- Volume 85, Issue 1, 2004

A novel simian immunodeficiency virus (SIV) was characterized from a Schmidt's guenon (Cercopithecus ascanius schmidti), which was housed in a local zoo. The virus infection was detected during a routine serological screening for antibodies that were cross-reactive with SIVmac antigens. Infection with an immunodeficiency virus was confirmed using an INNO-LIA HIV Confirmation assay. Using DNA isolated from a blot clot, a 1895 nt partial pol sequence was amplified and sequenced. Phylogenetic analysis showed that this virus, designated SIVschm, shares a distant relationship with SIVgsn, isolated from greater spot-nosed monkeys, and is one of the most divergent SIVs identified to date.

A serological survey for human T cell leukaemia virus (HTLV)/simian T cell leukaemia virus (STLV) antibodies was performed in 61 wild-caught African apes, including five gorillas and 56 chimpanzees originating from south Cameroon. Two young animals, a gorilla (Gorilla gorilla gorilla) and a chimpanzee (Pan troglodytes vellerosus), exhibited a pattern of complete HTLV-I seroreactivity. Sequence comparison and phylogenetic analyses using the complete LTR (750 bp) and a 522 bp fragment of the env gene indicated the existence of two novel STLV-I strains, both of which belonged to HTLV-I/STLV-I molecular clade subtype B, specific to central Africa. These first STLV-I strains to be characterized in gorilla and chimpanzee were closely related to each other as well as to several HTLV-I strains originating from inhabitants of south Cameroon, including pygmies. Such findings reinforce the hypothesis of interspecies transmission of STLV-I to humans, leading to the present day distribution of HTLV-I in central African inhabitants.

Hepatitis C virus (HCV) has high genomic variability and, since its discovery, at least six different types and an increasing number of subtypes have been reported. Genotype 1 is the most prevalent genotype found in South America. In the present study, three different genomic regions (5′UTR, core and NS5B) of four HCV strains isolated from Peruvian patients were sequenced in order to investigate the congruence of HCV genotyping for these three genomic regions. Phylogenetic analysis using 5′UTR–core sequences found strain PE22 to be related to subtype 1b. However, the same analysis using the NS5B region found it to be related to subtype 1a. To test the possibility of genetic recombination, phylogenetic studies were carried out, revealing that a crossover event had taken place in the NS5B protein. We discuss the consequences of this observation on HCV genotype classification, laboratory diagnosis and treatment of HCV infection.

Hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) genome was directly sequenced from 12 chronically infected patients who had not responded to interferon (IFN) treatment. Due to the quasispecies nature of HCV circulating genomes, serum samples from four patients showing different evolutionary characteristics were further analysed. Serial samples from each patient were taken before, soon after and 14–23 months after a 6 month IFN treatment. HVR1 from each sample was amplified, cloned and the clones sequenced. For each patient, a phylogenetic analysis of the clones was performed and quasispecies complexity and genetic distances were calculated. The amino acid sequences and predicted antigenic profiles were analysed. The pre-treatment samples of the different patients presented dissimilar genetic quasispecies composition. For three of the patients, we showed that, regardless of the complexity or diversity of the viral populations before treatment, they evolved towards genetic diversification following selective pressure. Once the environment became stable, the entire population tended towards homogeneity. The fourth patient represented a case where different components of the quasispecies coexisted for long periods without replacement. We propose herein that the evolution of HVR1 of E2 is more likely to be directed by selection of clearly different subpopulations (modification of quasispecies equilibrium) than by a continuous mechanism related to the successive accumulation of point mutations. The prevalence of a quasispecies shift mechanism was revealed by the cloning analysis during the follow-up period of the evolutionary process.

Hepatitis C virus (HCV) has been reported to replicate in monocytes/macrophages in infected patients. However, it is unclear whether macrophages are susceptible to infection in vitro and whether such an infection is consequential. Sera from 26 HCV-infected patients were incubated with primary human macrophages collected from healthy donors. Virus negative strand was detected by a Tth enzyme-based strand-specific assay and virus sequences were analysed by single strand conformation polymorphism (SSCP) and sequencing. Concentrations of the cytokines tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β, IL-6, IL-8, IL-10 and IL-12p70 were measured in culture supernatants and respective mRNAs were analysed in cell extracts by quantitative RT-PCR. For 15 sera, HCV RNA was detectable in 2- and 3-week cultures from at least one donor. Virus negative strand was detected in 29 % of macrophage samples in this group. In four cases, HCV RNA sequences amplified from macrophages differed from those amplified from sera suggesting evolution during infection. Concentrations of TNF-α and IL-8 were found to be significantly higher in supernatants from HCV-infected cultures. In conclusion, these preliminary data suggest that primary human macrophages are susceptible to HCV infection in vitro and this infection is associated with the induction of cytokines TNF-α and IL-8.

The three South African Territories (SAT) types of foot-and-mouth disease virus (FMDV) display great genetic and antigenic diversity, resulting from the independent evolution of these viruses in different geographical localities. For effective control of the disease in such areas, the use of custom-made vaccines is required. To circumvent the tedious process of vaccine strain selection, an alternative in the control process is being investigated. Specifically, it is proposed to replace the antigenic determinants of an infectious genome-length cDNA copy of a good SAT vaccine strain with those of appropriate field strains, producing custom-made FMDV chimeras for use in vaccine production. Here the construction of an infectious genome-length cDNA copy of the SAT2 vaccine strain, ZIM/7/83, is described, created utilizing an exchange-cassette strategy with an existing A12 genome-length cDNA clone. The virus derived from this cDNA (designated vSAT2) displayed excellent growth properties in cell culture, indicating its potential usefulness in the production of custom-made vaccine strains. Evaluation of the growth of various SAT2/A12 chimeras created during the derivation of SAT2 infectious cDNA suggested incompatibilities between the non-structural proteins of ZIM/7/83 and the 5′ UTR of A12.

Enterovirus 71 (EV71) infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal meningoencephalitis complicated with neurogenic pulmonary oedema. Its pathogenesis, especially the CNS involvement, is not clearly understood. The aim of this study was to set up a mouse EV71 infection model with CNS involvement. EV71 virus was administrated orally to neonatal mice. The EV71-infected mice manifested a skin rash at an early stage and hind limb paralysis or death at a later stage. Immunohistochemical staining and virus isolation demonstrated that EV71 replicated in the small intestine, induced viraemia and spread to various organs. Kinetic studies showed that EV71 antigen was first detected in the intestine at 6 h, in the thoracic spinal cord at 24 h, in the cervical spinal cord at 50 h and in the brain stem at 78 h post-infection. Leukocyte infiltration was evident in the spinal cord and brain stem. Furthermore, EV71 virus could be transmitted to littermates within the same cage.

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide and are recognized as the foremost cause of foodborne illness. Despite numerous efforts, routine cell cultures have failed to yield replicating NoV. This paper describes methods used to try to grow NoV in vitro in two laboratories. Cells (A549, AGS, Caco-2, CCD-18, CRFK, CR-PEC, Detroit 551, Detroit 562, FRhK-4, HCT-8, HeLa, HEC, HEp-2, Ht-29, HuTu-80, I-407, IEC-6, IEC-18, Kato-3, L20B, MA104, MDBK, MDCK, RD, TMK, Vero and 293) were cultured on solid or permeable surfaces. Differentiation was induced using cell culture supplements such as insulin, DMSO and butyric acid. In some cases, the cells and the NoV-containing stool samples were treated with bioactive digestive additives. Variables evaluated in cultivation experiments included the method of preparation of the virus inoculum, the genotype of the virus, conditions for maintenance of cell monolayers, additives in the maintenance medium and the method of inoculation of the cells. Serial blind passage studies were performed routinely. In addition to evaluation for CPE, evidence of virus replication was sought using immunofluorescent assays to detect newly produced viral capsid antigen and RT-PCR assays to detect the viral genome. Although some infected cultures remained NoV positive by RT-PCR for up to five passages and an occasional cell in a monolayer showed evidence of specific immunofluorescence, no reproducible NoV-induced CPE was observed and all RT-PCR results that were positive initially were negative following continued passaging. Thus, attempts to develop a method for the cultivation of NoV were unsuccessful.

Endemic circulation of hepatitis E virus (HEV) in Namibia was suspected from serological data during an outbreak of non-A, non-B hepatitis in Rundu in 1995. The source of the outbreak was suspected to be the water supply, which had been compromised approximately 6 months earlier. Four HEV isolates from four different persons in this outbreak were successfully amplified, sequenced and analysed over a 451 bp region of a subgenomic fragment from the 3′ end of the genome in ORF2. Phylogenetic analysis showed that the four Namibian HEV isolates clustered with a Mexican isolate in genotype II and shared 85·8–86·3 % nucleotide identity with the 1987 Mexican isolate, but were only 77·6–79·6 % similar to other African isolates. HEV isolated from the same region of Namibia in 1983 was reported to cluster in genotype I. However, virus isolates from sporadic cases of HEV isolated in 1997/8 in Nigeria were also from genotype II.

To investigate the genetic changes in hepatitis E virus (HEV) strains in the Kathmandu valley of Nepal, we compared the 412 nt sequence within open reading frame 2 of HEV among HEV isolates recovered from 16 patients in 1999, 14 patients in 2000 and 38 patients in 2002, and additional isolates recovered from 48 patients in 1997 whose nucleotide sequences have been previously published. All 116 HEV-viraemic samples were genotyped as 1 and subtyped further as 1a (n=85, 73 %), 1c (n=29, 25 %) and mixed infection of 1a and 1c (n=2, 2 %): subtype 1c was detected only in 1997. Among the 1a isolates, nucleotide sequence identity with the representative 1a isolate of Ne131-1997 was 96·4±2·4 % (mean±SD) in 1997, 93·9±1·7 % in 1999, 92·2±1·0 % in 2000 and 91·7±0·5 % in 2002, indicating gradual diversification of HEV sequences. When phylogenetic analysis of the 87 subtype 1a isolates was performed, they further segregated into five clusters, with two predominant clusters of 1a-2 and 1a-3: the annual frequency of cluster 1a-2 isolates decreased from 63 % in 1997, to 50 % in 1999, to 7 % in 2000 and no cases in 2002; cluster 1a-3 isolates were observed in all four years and its annual frequency increased from 5 % in 1997 to 95 % in 2002. Of the remaining three clusters, cluster 1a-1 was detectable only in 1997 and clusters 1a-4 and 1a-5 emerged in 2000 and 2002, respectively. These results indicate that genetic changes and takeover of HEV strains may contribute to the genetic variability of HEV in the community.

The evolutionary relationships of 26 sequenced members of the poxvirus family have been investigated by comparing their genome organization and gene content and by using DNA and protein sequences for phylogenetic analyses. The central region of the genome of chordopoxviruses (ChPVs) is highly conserved in gene content and arrangement, except for some gene inversions in Fowlpox virus (FPV) and species-specific gene insertions in FPV and Molluscum contagiosum virus (MCV). In the central region 90 genes are conserved in all ChPVs, but no gene from near the termini is conserved throughout the subfamily. Inclusion of two entomopoxvirus (EnPV) sequences reduces the number of conserved genes to 49. The EnPVs are divergent from ChPVs and between themselves. Relationships between ChPV genera were evaluated by comparing the genome size, number of unique genes, gene arrangement and phylogenetic analyses of protein sequences. Overall, genus Avipoxvirus is the most divergent. The next most divergent ChPV genus is Molluscipoxvirus, whose sole member, MCV, infects only man. The Suipoxvirus, Capripoxvirus, Leporipoxvirus and Yatapoxvirus genera cluster together, with Suipoxvirus and Capripoxvirus sharing a common ancestor, and are distinct from the genus Orthopoxvirus (OPV). Within the OPV genus, Monkeypox virus, Ectromelia virus and Cowpox virus strain Brighton Red (BR) do not group closely with any other OPV, Variola virus and Camelpox virus form a subgroup, and Vaccinia virus is most closely related to CPV-GRI-90. This suggests that CPV-BR and GRI-90 should be separate species.

The predicted extracellular domain of the CD2v protein of African swine fever virus (ASFV) shares significant similarity to that of the CD2 protein in T cells but has a unique cytoplasmic domain of unknown function. Here we have shown that CD2v is expressed as a glycoprotein of approximately 105 kDa in ASFV-infected cells. In the absence of an extracellular ligand, the majority of CD2v appears to localize to perinuclear membrane compartments. Furthermore, we have shown using the yeast two-hybrid system and by direct binding studies that the cytoplasmic tail of CD2v binds to the cytoplasmic adaptor protein SH3P7 (mAbp1, HIP55), which has been reported to be involved in diverse cellular functions such as vesicle transport and signal transduction. A cDNA clone encoding a variant form of SH3P7 could also be identified and was found to be expressed in a wide range of porcine tissues. Deletion mutagenesis identified proline-rich repeats of sequence PPPKPC in the ASFV CD2v protein to be necessary and sufficient for binding to the SH3 domain of SH3P7. In ASFV-infected cells, CD2v and SH3P7 co-localized in areas surrounding the perinuclear virus factories. These areas also stained with an antibody that recognizes a Golgi network protein, indicating that they contained membranes derived from the Golgi network. Our data provide a first molecular basis for the understanding of the immunomodulatory functions of CD2v in ASFV-infected animals.

A murine gammaherpesvirus-68 (MHV-68) mutant with deregulated transcription of its ORF50 transactivator was severely impaired in latency establishment. The deregulated virus showed reduced immunogenicity, probably reflecting a lower antigen load. However, it still elicited effective immunity to a subsequent wild-type (WT) virus challenge. Infection was not completely prevented, but was very substantially reduced in extent and the long-term level of WT viral DNA in lungs and spleens remained low. Thus latency-deficient MHV-68 illustrates a possible general approach to creating attenuated gammaherpesvirus vaccines that can protect against pathogenic WT infections.

A regulated switch between latent and lytic gene expression is common to all known herpesviruses. However, the effects on host colonization of altering this switch are largely unknown. We deregulated the transcription of the gene encoding the major lytic transactivator of murine gammaherpesvirus-68, ORF50, by inserting a new and powerful promoter element in its 5′ untranslated region. In vitro, the mutant virus (M50) transcribed ORF50 at a high level and showed more rapid lytic spread in permissive fibroblast cultures, but in vivo, the M50 virus showed a severe deficit in latency establishment, with no sign of the infectious mononucleosis-like illness normally associated with wild-type infection. Although a low level of M50 viral DNA was detectable by PCR in spleens, replication-competent virus could not be recovered beyond 10 days post-infection. The M50 virus was also attenuated in immunocompromised mice. Thus a gammaherpesvirus unable to shut off lytic cycle gene expression showed severely restricted host colonization.

The herpesvirus saimiri (HVS) ORF73 gene product shares limited homology with the ORF73 protein of Kaposi's sarcoma-associated herpesvirus (KSHV). ORF73 is expressed in an in vitro model of HVS latency, where the genome persists as a non-integrated circular episome. This suggests it may have a similar role to KSHV ORF73 in episomal maintenance, by tethering viral genomes to host-cell chromosomes. Here, the association of ORF73 with host mitotic chromosomes is described. Deletion analysis demonstrates that the distal 123 aa of the ORF73 protein are required for mitotic chromosomal localization and for self-association. Moreover, deletion of the extreme C terminus disrupts both self-association and host mitotic chromosome colocalization. This suggests that HVS ORF73 has a similar role to KSHV ORF73 in episomal maintenance and that association of ORF73 to host mitotic chromosomes is dependent on its ability to form multimers.

Molecular epidemiological studies of Kaposi's sarcoma-associated herpesvirus (KSHV) have concentrated on characterization of viral strains in tumour biopsy samples from Kaposi's sarcoma (KS) patients, mostly obtained in the United States and Europe. Tumour biopsies are a convenient source of viral DNA, as they have a high viral load compared to peripheral blood. However, sequences obtained from biopsies may not be representative of viral strains in asymptomatic subjects and information on ethnicity is often not available. Here, a population-based approach has been used to study the molecular and seroepidemiology of KSHV in isolated populations in Ecuador and Botswana. Amerindians in Ecuador had a variable prevalence of KSHV and all strains characterized were of subtype E, based on K1 sequencing. All Amerindian strains had predominant (P)-type K15 alleles and had sequences in both T0.7 and ORF 75 that appeared to be characteristic of these strains. The prevalence of KSHV in two ethnic groups in Botswana was extremely high. K1 sequences from both Bantu and San subjects were mostly of subtypes B and A5, which are typical of African KSHV strains, but the sequence from one San subject did not cluster with any known subtype. Considerable heterogeneity was seen in the T0.7 and ORF 75 genes in the San subjects and one had a minor (M)-type K15 allele. The heterogeneity of the KSHV strains found in these subjects from Botswana contrasts with the homogeneity of KSHV strains in Amerindians, reflecting differences in the evolutionary history of these populations.

Epstein–Barr virus nuclear antigen (EBNA)-6 is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. Previous studies have shown that EBNA-6 acts as a transcriptional regulator of viral and cellular genes; however at present, few functional domains of the 140 kDa EBNA-6 protein have been completely characterized. There are five computer-predicted nuclear localization signals (NLS), four monopartite and one bipartite, present in the EBNA-6 amino acid sequence. To identify which of these NLS are functional, fusion proteins between green fluorescent protein and deletion constructs of EBNA-6 were expressed in HeLa cells. Each of the constructs containing at least one of the NLS was targeted to the nucleus of cells whereas a construct lacking all of the NLS was cytoplasmic. Site-directed mutation of these NLS demonstrated that only three of the NLS were functional, one at the N-terminal end (aa 72–80), one in the middle (aa 412–418) and one at the C-terminal end (aa 939–945) of the EBNA-6 protein.

Porcine herpesvirus entry mediator C (HveC) is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD). Porcine HveC mediates entry of pseudorabies virus (PRV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and bovine herpesvirus type 1 (BHV-1). In order to assess the antiviral potential of a soluble form of porcine HveC, Vero cells were transformed with the chimeric gene expressing a fusion protein (PHveCIg) consisting of an extracellular domain of porcine HveC and the Fc portion of human IgG1. The transformed cell lines expressing PHveCIg showed marked resistance to PRV infection. Resistance to infection by other alphaherpesviruses (HSV-1 and BHV-1) was also observed in the transformed cell line. The present results demonstrate that a soluble form of porcine HveC is able to exert a significant antiviral effect against pseudorabies virus and other alphaherpesvirus infection in vitro.