- Volume 78, Issue 4, 1997

Forty-two ovine pestivirus isolates, collected over a period of 18 years, were compared by phylogenetic analysis. The viruses were mostly field isolates from Britain; two others originated from Sweden and two from New Zealand. RT-PCR products were obtained from two genomic regions, one within the 5′- noncoding (5′-NC) region, and the other encompassing parts of the p20 (Npro) and C coding regions. Direct sequencing of the 5′-NC PCR products, followed by computer-assisted phylogenetic analysis, divided the ovine pestiviruses into three main genotypes. The results demonstrated that sheep may naturally be infected not only with border disease virus (BDV), but also with bovine viral diarrhoea virus (BVDV) types I and II. The BDV isolates segregated into two principal subtypes represented by the Moredun strain from Scotland and the 137/4 strain from England. The BVDV-I group was composed of three clusters, two of them represented by BVDV reference strains NADL and Osloss, respectively, and the third by ovine isolates D1120/1 and D1432/P. The grouping of ovine pestiviruses, based on comparative nucleotide sequence analysis of the 5′-NC region, was confirmed by comparative analysis of the p20 (Npro) and C coding regions, performed both at the nucleotide and at the amino acid level. The presence of three genotypes in sheep, including BVDV-I and BVDV-II, indicates the inadequacy of the current host- species-based nomenclature and classification of pestiviruses.

Recently, putative viral agents responsible for human non-Ato E hepatitis have been independently reported by two groups of investigators and designated GB virus C (GBV-C) and hepatitis G virus (HGV), respectively. The entire nucleotide sequences were determined for two viral genomes isolated from Japanese blood donors with GBV-C RNA. One of them (GT230) had a total genomic length of 9390 nucleotides (nt) with 5′ and 3′ untranslated regions of 551 and 313 nt, while the other (GT110) had genomic lengths of 9395, 281 and 315 nt, respectively. They both had a single long open reading frame, encoding 2842 amino acids (aa) in GT230 and 2933 aa in GT110. Surprisingly, they both lacked a clearly identifiable core gene, and possessed the E1/E2 gene with only four potential N-linked glycosylation sites. Pairwise comparison and phylogenetic analysis of the entire sequence indicated that the prototype GBV-C and two HGV isolates reported, as well as GT230 and GT110, are the same virus possibly of different genotypes. The five GBV-C/HGV isolates were variable up to 13 8% in the genomic nucleotide sequence, and contained deletions and insertions within the 5 -terminal 518-593 nt, which resulted in four different sizes of predicted polyproteins encoded by genomes of individual isolates. By contrast, the 3′ untranslated region was well conserved. The high degree of sequence conservation within this region would favour it as a target for sensitive detection of GBV- C/HGV RNA.

The surface glycoprotein S (spike) of coronaviruses is believed to be an important determinant of virulence and displays extensive genetic polymorphism in cell culture isolates. This led us to consider whether the observed heterogeneity is reflected by a quasispecies distribution of mutated RNA molecules within the infected organ. Corona- virus infection of rodents is a useful model system for investigating the pathogenesis of virus-induced central nervous system (CNS) disease. Here, we investigated whether genetic changes in the S gene occurred during virus persistence in vivo. We analysed the variability of S gene sequences directly from the brain tissue of Lewis rats infected with the coronavirus mouse hepatitis virus (MHV) variant JHM-Pi using RT-PCR amplification methods. The S gene sequence displayed a remarkable genetic stability in vivo. No evidence for a quasispecies distribution was found by sequence analysis of amplified S gene fragments derived from the CNS of Lewis rats. Furthermore, the S gene also remained conserved under the selection pressure of a neutralizing antibody. Only a few mutations predicted to result in amino acid changes were detected in single clones. The changes were not represented in the consensus sequence. These results indicate that to retain functional proteins under the constraints of a persistent infection in vivo, conservation of sequence can be more important than heterogeneity.

Cynomolgus macaque monkeys (Macaca fascicu- laris) were immunized twice intramuscularly, either with a conventional non-adjuvanted subunit vaccine or with a candidate immune-stimulating complex (iscom) vaccine, each containing 10 µg envelope glycoprotein of a recent human influenza A(H3N2) virus (A/Netherlands/18/94). In contrast to the macaques vaccinated with the classical subunit vaccine, those immunized with the iscom vaccine developed high titres of specific IgM, IgA and IgG serum antibodies, as well as high titres of haemag- glutination-inhibiting and virus-neutralizing serum antibodies. Also, specific proliferative T cell responses were only found in the iscom-vaccinated monkeys and their levels were similar to those found in monkeys experimentally infected with the homologous virus. Upon intratracheal challenge with the homologous virus, the iscom-vaccinated monkeys were completely protected from detectable virus replication in lungs, pharynx and nose, whereas those vaccinated with the classical subunit vaccines were not, or were only partially protected. The kinetics of specific serum antibody development in the iscom-vaccinated monkeys after challenge were quite similar to those of monkeys after secondary infection with the same virus. In contrast, the post-challenge kinetics of serum antibody development in the monkeys vaccinated with the classical subunit vaccines resembled those of naive monkeys, confirming that these vaccines only provided limited protection in such animals.

The influenza virus M2 protein, target of the antiviral drugs amantadine and rimantadine, forms a proton channel which functions during virus uncoating and maturation by modifying the pH in virions as well as in trans-Golgi vesicles. We studied the influence of different ionic gradients on the inhibition of the proton translocation activity of isolated, baculo- virus-expressed M2 protein reconstituted into liposomes. Two distinct patterns of inhibition were observed. A group of amphiphilic amines including amantadine, cyclooctylamine and rimantadine inhibited M2 effectively in the presence of physiological Na concentrations. The 10-fold greater activity of rimantadine over amantadine and the 100-fold stronger effect of cyclooctylamine compared to cyclopentylamine matched the relative activities in influenza virus-infected cells. A com-pletely different inhibitory pattern emerged for the polyamines spermine, spermidine and putrescine. Polyamines have recently been identified as the ‘intrinsic’ rectifiers of a class of potassium channels and shown to interact with acidic amino acid residues lining and flanking the channel pore. In the presence of a physiological Na /K gradient their minimal inhibitory concentrations for influenza virus M2 protein were 100, 400 and 500 µM, polyamine levels reported to exist in oocytes. In conditions depleted for Na , polyamines inhibited M2 at concentrations two to three orders of magnitude lower. The data suggest that influenza virus M2 protein possesses a binding site for polyamines, distinct from the amantadine binding site, which is normally masked by Na and which could be targeted by selective antiviral inhibitors.

Syncytium formation and subsequent generalized cell fusion have been reported as potentially important mechanisms of virus-induced cytotoxic effects. We tried to clarify the roles of fusion regulatory factor-1 (FRP-1) in virus-induced cell fusion. Two mutated human FRP-1/CD98 proteins [FRP-1/ HN, in which the cytoplasmic domain was replaced with the cytoplasmic domain of human parainfluenza virus type 2 (HPIV-2) haemagglutinin- neuraminidase (HN), and FRP-1/330 (serine), in which a cysteine at amino acid 330 was mutated to serine], when expressed stably in L929 cells, were lacking in cell-fusion-enhancing activity stimulated by anti-FRP-1 antibodies. Anti-FRP-1 antibodies enhanced Newcastle disease virus (NDV)-mediated polykaryocyte formation in parent HeLa cells, while anti-FRP-1 antibodies showed no/low effect on polykaryocyte formation in NDV-infected HeLa cells constitutively expressing FRP-1/HN (HeLa-FRP-1/ HN cells), indicating that the FRP-1/HN molecule is capable of acting as a dominant negative inhibitor. Furthermore, when HeLa-FRP-1/HN cells were infected with various rubulaviruses (HPIV-2, mumps virus, simian viruses 5 and 41), virus-induced cell fusion was also suppressed, although virus replication was not inhibited in these cells, showing that FRP-1 molecules are required for virus-induced cell fusion. Therefore, FRP-1 is considered to be related to the pathogenesis of paramyxoviruses.

Fv4 is an endogenous defective murine leukaemia virus (MuLV) which expresses high levels of an envelope protein (Env) closely related to that of the ecotropic class of MuLVs. Mice bearing the natural Fv4 gene or a transgenic version are resistant to infection by ecotropic MuLVs. Fv4 mice secrete the surface peptide (SU) of the Fv4 Env in their serum and this secreted Env can block infection of NIH3T3 cells. To study the secretion of Fv4, we metabolically labelled cells expressing Fv4 Env or Env from infectious MuLVs and followed synthesis, glycosyl- ation, proteolytic processing and secretion of Env species. We found no difference in the kinetics of synthesis or processing of Fv4 Env compared to the envelopes of infectious MuLVs, but Fv4 Env associated more weakly with its transmembrane anchor and was shed from the surface of cells.

Infection of domestic cats with feline immunodeficiency virus (FIV) causes progressive immunological deterioration similar to that caused by human immunodeficiency virus (HIV). Lentiviruses related to but phylogenetically distinct from FIV have been detected in several non-domestic feline species. Serological cross-reactivity of these viruses raises the question as to whether inter-species transmission may occur. To address this issue, we asked whether lion lentivirus (FIV-Ple) or two strains of puma lentivirus (FIV-Pco) could replicate or cause disease in domestic cats. We found that domestic cats inoculated with FIV-Ple developed persistent cell-associated viraemia, transient cell-free viraemia and antiviral antibody. Clinical disease was not detected throughout a 6 month observation period. Two of four cats inoculated with FIV-Pco developed cell-associated viraemia, seroconverted and exhibited transient lymphadenopathy. No changes in white blood cell parameters or other haematological abnormalities were detected in any of the infected cats. Virus-specific RNA was detected in cocultivated lymphocytes of all infected cats by RT- PCR. These findings reveal that non-domestic cat lentiviruses are infectious for domestic cats and can establish persistent infection in the absence of disease.

Caprine arthritis-encephalitis virus (CAEV), an ungulate lentivirus, causes a natural infection in goats. The present report demonstrates that in vitro, CAEV infection is associated with apoptosis, characterized by morphological changes such as condensation of chromatin and the appearance of apoptotic bodies. The presence of DNA fragments was documented by the appearance of a DNA ‘ladder’ in agarose gel electrophoresis, as well as by in situ end-labelling of DNA ends. In addition, flow cytometric analyses revealed the presence of a hypodiploid peak that gradually appeared as virus infection progressed.

We have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (EIAV), designated V70. Five viruses (F1V, F2V, F3V, F4V and F5V) were recovered during periodic febrile episodes. Crossneutralization tests revealed that all of these variants and the parental V70 were antigenically distinct. Sequencing of their full-length env gp90 genes and gp45 5′ sequences revealed novel mutations at a limited number of nucleotide positions, consisting of insertions and duplications in the gp90 principal neutralizing domain (PND) in F1V, F3V and F5V. Parts or all of small units (6, 9 and 12 nucleotides) located just before the insertion site were used for the duplications. Furthermore, amino acid substitutions in the env PND and hypervariable region were also observed in all five viruses. These mutations may contribute to the generation of serial variants. Consequently, the full-length gp90 sequences showed close relationships between V70, F2V and F4V, and between F1V, F3V and F5V. In addition to the two domains (PND and hypervariable region), a comparison of these viruses with the reported env gp90 sequences revealed four additional variable domains, although these four domains were highly conserved among the five variants.

A detailed analysis of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses and the identification of the proteins and epitopes they target may improve the design of immunotherapeutic interventions and provide insights into AIDS pathogenesis. Here, we identified a new CTL epitope in the SIV Gag protein, recognized by CD8 and MHC class I-restricted CTL clones from a long-term asymptomatic cynomolgus macaque (Macaca fascicularis) infected with SIVmac32H-J5. Using overlapping synthetic peptides, the optimal minimal epitope was characterized as a nine amino acid peptide representing amino acids 242–250 of p26 (SVDEQIQWM). CTL recognition was shown to be abolished by amino acid substitutions observed within homologous human immunodeficiency virus (HIV)-1 and HIV-2 sequences.

SDZ NIM 811 is a cyclosporin A (CsA) analogue that is completely devoid of immunosuppressive capacity but exhibits potent and selective antihuman immunodeficiency virus type 1 (HIV-1) activity. Binding to cyclophilin A, the intracellular receptor for cyclosporins, is a prerequisite for HIV-1 inhibition by cyclosporins. Cyclophilin A was demonstrated to bind to HIV-1 p24 gag and this cyclophilin-Gag interaction leads to the incorporation of cyclophilin Ainto HIV-1 virions. SDZ NIM 811 inhibitsthis protein interaction, and this is likely to be the molecular basis for its antiviral activity. Here, we show that in activated primary T cells SDZ NIM 811 interferes with two stages of the virus replication cycle: (i) translocation of pre-integration complexes into the nucleus and (ii) production of infectious virus particles. SDZ NIM 811 not only inhibits translocation of HIV-1 pre-integration complexes in primary T cells, but also in a growth-arrested T cell line. In vivo, most T lymphocytes are quiescent, but serve nevertheless as a major and inducible HIV-1 reservoir in infected individuals. Significant amounts of cyclophilin A were found to be associated with virus particles propagated in primary T cells. SDZ NIM 811 caused a strong reduction in the amount of incorporated cyclophilin A, thereby reducing infectivity. Thus, cyclophilin A seems to be necessary for HIV-1 replication in primary T cells.

Retroviruses use a cellular tRNA molecule as primer for reverse transcription. The complementarity between the 3′ end of this tRNA and a sequence near the 5′ end of the viral RNA, the primer-binding site (PBS), allows the primer to anneal onto the viral RNA. During reverse transcription 18 nucleotides of the tRNA primer are copied into the viral cDNA, thereby regenerating the PBS sequence of the progeny. Thus, the PBS sequence reveals which primer was used. Human immunodeficiency viruses are known to replicate efficiently with tRNALys3 as primer. Examination of the PBS sequence in natural and laboratory isolates indicates that a variant tRNALys is occasionally used as primer. This variant, for which the murine genomic sequence was described previously, was termed tRNALys5 and differs from tRNALys3 at five nucleotide positions. These results suggest that HIV uses both tRNALys3 and tRNALys5 molecules as primer, causing a switch of the PBS sequence.

We characterized two human immunodeficiency virus type 1 (HIV-1) strains, HIVMCK and HIV213, which have different cytopathic effects in infected cells. HIV213 was highly cytopathic, whereas HIVMCK was not. Biological analyses of chimeric viruses from the cloned infectious DNAs of HIVMCK and HIV213 showed that the Vpu region was responsible for the differing cytopathicity of these viruses. Although HIVMCK expressed Vpu protein, HIV213 did not because of a mutation at the start codon for Vpu. The amounts of envelope glycoprotein and virus particles associated with the cell surface were significantly increased on cells infected with Vpu-deficient viruses compared with Vpu-positive viruses. These data suggest that the highly cytopathic effects of HIV213 (Vpu-deficient) are due to an accumulation of envelope glycoprotein at the infected-cell surface, which would be caused by the retention of progeny virions in the absence of Vpu-facilitated virion release.

Novel herpesviruses have been described recently. These include human herpesviruses 6, 7 and 8 (HHV-6, -7, -8). HHV-6 has at least two strain groups, variants A and B. The B strains are predominant in the West and can account for over 97% of infections in infants. In contrast, the A strains are rare and the few well-characterized isolates have been from adult African AIDS patients. It is not clear whether the HHV-6 variant A strains are AIDS-related and/or whether they can also be acquired as childhood infections and may reactivate later during adulthood. What contribution geographical variation plays has yet to be assessed. HHV-8 has been associated with AIDS-related epidemic Kaposi’s sarcoma (KS), but has also been identified in endemic KS. In regions of Africa where KS is endemic, the onset of AIDS has led to increased prevalence of KS. In this report, we examine in Zambia, an AIDS epidemic and KS endemic region, infection with these novel herpesviruses during infancy. In blood samples from human immunodeficiency virus-negative infants with first febrile episode, both semi-quantitative PCR and sequence analyses were used to identify HHV-8 in 8% and HHV-6 in 30%, with 44% of these variant A; in childhood endemic KS biopsies HHV-8 was detected in 100% and HHV-6 in none. The high viral-DNA loads in the infant blood samples were consistent with viraemia. This is the first demonstration that HHV-6 variant A and HHV-8 may be acquired as common childhood infections.

The herpes simplex virus type 1 (HSV) singlestranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.

Persistence of herpes simplex virus (HSV) DNA in mouse footpad keratinocytes was studied by nonisotopic in situ hybridization. HSV DNA was retained in keratinocyte nuclei for more than 2 weeks after disappearance of infectious virus and viral antigens. The anatomical location of viral DNA became more superficial with increasing time post-infection, reflecting the migration of cells from the basal layer of the epidermis towards the stratum granulosum. Latency-associated transcripts (LATs) were not detected in footpad cells at any of the times studied. In contrast, LATs were detected readily in the nuclei of lumbar ganglionic neurons innervating HSV DNA positive footpads. It was concluded that, after termination of productive infection in the skin, HSV DNA persists transiently in keratinocyte nuclei, in the absence of abundant latency-associated transcription. An implication of these data is that detection of HSV DNA in the skin may reflect recent, but not necessarily current, cutaneous virus replication.

The complete DNA sequence of a field strain of canine adenovirus type 1 was determined by sequencing random fragments of viral DNA cloned into pBluescript. The virus has a genome of 30536 bp flanked by two identical 161 bp inverted terminal repeats. Thirty ORFs have been identified, based on genomic location or sequence identity with published adenoviruses. These are arranged into similar discrete regions found in the human adenoviruses. ORFs in the late region show greatest identity with published human adenovirus sequences, whereas the E3 and E4 ORFs show little or none.

Transcription of the E1A gene of the highly oncogenic adenovirus 12 (Ad12) initiates at two start sites (TS1 and TS2). We have previously shown that the E2F and ATF motifs distal of TS1 co-operatively participate in E1A autostimulation from the TS1 promoter region. Here we report the identification of a second E2F-like target region (E2FII) immediately upstream of the E1A-stimulating factor 1 binding site (ESF-1), important for 13S-mediated autoactivation from TS2. Reporter constructs lacking distinct TS2 c/s-acting elements were analysed for their levels of CAT expression in the absence and presence of the E1A 13S protein in transient expression assays. In the absence of 13S, full promoter activity was observed only for a construct containing all elements (the E2F-like motif, an E-Box and the TATA element). Promoter activation increased significantly in Ad12 E1A-co-transfected cells. Induction by the 13S protein was also detected for the construct containing a non-functional ESF-1 sequence. Our results indicate that the E2F-like motif is responsible for activation mediated by the 13S protein from TS2, while ESF-1-or TATA-binding protein activity were not involved. Additionally, the TATA sequence appeared to be dispensable for transactivation. Gel-shift experiments using the E2F-like promoter element as a probe indicated the binding of an E2F-5 or E2F-5-like transcription factor to this region. We conclude that transcription through the TS1 as well astheTS2 promoter region is stimulated by the Ad12 13S protein. Moreover, transfection of the construct including bothTS1 and TS2 indicates an E2F-site-mediated synergism between both regions with respect to E1A-induced transactivation.