistent, low levels of virus replication in the absence of clinical disease. A virus neutralization (VN) assay was developed to examine the role of VN antibodies in controlling virus replication in cattle experimentally infected with the BIVR29 isolate of BIV. All animals developed VN antibody, but there was no correlation between VN titres and restriction of virus replication in vivo. BIV infection did not induce high-titred, cross-neutralizing antibody and there was no evidence for antigenic variation through more than 4 years in vivo. Genetic comparisons among the BIVR29 inoculum virus and viruses isolated from infected animals identified only limited genetic variation during 4 years in vivo. Moreover, there was no evidence that the observed variation was due to selection. Analyses of genetic diversity in the virus stock used for inoculation indicated a fairly homogeneous population. In the absence of high levels of virus replication and overt clinical disease, there appeared to be little selection of virus variants, resulting in antigenic and genetic stability of BIVR29 during long-term, persistent infection.

A 20089 nucleotide (nt) sequence was determined for the 5′ end of the (+)-ssRNA genome of gill-associated virus (GAV), a yellow head-like virus infecting Penaeus monodon prawns. Clones were generated from a ∼22 kb dsRNA purified from lymphoid organ total RNA of GAV-infected prawns. The region contains a single gene comprising two long overlapping open reading frames, ORF1a and ORF1b, of 4060 and 2646 amino acids, respectively. The ORFs are structurally related to the ORF1a and ORF1ab polyproteins of coronaviruses and arteriviruses. The 99 nt overlap between ORF1a and ORF1b contains a putative AAAUUUU ‘slippery’ sequence associated with −1 ribosomal frameshifting. A 131 nt stem–loop with the potential to form a complex pseudoknot resides 3 nt downstream of this sequence. Although different to the G/UUUAAAC frameshift sites and ‘H-type’ pseudoknots of nidoviruses, in vitro transcription/translation analysis demonstrated that the GAV element also facilitates read-through of the ORF1a/1b junction. As in coronaviruses, GAV ORF1a encodes a 3C-like cysteine protease domain located between two hydrophobic regions. However, its sequence suggests some structural relationship to the chymotrypsin-like serine proteases of arteriviruses. ORF1b encodes homologues of the ‘SDD’ polymerase, which among (+)-RNA viruses is unique to nidoviruses, as well as metal-ion-binding and helicase domains. The presence of a dsRNA replicative intermediate and ORF1a and ORF1ab polyproteins translated by a −1 frameshift suggests that GAV represents the first invertebrate member of the Order Nidovirales.

Hepatitis C virus (HCV) sequences from throughout the world have been grouped into six clades, based on recently proposed criteria. Here, the partial sequences and clade assignment are reported for three HCV isolates from chronic hepatitis C patients from Somalia, for whom conventional assays failed to identify the genotype. Phylogenetic analysis of the sequences of the core, envelope 1 and part of the non- structural 5b regions suggests that all three isolates belong to a distinct HCV genetic group, tentatively classified as subtype 3h. This novel HCV subtype shows the highest sequence similarity with HCV isolates from Indonesia. Despite the fact that these patients were infected with HCV clade 3, none of them responded to standard interferon treatment.

The crystal structure of a 15 amino acid synthetic peptide, corresponding to the sequence of the major antigenic site A (G–H loop of VP1) from a multiple variant of foot-and-mouth disease virus (FMDV), has been determined at 2·3 Å resolution. The variant peptide includes four amino acid substitutions in the loop relative to the previously studied peptide representing FMDV C-S8c1 and corresponds to the loop of a natural FMDV isolate of subtype C1. The peptide was complexed with the Fab fragment of the neutralizing monoclonal antibody 4C4. The peptide adopts a compact fold with a nearly cyclic conformation and a disposition of the receptor-recognition motif Arg–Gly–Asp that is closely related to the previously determined structure for the viral loop, as part of the virion, and for unsubstituted synthetic peptide antigen bound to neutralizing antibodies. New structural findings include the observation that well-defined solvent molecules appear to play a major role in stabilizing the conformation of the peptide and its interactions with the antibody. Structural results are supported by molecular-dynamic simulations. The multiply substituted peptide developed compensatory mechanisms to bind the antibody with a conformation very similar to that of its unsubstituted counterpart. One water molecule, which for steric reasons could not occupy the same position in the unsubstituted antigen, establishes hydrogen bonds with three peptide amino acids. The constancy of the structure of an antigenic domain despite multiple amino acid substitutions has implications for vaccine design.

Arboviruses with genomes composed of 12 segments of double-stranded (ds) RNA have previously been classified as members or probable members of the genus Coltivirus within the family Reoviridae. A number of these viruses have been isolated in North America and Europe and are serologically and genetically related to Colorado tick fever virus, the Coltivirus type species. These isolates constitute subgroup A of the coltiviruses. The complete genome sequences are now presented of two Asian arboviruses, Kadipiro virus (KDV) and Banna virus (BAV), which are currently classified as subgroup B coltiviruses. Analysis of the viral protein sequences shows that all of the BAV genome segments have cognate genes in KDV. The functions of several of these proteins were also indicated by this analysis. Proteins with dsRNA-binding domains or with significant similarities to polymerases, methyltransferases, NTPases or protein kinases were identified. Comparisons of amino acid sequences of the conserved polymerase protein have shown that BAV and KDV are only very distantly related to the subgroup A coltiviruses. These data demonstrate a requirement for the subgroup B viruses to be reassigned to a separate new genus, for which the name Seadornavirus is proposed.

We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6+ CD8bright+ CD4− (CTL phenotype), CD6+ CD8dull+ CD4+ (the fraction containing memory T helper cells), CD6+ CD8− CD4+ (T helper cell phenotype), CD6− CD8dull+ CD4− γδ-T+ ( γδ-T cell phenotype), CD6− CD8dull+ CD4− γδ-T− (NK phenotype) and CD6− CD8− CD4− γδ-T− or  γδ-T+. Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6+ CD8+ and CD4+ CD8+ and fewer CD6− CD8+ and γδ-T+ CD8+ lymphocytes than IL-2 stimulation of naive PBMC (P<0·05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6+ CD8+ T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6− CD8+ T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.

The DNA sequence of a 2·4 kbp fragment located in the internal and terminal inverted repeat sequences of the pseudorabies virus genome determined in this study closes a gap between the previously described genes for the ICP4 and ICP22 homologues. The novel sequence contains no conserved herpesvirus open reading frames. Northern blot and cDNA analyses revealed a viral immediate-early transcript of 1·8 kb, which is spliced by the removal of two small introns close to its 5′ end and which presumably represents the mRNA of the downstream open reading frame encoding the ICP22 homologue. Upstream of the transcribed region, an imperfect set of three directly repeated sequences was identified. Each of them contains a complementary pair of the alphaherpesvirus origin-binding protein recognition motif GTTCGCAC, spaced by AT-rich sequences. In vitro studies confirmed that the DNA fragment analysed includes a functional origin of viral DNA replication.

Varicella-zoster virus (VZV) glycoproteins gH and gL were examined in a recombinant vaccinia virus system. Single expression of glycoprotein gL produced two molecular forms: an 18 kDa form and a 19 kDa form differing in size by one endoglycosidase H-sensitive N-linked oligosaccharide. Coexpression of gL and gH resulted in binding of the 18 kDa gL form with the mature form of gH, while the 19 kDa gL form remained uncomplexed. The glycosylation processing of gL was not dependent on gH; however, gL was required for the conversion of precursor gH (97 kDa) to mature gH (118 kDa). Subsequent analyses indicated that gL (18 kDa) was a more completely processed gL (19 kDa). Screening of the culture media revealed that gH and gL were secreted, but only if coexpressed and complexed together. The secreted form of gL was 18 kDa while that of gH was 114 kDa. The fact that secreted gH was smaller than intracytoplasmic gH suggested a proteolytic processing event prior to secretion. The 19 kDa form of gL was never secreted. These findings support a VZV gL recycling pathway between the endoplasmic reticulum and the cis-Golgi apparatus.

Terminal differentiation of embryonal carcinoma cells and monocytes has been shown to be important for their permissiveness for human cytomegalovirus (HCMV) infection, even though such terminally differentiated cells have withdrawn from the cell cycle and are, essentially, in G0 arrest. Recently, data from a number of laboratories have shown that productive infection with HCMV of quiescent fibroblasts held reversibly in G0 of the cell cycle can result in cell cycle progression, which results eventually in cycle arrest. In contrast to quiescent fibroblasts, the effect of HCMV on cells that have withdrawn irreversibly from the cell cycle due to terminal differentiation has not, so far, been addressed. Here, it is shown that, in cells that have arrested in G0 as a result of terminal differentiation, HCMV is able to induce cell functions associated with progression of the cell cycle through G1 into early S phase. This progression is correlated with a direct physical and functional interaction between the HCMV 86 kDa major immediate-early protein (IE86) and the cyclin-dependent kinase inhibitor p21Cip1.

The pleiotropic cytokine TGF-β1 is a member of a large family of related factors involved in controlling cell proliferation, differentiation and apoptosis. TGF-β ligands interact with a complex of type I and type II transmembrane serine/threonine kinases and they transmit their signals to the nucleus via a family of Smad proteins. A panel of over 20 Burkitt’s lymphoma (BL) cell lines has been compiled including those that are Epstein–Barr virus (EBV) negative, those that carry EBV with a restricted pattern of EBV latent gene expression (group I) and those that express the full range of latent EBV genes (group III), together with selected EBV-transformed lymphoblastoid cell lines (LCLs). Most of the EBV-negative and group I BL cell lines underwent apoptosis or a G1 arrest in response to TGF-β1 treatment. In contrast, group III cell lines and LCLs were completely refractory to these effects of TGF-β1. All of the cell lines expressed the TGF-β pathway Smads and the TGF-β type I receptor. Lack of responsiveness to TGF-β1 appears to correlate with a down-regulation of TGF-β type II receptor expression. Studies of EBV-converted and stably transfected BL cell lines demonstrated that the EBV gene LMP-1 is neither necessary nor sufficient to block the TGF-β1 response.

DNA helicases of baculoviruses are essential for virus replication and have been implicated as molecular determinants of host range. Although these proteins contain seven motifs (I, Ia, II–VI) characteristic of DNA helicases, the two most important characteristics of helicases – duplex-DNA unwinding and ATPase activity – have not been demonstrated. In the present study, a recombinant putative DNA helicase (rP137) of Trichoplusia ni granulovirus (TnGV) was purified from insect cells infected with a recombinant Autographa californica multicapsid nucleopolyhedrovirus that overproduced rP137. The rP137 protein exhibited an intrinsic DNA-independent ATPase activity that required Mg2+ as a co-factor, an activity that was reduced in the presence of TnGV and phage λ DNAs. These results provide further evidence that baculovirus helicase genes encode proteins with biochemical properties similar to those of classical DNA helicases.

The coding sequences of four overlapping polypeptides starting at four different in-frame AUG codons and co-terminating at the stop codon of the cap gene of Junonia coenia densovirus (JcDNV) were inserted under the control of the p10 promoter of Autographa californica nucleopolyhedrovirus (AcMNPV) to generate AcMNPV-VP1 (four polypeptides), AcMNPV-VP2 (three polypeptides), AcMNPV-VP3 (two polypeptides), and AcMNPV-VP4 (one polypeptide) recombinant viruses. In all cases, infection of Spodoptera frugiperda cells (Sf9) by each of the four recombinant viruses resulted in the production of virus-like particles (VLPs) 22–25 nm in diameter. The VLPs produced by the three recombinants AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-VP4 were abundant and contained three, two and one polypeptides, respectively. VP4, the shortest polypeptide, thus appears to be sufficient for assembly of VLPs morphologically similar to those formed with two to four polypeptides. The ratio of VPs did not appear to be critical for assembly of the particles. The polypeptide starting at the first AUG immediately downstream from the p10 promoter was always the most abundantly expressed in infected cells, regardless of the construct. In contrast, plaque-purified AcMNPV-VP1 recombinants were unstable and produced less than one-twentieth of the VLPs produced by the others. All VP transcripts started at the TAAG late motif of the p10 promoter and had a poly(A) tail 14 nt downstream of a poly(A) addition signal located 98 nucleotides downstream of the common stop codon. No significant transcription initiation inside the cap sequence of AcMNPV-VP2, AcMNPV-VP3 and AcMNPV-PV4 was observed.