- Volume 79, Issue 5, 1998

The nucleotide and deduced amino acid sequences of louping ill (LI) virus isolates, collected from representative regions of the British Isles and Norway, were determined for either the entire envelope gene (20 isolates) or for a portion of the envelope gene that spans a hypervariable region and includes an LI virus specific marker sequence (53 isolates). Phylogenetic analysis reveals the presence of three major geographical populations of LI virus in the British Isles, viz. Irish, Welsh and British LI viruses, which all cause encephalomyelitis in animals, predominantly sheep, and co-habit the same tick population. British LI virus occurs throughout Scotland, England, Ireland and Norway. Irish and Welsh LI viruses occur only in Ireland and Wales, respectively. Phylogenetic analysis also predicts that LI virus initially emerged in Ireland and that a descendant was introduced into Great Britain via Wales and was subsequently transported to the borders of Scotland, from where it was dispersed throughout Scotland, northern England and Norway. More recently, the British LI virus was reintroduced into Ireland and also into south-west England. Dates of lineage divergence, calculated from the synonymous substitution rate, indicate that LI virus emerged in the British Isles less than 800 years ago and most LI virus dispersal occurred during the last 300 years. By combining these data with historical records it appears that livestock movement can be implicated in the dispersal of LI virus.

The ORF5-encoded major envelope glycoprotein (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is one of the three major structural proteins of this virus. While some porcine convalescent sera and monoclonal antibodies directed against GP4 and GP5 have the capacity to neutralize the virus in vitro, the protein specificity of porcine neutralizing sera has not yet been established. DNA immunization with a plasmid encoding GP5 of PRRSV, under the control of a human cytomegalovirus promoter, induced anti-GP5-specific neutralizing antibodies in pigs and BALB/c mice. The GP5 protein specificity of neutralizing sera was confirmed by immunoblotting and ELISA. Peripheral blood mononuclear cells obtained from DNA-vaccinated pigs underwent blastogenic transformation in the presence of E. coli-expressed recombinant ORF5-encoded protein, indicating the specificity of the cellular immune response to GP5. Following a massive intratracheal challenge with the virulent IAF-Klop strain of PRRSV, DNA-vaccinated pigs were protected from generalized viraemia and the development of typical macroscopic lung lesions that were observed in unvaccinated, virus-challenged controls, as well as in pigs that were immunized with E. coli-expressed GST-ORF5 recombinant fusion protein. Interstitial pneumonitis and broncho-alveolitis were remarkably milder in DNA-vaccinated animals. These results suggest that the GP5 of PRRSV is a good candidate for a subunit recombinant-type vaccine.

Virus recombinants constructed from Theiler's murine encephalomyelitis virus (TMEV) strain GDVII, which causes a rapidly fatal encephalitis in mice, and the less virulent BeAn, which persists in the murine central nervous system (CNS) and causes inflammatory demyelination, and a GDVII mutant deleted of 46 of 76 leader protein amino acids were analysed for virus persistence in the CNS. The two recombinant and mutant viruses principally contain GDVII sequences including the nucleotides encoding the polyprotein and 3' untranslated region. These viruses were found to replicate in the CNS of mice but they did not produce acute encephalitis or paralysis, i.e. they were attenuated in neurovirulence compared to the GDVII parent. More important, none of the viruses persisted in the mouse CNS nor caused chronic demyelination. Thus, attenuation of GDVII neurovirulence alone is not sufficient to establish TMEV persistence. This result is discussed in the context of a genomic determinant for persistence.

In rabies virus, the ribonucleoprotein complex (RNP), the RNA genome (-) and the antigenome (+) are specifically coated by the viral nucleoprotein (N protein), forming the template for transcription and replication bythe viral RNA polymerase. This specific encapsidation starts at the 5' ends of the RNAs. To investigate domains of the N protein that govern binding specificity, we tested in vitro the ability of both full-length and truncated forms of the N protein to interact with a synthetic RNA probe corresponding to the 5' end of the antigenome. UV-LASER cross-linking, which covalently links RNA and proteins in intimate contact, showed that the entire N protein (450 aa) and the NH2-terminal 376 aa (t42) contained all of the determinants for specific interaction. It was demonstrated by affinity chromatography that a peptide near the COOH terminus of t42 (position 298352), which is located in the most conserved region of Rhabdoviridae N proteins, bound directly to the viral RNA. However, no significant sequence similarity was detected between this peptide and known RNA binding proteins in the databases. This suggests both that N proteins may possess a new type of RNA binding motif and that protein folding contributes to the architecture of the RNA binding site.

Recently, we demonstrated that infection of cells with all measles virus (MV) strains tested was inhibited by antibodies against CD46, although not all strains caused downregulation of the MV receptor CD46 from the surface of human cells. We now show that infection of cells with MV strain WTFb, a variant of wild-type isolate WTF which has been isolated and propagated on human BJAB cells, is not inhibited by antibodies against CD46. In contrast, infection of cells with the closely related strain WTFv, a Vero cell-adapted variant of WTF, is inhibited by antibodies against CD46. This observation led us to investigate the interaction of these viruses and the vaccine strain Edmonston (Edm) with CD46 and target cells. Cellular receptors with high affinity binding for WTFb are present on BJAB cells, but not on transfected CD46-expressing CHO cells. In contrast to the Edm strain, virus particles and solubilized envelope glycoproteins of WTFb have a very limited binding capacity to CD46. Furthermore, we show that recombinant soluble CD46 either does not bind, or binds very weakly, to WTFb glycoproteins expressed on the cell surface. Our findings indicate that wild-type MV strain WTFb and vaccine strain Edm use different binding sites on human cells. In addition, the results suggest that MV strains may alternatively use CD46 and an unknown molecule as receptors, and that the degree of usage of both receptors may be MV strain-specific.

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

The ATP requirement of influenza A virus RNA-dependent RNA polymerase was studied during in vitro transcription reactions. In complete transcription reactions, the Km for ATP was 10-fold higher than the Km values for the other NTPs. However, during transcription elongation the Km for ATP was as low as the Km values for the other NTPs, suggesting a special requirement for ATP during transcription initiation. Gel analysis of RNA products of transcription initiation reactions showed that the incorporation of AMP into nascent RNA was more efficient at positions 4, 6 and 7 relative to the template RNA than at position 5. The polymerase produced short, abortive transcripts with lengths corresponding to positions 3 and 4 relative to the template but never to position 5 or longer. These results suggest that incorporation of AMP at position 5 induces the influenza A virus polymerase to go through a transition from a transcription initiation to an elongation complex. This functional change of the polymerase complex rather than a requirement for ATP beta-gamma bond hydrolysis is the most likely reason for the particularly high Km for ATP during the early phase of transcription. This conclusion is supported by the fact that the ATP analogue ATPgammaS [adenosine 5'-O-(3-thiotriphosphate)] can efficiently replace ATP in in vitro transcription reactions and shows a comparable drop of Km between transcription initiation and elongation.

A recombinant vaccinia virus inducibly expressing ORF A1 of infectious bursal disease virus (IBDV) has been constructed and characterized. Cells infected with this recombinant virus express the IBDV polyprotein, which is proteolytically processed to give mature VP2, VP3, and VP4 polypeptides. An electron microscopy study revealed that the cytoplasm of cells infected with the recombinant virus contains abundant IBDV-like particles (VLP). These VLP form close-packed paracrystalline arrays that are specifically recognized by anti-IBDV antibodies. The size and morphology of purified VLP were found to be akin to those of authentic IBDV particles.

In order to investigate the transmission of human immunodeficiency virus type 1 (HIV-1) from mother-to-child we have examined serial plasma RNA samples obtained from a mother over an eight year period spanning four pregnancies. Child 1 and 2 (born January 1987 and June 1990) were uninfected whilst child 3 and 4 (born July 1992 and February 1994) were HIV positive. Genetic variation was examined within the viral population of the mother and her two infected children for both the V3 loop and flanking regions of the env gene and the p17 region of the gag gene. In one child (child 4) a highly homogeneous virus population was observed within both env and gag in contrast to the more heterogeneous virus population observed within the mother. Viral sequences of child 4 clustered within a single branch within the reconstructed phylogenetic tree. This is consistent with the transmission of a single maternal variant to the child in this case, which may indicate a selective process. By contrast, child 3 showed substantial genetic heterogeneity even within the first samples obtained shortly after birth. Sequences of child 3 clustered in two distinct groups within the phylogenetic tree and were separated by sequences of the mother. These results are not consistent with the selective transmission of a single maternal variant to the child in this case and we therefore propose that the infection within child 3 is the result of the transmission of multiple sequence variants to the child. All transmitted sequence variants were predicted to be of the macrophage-tropic, nonsyncytium-inducing (NSI) phenotype.

Human immunodeficiency virus type 1 (HIV-1) wild-type (WT) virion infectivity factor (Vif) protein (Vifwt) and full-length Gag precursor (Pr55Gag) were found to be co-encapsidated into extracellular, membrane-enveloped virus-like particles released by budding from Sf9 cells co-expressing the two recombinant proteins in trans, with an average copy number of 3.5+/-0.6 Vifwt per 100 Pr55Gag molecules. No preferential localization at the plasma membrane was observed for recombinant Vif in the absence of Gag expression, and a significant proportion of Vif accumulated within the nucleus. Two conserved motifs, W89RKRRY94 and P156KKIKP161, seemed to act as nuclear addressing signals. The Pr55Gag and Vifwt interacting domains were analysed by biopanning of a phage-displayed hexapeptide library. The Vif-binding domain, which spanned residues H421-T470 in Pr55Gag, corresponded to the C-terminal region of nucleocapsid (NC), including the second zinc finger, the intermediate spacer peptide sp2 and the N-terminal half of the p6 domain. Deletions in these Gag domains significantly decreased the Vif encapsidation efficiency, and complete deletion of NC abolished Vif encapsidation. In Vif, four discrete Gag-binding sites were identified, within residues T68-L81 (site I) and W89-P100 (site II) in the central domain, and within residues P162-R173 (III) and P177-M189 (IV) at the C terminus. Substitutions in site I and deletion of site IV were detrimental to Vif encapsidation, whereas substitution of basic residues for alanine in sites III and IV had a positive effect. The data suggest a direct intracellular Gag-Vif interaction and the occurrence of a Pr55Gag-mediated membrane-targeting pathway for Vif in Sf9 cells.

Studies analysing human immunodeficiency virus type 1 replication in primary cells have demonstrated that Vpr, although dispensable, plays a role along with the matrix (MA) protein in allowing nuclear localization of viral preintegration complexes in non-dividing monocyte-derived macrophages (MDMs). In the current study, experimental infection conditions to analyse the role of Vpr, independently of MA, during infection of PHA/IL-2-stimulated peripheral blood mononuclear cells (PBMC) were designed. It was shown that the absence of Vpr results in a subtle effect on virus production in long-term infection. PCR analysis of the steps of virus retrotranscription during a single cycle of replication in stimulated PBMC revealed that the absence of Vpr alone correlates with an impairment in the nuclear localization of viral DNA. Our data indicate that Vpr is involved in the virus life-cycle during infection of dividing PBMC, presumably as it is during infection of MDMs.

The question of whether consensus nucleotide substitutions in the long terminal repeat (LTR) region of simian immunodeficiency virus strain mac (SIVmac) are important for neurovirulence was investigated in this report. Brains and lymph nodes from two macaques that developed AIDS and encephalitis following inoculation with two strains of neurovirulent SIVmac, and from one animal with AIDS but no neurological disease after inoculation with non-neurovirulent SIVmac239 were used. The 5' LTR regions from neurovirulent SIVmacR71/17E and SIVmac7F-Lu were amplified, cloned and sequenced and these sequences were compared to the LTRs amplified from three regions of the respective encephalitic brains and lymph nodes from macaques inoculated with each virus. The SIVmac7F-Lu and SIVmacR71/17E viruses had zero and three consensus substitutions, respectively, in the U3, R and U5 regions of the LTR compared to that of SIVmac239. The only consensus substitution in the LTR-gag region of the genome was a T to C change at position 829 within the tRNA binding site. The sequences amplified from the brain and lymph nodes of the two animals with AIDS and encephalitis were identical. This single common substitution in this region of the virus genome, the T to C substitution at position 829, was also found in the LTRs isolated from the brain and lymphoid organs from the macaque inoculated with SIVmac239. The virtual identity in nucleotide sequences in the LTR of the neurovirulent and non-neurovirulent viruses and in CNS and lymph tissues of animals inoculated with the viruses suggests that the LTR has no effect on the tissue tropisms of the viruses.

In T cells, transcriptional activation by the long terminal repeat (LTR) of the mink cell focus-forming murine leukaemia virus requires some spacing between the enhancer and promoter. A large size-range of intervening sequences (11-93 bp) is able to activate transcription efficiently. Neither a specific nucleotide sequence nor stereospecific alignment of the spacer is important.

The hepatitis D virus (HDV) genotypes in 46 HDV-infected patients and 12 prostitutes were screened with Xhol restriction fragment length polymorphism (RFLP) analysis of reverse transcription PCR products of viral genomes and verified by phylogenetic analysis. The amplificates of three (6.5%) patients and two (17%) prostitutes showed a novel RFLP pattern different from those of the three known genotypes. Complete HDV genomic sequence identities between isolates with a novel RFLP and the HDV genotypes I, II and III were 72.3, 77.2 and 63.0%, respectively. Importantly, divergence was mostly seen in various regions related to replication or packaging. The novel isolates formed a monophyletic group (P < 0.05) and were most closely related to genotype II.

Hepatitis delta virus (HDV) packaging requires prenylation of the HDV large protein (p27), as well as a direct protein-protein interaction between HDV proteins and hepatitis B virus (HBV) envelope protein domains. To investigate this interaction, we have analysed the binding capacity of baculovirus-expressed delta p24 and p27 proteins to synthetic peptides specific for the HBV envelope. Although a higher degree of binding was observed with p27, both p24 and p27 could bind HBV envelope peptides. One such peptide corresponded to residues 56-80 located in the cytosolic loop of the small HBV envelope protein, and another corresponded to 23 carboxy-terminal residues of the pre-S1 specific to the large HBV envelope protein. This indicates that in addition to p27, p24 may contribute to packaging of HDV through a protein-protein interaction with HBV envelope domains, and that an interaction between the pre-S1 polypeptide and delta proteins may play a role in infectivity.

Hepadnaviruses have a complex replication cycle which includes reverse transcription of the pregenomic RNA. The initial step in this process in hepatitis B virus (HBV) requires the viral polymerase to engage a highly stable region of secondary structure within the pregenomic RNA termed the epsilon stem-loop. While reverse transcriptases belonging to the retrovirus family use a specific cellular tRNA as primer, HBV polymerase utilizes a tyrosine residue located within its own N terminus. Therefore, the first deoxyribonucleotide is covalently coupled to HBV polymerase prior to extension of the DNA strand by conventional reverse transcription. We have expressed HBV polymerase in a baculovirus and following purification have found it to be active with respect to protein-priming and reverse transcription of copurified RNA. Importantly, we found both of these processes to be critically dependent on the presence of the epsilon stem-loop. The metal ion preferences of HBV polymerase were also investigated for both the protein-priming and reverse transcription activities of this enzyme. Reverse transcription was dependent on magnesium, with an optimal concentration of 5 mM. However, protein-priming was strongly favoured by manganese ions and was optimal at a concentration of 1 mM. Thus, using manganese as sole source of metal ions our activity assay is restricted to the protein-priming event and will allow the search for novel antivirals specifically blocking this unique mechanism.

Papillomaviruses are small DNA viruses which infect and induce benign warts and sometimes malignant tumours in the epithelium of the skin or mucosa. The viruses do not replicate in conventional tissue culture systems and little is known about the requirements for virus assembly. We investigated the effect of ethylene glycol-bis(aminoethyl ether)-tetraacetic acid (EGTA) and dithiothreitol (DTT) treatment on the stability of bovine papillomavirus type 1 (BPV-1) particles in vitro. Removal of calcium ions by 11 mM EGTA at pH 8.0 together with reduction of disulfide bonds by 15 mM DTT destabilized BPV particles. Electron microscopy examination of treated particles showed that the BPV particles had been disrupted to capsomeres. Addition of exogenous calcium ions to the disruption buffer prevented virus destabilization. Adding calcium to the disrupted BPV particles resulted in the reassembly of disrupted particles. The reassembled particles were morphologically similar to intact BPV virions. We further quantified the efficiency of reassembly by focus formation assay. We recorded 500-fold less infectivity for reassembled BPV and 4-fold less haemagglutination activity compared to untreated BPV, pointing towards a decrease in the amount of reassembled particles recovered.

Five major genotypes of JC virus (JCV) have been defined based on nucleotide differences in the VP1 gene of the DNA sequence. These types are probably a result of virus evolution in geographically isolated population groups. One of the first genotypes identified, Type 2, was found to represent strains of Asian origin. In order to further define the spectrum within Type 2 strains, the entire 5.1 kb genome of nine urinary strains of JCV was amplified by PCR with one pair of primers. These urine samples were obtained in the USA (California and New Mexico) from three European Americans, three Native Americans, two African Americans and one Hispanic American. The complete genome of an Asian JCV strain (Tokyo-1) isolated from progressive multifocal leukoencephalopathy (PML) brain in Japan was also sequenced. Here, we report the analysis of these ten DNA sequences and their deduced protein translations. Two phylogenetically distinct subtypes of Type 2 were found, 2A and 2B, which differ from each other by 0.8-1.1% of the coding region sequence. A 215 bp product amplified with primers in the VP1 gene contains enough sequence information to distinguish the major types and subtypes of JCV and is suitable for application in viral epidemiological studies. The investigation of these genomic variations is of special interest because JCV Type 2 strains are found at a significantly higher frequency in brain tissue of patients with PML than would be predicted from their excretion in a control population.