- Volume 85, Issue 11, 2004

Bovine enteroviruses are members of the family Picornaviridae, genus Enterovirus. Whilst little is known about their pathogenic potential, they are apparently endemic in some cattle and cattle environments. Only one of the two current serotypes has been sequenced completely. In this report, the entire genome sequences of bovine enterovirus 2 (BEV-2) strain PS87 and a recent isolate from an endemically infected herd in Maryland, USA (Wye3A) are presented. The recent isolate clearly segregated phylogenetically with sequences representing the BEV-2 serotype, as did other isolates from the endemic herd. The Wye3A isolate shared 82 % nucleotide sequence identity with the PS87 strain and 68 % identity with a BEV-1 strain (VG5-27). Comparison of BEV-2 and BEV-1 deduced protein sequences revealed 72–73 % identity and showed that most differences were single amino acid changes or single deletions, with the exception of the VP1 protein, where both BEV-2 sequences were 7 aa shorter than that of BEV-1. Homology modelling of the capsid proteins of BEV-2 against protein database entries for picornaviruses indicated six significant differences among bovine enteroviruses and other members of the family Picornaviridae. Five of these were on the ‘rim’ of the proposed enterovirus receptor-binding site or ‘canyon’ (VP1) and one was near the base of the canyon (VP3). Two of these regions varied enough to distinguish BEV-2 from BEV-1 strains. This is the first report and analysis of full-length sequences for BEV-2. Continued analysis of these wild-type strains should yield useful information for genotyping enteroviruses and modelling enterovirus capsid structure.

Sequencing of the gene that encodes the capsid protein VP1 has been used as a surrogate for antigenic typing in order to distinguish enterovirus serotypes; three new serotypes were identified recently by this method. In this study, 14 enterovirus isolates from six countries were characterized as members of two new types within the species Human enterovirus B, based on sequencing of the complete capsid-encoding (P1) region. Isolates within each of these two types differed significantly from one another and from all other known enterovirus serotypes on the basis of sequences that encode either VP1 alone or the entire P1 region. Members of each type were ⩾77·2 % identical to one another (89·5 % amino acid identity) in VP1, but members of the two different types differed from one another and from other enteroviruses by ⩾31 % in nucleotide sequence (25 % amino acid sequence difference), indicating that the two groups represent separate new candidate enterovirus types. The complete P1 sequences differed from those of all other enterovirus serotypes by ⩾31 % (26 % amino acid sequence difference), but were highly conserved within a serotype (<8 % amino acid sequence difference). Phylogenetic analyses demonstrated that isolates of the same serotype were monophyletic in both VP1 and the capsid as a whole, as shown previously for other enterovirus serotypes. This paper proposes that these 14 isolates should be classified as members of two new human enterovirus types, enteroviruses 74 and 75 (EV74 and EV75).

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is one of the most dangerous diseases of cloven-hoofed animals and is a constant threat to the dairy and beef industries in the Middle East and other regions of the world, despite intensive vaccination programmes. In this work, the ability of specific small interfering (si)RNAs to inhibit virus replication in BHK-21 cells was examined. By using bioinformatic computer programs, all FMDV sequences in public-domain databases were analysed. The analysis revealed three regions of at least 22 bp with 100 % identity in all FMDV entries. From these sequences, three specific siRNA molecules were prepared and used to test the ability of siRNAs to inhibit virus replication. By using real-time quantitative PCR to measure the amount of viral RNA in infected cells, it was shown that virus replication was inhibited in cells that were transfected with siRNAs. When viral titres were examined, 100 % inhibition of growth could be demonstrated in cells transfected with a mixture of all three anti-FMDV siRNAs, compared with control cells transfected with anti-LacZ siRNA.

Avian pneumovirus (APV) is a member of the genus Metapneumovirus of the subfamily Pneumovirinae. This study describes the development of a reverse-genetics system for APV. A minigenome system was used to optimize the expression of the nucleoprotein, phosphoprotein, M2 and large polymerase proteins when transfected into Vero cells under the control of the bacteriophage T7 promoter. Subsequently, cDNA was transcribed from the virion RNA to make a full-length antigenome, which was also cloned under the control of the T7 promoter. Transfection of the full-length genome plasmid, together with the plasmids expressing the functional proteins in the transcription and replication complex, generated APV in the transfected cells. The recombinant virus was passaged and was identified by cytopathic effect (CPE) that was typical of APV, the presence of a unique restriction-endonuclease site in the cDNA copy of the genome and immunofluorescence staining with anti-APV antibodies. Replacement of the full-length wild-type antigenome with one lacking the small hydrophobic (SH) protein and the attachment (G) genes generated a virus that grew more slowly and produced atypical CPE with syncytia much larger than those seen with wild-type virus.

Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2Kd-restricted CTL epitopes (F85–93 and F92–106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85–93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249–258, which is presented by Kd as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous Kd ligand. The CD8+ T-lymphocyte responses to epitopes F85–93 and F249–258 present in the F protein of RSV Long were found to be strongly skewed to F85–93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8+ T-lymphocyte responses to F85–93 and F249–258 epitopes was observed in vivo during a primary response.

Interference of measles virus (MV) with dendritic-cell (DC) functions and deregulation of T-cell differentiation have been proposed to be central to the profound suppression of immune responses to secondary infections up to several weeks after the acute disease. To address the impact of MV infection on the ability of DCs to promote Th-cell differentiation, an in vitro system was used where uninfected, tumour necrosis factor alpha/interleukin (IL) 1β-primed DCs were co-cultured with CD45RO− T cells in the presence of conditioned media from MV-infected DCs primed under neutral or DC-polarizing conditions. It was found that supernatants of DCs infected with an MV vaccine strain strongly promoted Th1 differentation, whereas those obtained from wild-type MV-infected DCs generated a mixed Th1/Th0 response, irrespective of the conditions used for DC priming. Th-cell commitment in this system did not correlate with the production of IL12 p70, IL18 or IL23. Thus, a combination of these or other, as yet undefined, soluble factors is produced upon MV infection of DCs that strongly promotes Th1/Th0 differentiation.

Influenza B virus is one of two types of influenza virus that cause substantial morbidity and mortality in humans, the other being influenza A virus. The inability to provide lasting protection to humans against influenza B virus infection is due, in part, to antigenic drift of the viral surface glycoprotein, haemagglutinin (HA). Studies of the antigenicity of the HA of influenza B virus have been hampered by lack of knowledge of its structure. To address this gap, two possible models have been inferred for this structure, based on two known structures of the homologous HA of the influenza A virus (subtypes H3 and H9). Statistical, structural and functional analyses of these models suggested that they matched important details of experimental observations and did not differ from each other in any substantive way. These models were used to investigate two HA sites at which viral variants appeared to carry a selective advantage. It was found that each of these sites coevolved with nearby sites to compensate for either size or charge changes.

Hantaviruses are known to cause two severe human diseases: haemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. The mechanisms of pathogenesis of these two diseases are progressively becoming understood. Recently, two hantaviruses, Hantaan and Prospect Hill were reported to cause programmed cell death of Vero E6 cells. This study shows that Tula hantavirus (TULV) infection efficiently triggers an apoptotic programme in infected Vero E6 cells, and that the replication of TULV is required for the activation of caspase 3 and the cleavage of poly (ADP-ribose) polymerase, two molecular hallmarks of apoptosis. The enforced treatment of infected Vero E6 cells with tumour necrosis factor alpha (TNF-α), but not interferon alpha (IFN-α), advanced the time course of apoptosis. Furthermore, caspase 8 was activated on day 4 post-infection, the same day when caspase 3 was activated. TNF receptor 1 was induced during a late stage of TULV infection. These data suggest that, unlike during influenza A virus infection, TNF-α, but not type I IFN-α/β, may contribute significantly to apoptosis in a synergistic manner with TULV propagation. Interestingly, pretreatment with a broad-spectrum caspase inhibitor, z-VAD-fmk, efficiently inhibited apoptosis of TULV-infected Vero E6 cells. Taken together, these results suggest that TULV replication initiates a typical apoptotic programme involving caspase 8 activation.

The genome of Bunyamwera virus (BUN; family Bunyaviridae) consists of three segments of negative-sense, single-stranded RNA that are called L (large), M (medium) and S (small), according to their size. The genomic RNAs are encapsidated by the viral nucleocapsid protein to form ribonucleoprotein complexes (RNPs). The terminal 3′ and 5′ non-coding sequences are complementary and interact to give a panhandle-like structure to the RNP. Located within these non-coding sequences are elements that control replication and transcription. The sequences of the terminal 11 nt are conserved among the genome segments and are followed by shorter, complementary nucleotide motifs that are conserved on a segment-specific basis. Here, a detailed analysis of the 3′ and 5′ non-coding regions of the BUN S segment is presented. By using a mini-replicon system, it was shown that a functional BUN S promoter requires complementarity, as well as defined sequences, within the terminal 15 nt of either end. It was also shown that the minimal requirement for transcription is localized within the terminal 32 nt of the S segment. A comparison of known strong BUN promoters led to the prediction of a structural element outside the terminal 15 nt; introduction of this motif into the BUN S sequence resulted in increased antigenome and mRNA levels and increased expression of S segment proteins, as shown by mini-replicon assays, as well as recovery of a recombinant virus.

A novel, linear B-cell epitope has been identified at the N terminus of the rabies virus (RABV) glycoprotein. Screening of a phage-display library demonstrated that two glycoprotein-specific mAbs recognized a conserved sequence, WxxxDI, which aligned between aa 14 and 19 of the mature glycoprotein. Screening of truncated glycoprotein fragments with both mAbs confirmed the location of the epitope in the N-terminal region. Alignment of amino acid sequences from a range of RABV isolates indicated that the site was conserved in most viruses. Alignment with representatives of other lyssaviruses suggested that it is conserved within phylogroup I, which includes the European bat lyssaviruses, but not phylogroup II. A 12 aa synthetic peptide of this epitope was recognized by both mAbs and sera from a subset of rabies-vaccinated dogs. In a multimeric form, the peptide could induce an epitope-specific response following immunization in rabbits and mice.

Intracellular antibodies or intrabodies (ICAbs) have great potential in protein knockout strategies for intracellular antigens. In this study, they have been used to investigate the role of the rotavirus non-structural protein NSP5 in the virus replication cycle. Intracellular antibody-capture technology was used to select single-chain Fv format (scFv) ICAbs against an NSP5 mutant. Five different specific ICAbs were selected and expressed in MA104 cells, in the scFv format, as cytoplasmic- and nuclear-tagged forms. By confocal microscopy, it was found that three of these ICAbs recognized the full-length wild-type NSP5 specifically, forming antigen-specific aggresomes in the cytoplasm of cotransfected cells. Expression of the ICAbs in rotavirus-infected cells largely reduced the assembly of viroplasms and cellular cytopathic effect. Replication of dsRNA was partially inhibited, despite there being no reduction in virus titre. These results demonstrate for the first time a key role for NSP5 during the virus replicative cycle.

vpx genes of human immunodeficiency virus type 2 (HIV-2) and immunodeficiency viruses from macaques (SIVmac), sooty mangabeys (SIVsm) and red-capped mangabeys (SIVrcm) encode a 112 aa protein that is packed into virion particles via interaction with the p6 domain of p55gag. Vpx localizes to the nucleus when expressed in the absence of other viral proteins. Moreover, Vpx is necessary for efficient nuclear import of the pre-integration complex (PIC) and critical for virus replication in quiescent cells, such as terminally differentiated macrophages and memory T cells. Vpx does not contain sequence elements that are homologous to previously characterized nuclear localization signals (NLSs). Therefore, it is likely that Vpx-dependent import of the PIC is mediated by interaction of Vpx with cellular proteins that do not belong to the classical import pathways. By using a yeast two-hybrid screen, α-actinin 1, a cytoskeletal protein, was identified to interact with SIVmac239 Vpx. Interestingly, deletion of the proline-rich C-terminal domain (aa 101–112) of Vpx, which is important for nuclear localization, resulted in loss of interaction with α-actinin 1. These findings suggest that the interaction with α-actinin 1 may play an important role in the transport of Vpx to the nucleus and in Vpx-mediated nuclear import of the PIC.

Little information is available on the prevalence of retrovirus infections in populations of non-human primates living in their natural habitats. To gain such information, methods were developed to detect antibodies to simian T-lymphotropic virus type 1 (STLV-1) in urine from wild chimpanzees. Samples from more than 74 chimpanzees living in three communities in the Taï National Park, Côte d'Ivoire, were analysed. The prevalence of STLV-1 antibodies in adults and adolescents was significantly higher (35/49, 71·4 %) than that in infant and juvenile chimpanzees (3/31, 9·7 %).

A survey for the presence of simian foamy retroviruses (SFVs) was performed in 44 wild-caught apes and monkeys, including 27 gorillas, 11 mandrills and six drills, originating from south Cameroon or Gabon. Combined serological and/or nested-PCR assays indicated SFV infection among five Gorilla gorilla gorilla, seven Mandrillus sphinx and two Mandrillus leucophaeus. Sequences of a 425 bp fragment of the integrase gene were obtained for 11 animals. Phylogenetic studies indicated that strains from gorillas, mandrills and drills each formed a highly supported phylogenetic clade with, moreover, the existence of two different gorilla SFVs. This study demonstrates for the first time that these animals are naturally infected with specific SFVs. In the context of simian-to-human interspecies transmission, the results confirm that such viruses can also infect humans, as the SFVs identified in wild-caught animals were the same as those recently reported as infecting hunters living in the same geographical areas.

Ovine pulmonary adenocarcinoma (OPA) can be reproduced consistently in neonatal lambs by intratracheal injection of inocula containing jaagsiekte sheep retrovirus (JSRV). In this study, clinical disease, confirmed pathologically as OPA, was induced in a high proportion of lambs that had been inoculated intratracheally with infectious lung fluid at 1, 3 and 6 months of age. The incubation periods, however, were longer in these three age groups than in 1-week-old lambs that were used as controls. Viraemia was detected in all age groups before onset of clinical signs, but occurred later in older animals. These results suggest an age-dependent susceptibility to OPA that could be determined by the availability of JSRV target cells in the ovine lung. The feasibility of inducing OPA in older lambs and detecting JSRV viraemia in preclinical stages enables improved studies on the pathogenesis, assessment of vaccines, diagnosis and control of the disease.

gypsy is the only endogenous retrovirus of Drosophila whose infectious properties have been reported. Previous studies have shown an unexpected relationship between the gene encoding the putative envelope glycoprotein (Env) of gypsy and genes encoding the fusion protein of several baculoviruses. The fact that fusion proteins mediate membrane fusion suggests that Env of insect retroviruses might also have fusogenic properties. The results reported here indicate that gypsy Env mediates cell-to-cell fusion. Cleavage of the Env precursor was also studied; it is shown that this polypeptide is cleaved at a furin-like cleavage site. This is the first report that the env-like gene of insect retroviruses encodes a fusion protein.

Human herpesvirus 6 (HHV-6) replication was evaluated during in vitro expansion of CD34-positive cells that were selected from 11 peripheral blood progenitor cell (PBPC) samples. In order to permit cellular differentiation towards the myeloid lineage, PBPCs were cultured for 14–21 days in a liquid, serum-free medium supplemented with interleukin 1 (IL1), IL3, IL6, granulocyte–macrophage colony-stimulating factor, granulocyte colony-stimulating factor and stem-cell factor. Among the 10 cultures from HHV-6-seropositive patients, the late, alternatively spliced U100 viral mRNA was detected in five of them after PBPC culture for 14 or 21 days. Recovery of infectious virus from one of the expansions, associated with an increase of HHV-6 viral load and detection of the U100 spliced messenger, confirmed the occurrence of a complete replicative cycle. These data thus demonstrate for the first time that haematopoietic differentiation can lead to HHV-6 reactivation.

Human cytomegalovirus (HCMV) is a frequent cause of major disease following primary infection or reactivation from latency in immunocompromised patients. It has also been suggested that there may be a link between HCMV and vascular disease. Both smooth muscle and endothelial cells are targets for primary infection with HCMV and have also been postulated as potential sites of HCMV latency. One of the most intensely studied sites of HCMV latency is the cells of the myeloid lineage; there is increasing evidence that the myeloid and endothelial lineages arise from a common precursor in the bone marrow, suggesting that endothelial cells could be another route of HCMV dissemination. However, using a highly sensitive PCR capable of detecting endogenous HCMV in myeloid cells, the HCMV genome in endothelial and smooth muscle cells isolated from the saphenous veins of seropositive patients was not detected. These data suggest that vascular endothelial and smooth muscle cells are unlikely to be important sites of HCMV latency in vivo.