- Volume 86, Issue 5, 2005

Swine vesicular disease virus (SVDV) evolved from coxsackie B virus serotype 5 (CVB5) in the recent past, crossing the species barrier from humans to pigs. Here, SVDV isolates from early and recent outbreaks have been compared for their capacity to utilize the progenitor virus receptors coxsackie–adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55). Virus titre of CVB5 and SVDV isolates It′66 and UK′72 on human HeLa cells was reduced by pre-incubation with either anti-DAF or anti-CAR antibodies; however, recent SVDV isolates R1072, R1120 and SPA′93 did not infect HeLa cells lytically. CVB5 and SVDV infection of the pig cell line IB-RS-2 was inhibited completely by anti-CAR antibodies for all isolates, and no reduction was observed following pre-incubation of cells with anti-pig DAF antibodies. Expression of human DAF in the pig cell line IB-RS-2 enhanced the virus titre of early SVDV isolates by 25-fold, but had no effect on recent SVDV isolate titre. Binding of radiolabelled CVB5 to IB-RS-2 cells was increased seven- to eightfold by expression of human DAF and binding of early SVDV isolates was increased 1·2–1·3-fold, whereas no increase in binding by recent SVDV isolates was mediated by human DAF expression. Addition of soluble hDAF-Fc inhibited CVB5, but not SVDV, infection of pig cells. Pre-incubation of all viruses with soluble hCAR-Fc blocked infection of IB-RS-2 pig cells completely; titration of the amount of soluble hCAR-Fc required to block infection revealed that early isolate UK′72 was the least susceptible to inhibition, and the most recent isolate, SPA′93, was the most susceptible.

HeLa and 16HBE14o− bronchial epithelium cells infected with human rhinovirus serotype 14 (HRV14) were found to exhibit typical apoptotic morphological alterations, such as cell contraction and nuclear condensation. These events coincided with high-molecular-weight DNA fragmentation, activation of caspase-9 and caspase-3 and poly(ADP–ribose) polymerase cleavage. Caspase activation was preceded by cytochrome c translocation from the mitochondria to the cytoplasm, indicating that apoptosis caused by HRV14 infection was triggered predominantly via the mitochondrial pathway. Apoptosis did not affect HRV14 replication per se, but it facilitated the release of newly formed virus from cells. As apoptosis was fully induced at the time of maximal accumulation of progeny HRV14, it is postulated that apoptosis contributed to the destabilization of the cell and facilitated viral progeny release.

Enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is sometimes associated with serious neurological disorders. In this study, an attempt was made to identify molecular determinants of EV71 attenuation of neurovirulence in a monkey infection model. An infectious cDNA clone of the virulent strain of EV71 prototype BrCr was constructed; temperature-sensitive (ts) mutations of an attenuated strain of EV71 or of poliovirus (PV) Sabin vaccine strains were then introduced into the infectious clone. In vitro and in vivo phenotypes of the parental and mutant viruses were analysed in cultured cells and in cynomolgus monkeys, respectively. Mutations in 3D polymerase (3Dpol) and in the 3′ non-translated region (NTR), corresponding to ts determinants of Sabin 1, conferred distinct temperature sensitivity to EV71. An EV71 mutant [EV71(S1-3′)] carrying mutations in the 5′ NTR, 3Dpol and in the 3′ NTR showed attenuated neurovirulence, resulting in limited spread of virus in the central nervous system of monkeys. These results indicate that EV71 and PV1 share common genetic determinants of neurovirulence in monkeys, despite the distinct properties in their original pathogenesis.

A series of 46 charged-to-alanine mutations in the yellow fever virus NS2B–NS3 protease, previously characterized in cell-free and transient cellular expression systems, was tested for their effects on virus recovery. Four distinct plaque phenotypes were observed in cell culture: parental plaque-size (13 mutants), reduced plaque-size (17 mutants), small plaque-size (8 mutants) and no plaque-formation (8 mutants). No mutants displayed any temperature sensitivity based on recovery of virus after RNA transfection at 32 versus 37 °C. Most small plaque-mutants were defective in growth efficiency compared with parental virus. However not all small plaque-mutants had defective 2B/3 cleavage, with some showing selective defects at other non-structural protein cleavage sites. Revertant viruses were recovered for six mutations that caused reduced plaque sizes. Same-site and second-site mutations occurred in NS2B, and one second-site mutation occurred in the NS3 protease domain. Some reversion mutations ameliorated defects in cleavage activity and plaque size caused by the original mutation. These data indicate that certain mutations that reduce NS2B–NS3 protease cleavage activity cause growth restriction of yellow fever virus in cell culture. However, for at least two mutations, processing defects other than impaired cleavage activity at the 2B/3 site may account for the mutant phenotype. The existence of reversion mutations primarily in NS2B rather than NS3, suggests that the protease domain is less tolerant of structural perturbation compared with the NS2B protein.

The hepatitis C virus (HCV) non-structural protein NS4B induces morphological changes in the endoplasmic reticulum (ER) membrane that may have a direct role in viral RNA replication. A chimeric GFP–NS4B fusion protein located to the ER membrane and to foci that were attached to the ER. These membrane-associated foci (MAFs) could be related to the membrane alterations observed in cells that replicate HCV RNA. The relationship of MAFs to pre-existing cellular structures is not known. Indirect immunofluorescence analysis demonstrated that they did not contain a cellular marker for vesicles, which have been implicated in the replication of other viruses. From photobleaching studies to examine diffusion of NS4B, the GFP-tagged protein had reduced mobility on MAFs compared with on the ER membrane. This slower mobility suggested that NS4B is likely to form different interactions on MAFs and the ER.

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.

Heparan sulphate and sialoadhesin were previously identified on porcine macrophages as receptors for porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the exact role and cooperation of heparan sulphate and sialoadhesin during PRRSV attachment and internalization was analysed. It was observed that both heparan sulphate and sialoadhesin mediate PRRSV attachment and that only these two receptors are involved in attachment. Analysis of attachment kinetics of PRRSV to macrophages revealed that early attachment is mediated mainly via an interaction with heparan sulphate, followed by a gradual increase in interaction with sialoadhesin. By using wild-type CHO and CHO deficient in heparan sulphate expression, it was shown that heparan sulphate alone is sufficient to mediate PRRSV attachment, but not entry, and that heparan sulphate is not necessary for sialoadhesin to function as a PRRSV internalization receptor, but enhances the interaction of the virus with sialoadhesin.

The aim of this study was to inhibit influenza virus M2 protein expression by mutating the splicing signal of the M gene. Mutations were introduced into the GU dinucleotide sequence at the 5′-proximal splicing site of the M gene (corresponding to nt 52–53 of M cRNA). Transfected cells expressing mutated M viral ribonucleoproteins failed to generate M2 mRNA. Interestingly, recombinant viruses with mutations at the dinucleotide sequence were viable, albeit attenuated, in cell culture. These recombinants failed to express M2 mRNA and M2 protein. These observations demonstrated that the GU invariant dinucleotide sequence at the 5′-proximal splicing site of M gene is essential for M2 mRNA synthesis. These results also indicated that the M2 ion-channel protein is critical, but not essential, for virus replication in cell culture. This approach may provide a new way of producing attenuated influenza A virus.

Influenza C virus contains two envelope glycoproteins: CM2, a putative ion channel protein; and HEF, a unique multifunctional protein that performs receptor-binding, receptor-destroying and fusion activities. Here, it is demonstrated that expression of HEF is sufficient to pseudotype replication-incompetent vesicular stomatitis virus (VSV) that lacks the VSV glycoprotein (G) gene. The pseudotyped virus showed characteristic features of influenza C virus with respect to proteolytic activation, receptor usage and cell tropism. Chimeric glycoproteins composed of HEF ectodomain and VSV-G C-terminal domains were efficiently incorporated into VSV particles and showed receptor-binding and receptor-destroying activities but, unlike authentic HEF, did not mediate efficient infection, probably because of impaired fusion activity. HEF-pseudotyped VSV efficiently infected polarized Madin–Darby canine kidney cells via the apical plasma membrane, whereas entry of VSV-G-complemented virus was restricted to the basolateral membrane. These findings suggest that pseudotyping of viral vectors with HEF might be useful for efficient apical gene transfer into polarized epithelial cells and for targeting cells that express 9-O-acetylated sialic acids.

Throughout North America, rabies virus (RV) is endemic in bats. Distinct RV variants exist that are closely associated with infection of individual host species, such that there is little or no sustained spillover infection away from the primary host. Using Bayesian methodology, nucleotide substitution rates were estimated from alignments of partial nucleoprotein (N) gene sequences of nine distinct bat RV variants from North America. Substitution rates ranged from 2·32×10−4 to 1·38×10−3 substitutions per site per year. A maximum-likelihood (ML) molecular clock model was rejected for only two of the nine datasets. In addition, using sequences from bat RV variants across the Americas, the evolutionary rate for the complete N gene was estimated to be 2·32×10−4. This rate was used to scale trees using Bayesian and ML methods, and the time of the most recent common ancestor for current bat RV variant diversity in the Americas was estimated to be 1660 (range 1267–1782) and 1651 (range 1254–1773), respectively. Our reconstructions suggest that RV variants currently associated with infection of bats from Latin America (Desmodus and Tadarida) share the earliest common ancestor with the progenitor RV. In addition, from the ML tree, times were estimated for the emergence of the three major lineages responsible for bat rabies cases in North America. Adaptation to infection of the colonial bat species analysed (Eptesicus fuscus, Myotis spp.) appears to have occurred much quicker than for the solitary species analysed (Lasionycteris noctivagans, Pipistrellus subflavus, Lasiurus borealis, Lasiurus cinereus), suggesting that the process of virus adaptation may be dependent on host biology.

Junín virus (JUNV), the causative agent of Argentine haemorrhagic fever, is a human pathogen that naturally enters the body through the epithelial cells of the respiratory and digestive tracts. The interaction of JUNV with two types of polarized epithelial cultures, Vero C1008 and A549, was investigated. Radioactive virus-binding assays showed that JUNV infects polarized lines preferentially through the apical surface. High-level expression of viral nucleoprotein was detected in polarized cell lines infected through the apical domain. Virus production from apical media was about 100-fold higher than that found into the basolateral medium. Confocal-immunofluorescence analysis revealed high-level expression of glycoprotein at the apical-membrane surface. Disruption of the microtubule network by colchicine impaired JUNV vectorial release. This is the first study to analyse the interaction between a member of the virus family Arenaviridae and polarized epithelial cells, showing preferential entry and release from the apical plasma membrane.

Rotavirus genomes contain 11 double-stranded (ds) RNA segments. Genome segment 11 encodes the non-structural protein NSP5 and, in some strains, also NSP6. NSP5 is produced soon after viral infection and localizes in cytoplasmic viroplasms, where virus replication takes place. RNA interference by small interfering (si) RNAs targeted to genome segment 11 mRNA of two different strains blocked production of NSP5 in a strain-specific manner, with a strong effect on the overall replicative cycle: inhibition of viroplasm formation, decreased production of other structural and non-structural proteins, synthesis of viral genomic dsRNA and production of infectious particles. These effects were shown not to be due to inhibition of NSP6. The results obtained strengthen the importance of secondary transcription/translation in rotavirus replication and demonstrate that NSP5 is essential for the assembly of viroplasms and virus replication.

Mammalian reoviruses exhibit a propensity to replicate in transformed cells. It is currently believed that the interferon-inducible RNA-dependent protein kinase (PKR), an intracellular host-cell resistance factor that is inhibited by an activated Ras-dependent pathway in transformed cells, is responsible for this discrimination. In the present study, reovirus isolates differing in their sensitivity to interferon were obtained by chemical mutagenesis, and examined for their replicative properties in parental and Ras-transformed mouse NIH-3T3 cells. It was observed that most isolates can bypass resistance mechanisms of parental cells at high m.o.i., and that there is a correlation between the ability to discriminate between transformed and parental cells, and interferon sensitivity. Most interestingly, an interferon-hypersensitive mutant virus was more dependent on Ras activation than any other viral isolate. Altogether, this suggests that optimal reovirus isolates could be selected to attack tumour cells depending on the nature of the alterations in interferon-inducible pathways found in these cells.

Evidence has been presented which shows that part of the C-terminal tail of the gp41 transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a neutralization epitope and is thus exposed on the external surface of the virion. Here, SAR1, a monoclonal antibody, which was stimulated by immunization with a plant virus expressing 60 copies of the GERDRDR sequence from the exposed gp41 tail, and has an unusual pattern of neutralization activity, giving little or no neutralization of free virions, but effecting modest post-attachment neutralization (PAN) of virus bound to target cells was investigated. Here, the properties of PAN were investigated. It was found that PAN could be mediated at 4 or 20 °C, but that at 20 °C maximum PAN required virus–cell complexes to be incubated for 3 h before addition of antibody. Further PAN appeared stable at 20 °C and could be mediated for at least 5 h at this temperature. In contrast, when virus–cell complexes formed at 20 °C but then shifted to 37 °C for various times before addition of SAR1, PAN was maximal after just 10 min, and was lost after 30 min incubation. Thus, PAN at 37 °C is transient and temperature-dependent. Since this scenario recalled the temperature requirements of virus–cell fusion, fusion of HIV-1-infected and non-infected cells was investigated, and it was found that SAR1 inhibited this process by up to 75 %, in a dose-dependent manner. However, antibodies to adjacent epitopes did not inhibit fusion. These data confirm the external location of the SAR1 epitope, implicate the gp41 C-terminal tail in the HIV-1 fusion process for the first time, and suggest that SAR1 mediates PAN by inhibiting virus-mediated fusion.

Nuclear export of unspliced and incompletely spliced human immunodeficiency virus type 1 mRNA is mediated by the viral Rev protein. Rev binds to a structured RNA motif known as the Rev-response element (RRE), which is present in all Rev-dependent transcripts, and thereby promotes entry of the ribonucleoprotein complex into the nuclear-export pathway. Recent evidence indicates that a dimerization interface and a genetically separable ‘trimerization’ interface are required for multimeric assembly of Rev on the RRE. In this report, the effect of mutations within the trimerization interface on Rev function was examined in mammalian cells. All trimerization-defective Rev molecules had profoundly compromised Rev function and a range of localization defects was observed. However, despite the potential for formation of heterodimers between functional and non-functional Rev proteins, trimerization-defective Rev mutants were unable to inhibit wild-type Rev function in a trans-dominant-negative manner.

The CD8+ lymphocyte response is a main component of host immunity, yet it is difficult to quantify its contribution to the control of persistent viruses. Consequently, it remains controversial as to whether CD8+ cells have a biologically significant impact on viral burden and disease progression in infections such as human immunodeficiency virus-1 and human T-lymphotropic virus type I (HTLV-I). Experiments to ascertain the impact of CD8+ cells on viral burden based on CD8+ cell frequency or specificity alone give inconsistent results. Here, an alternative approach was developed that directly quantifies the impact of CD8+ lymphocytes on HTLV-I proviral burden by measuring the rate at which HTLV-I-infected CD4+ cells were cleared by autologous CD8+ cells ex vivo. It was demonstrated that CD8+ cells reduced the lifespan of infected CD4+ cells to 1 day, considerably shorter than the 30 day lifespan of uninfected cells in vivo. Furthermore, it was shown that HTLV-I-infected individuals vary considerably in the rate at which their CD8+ cells clear infected cells, and that this was a significant predictor of their HTLV-I proviral load. Forty to 50 % of between-individual variation in HTLV-I proviral load was explained by variation in the rate at which CD8+ cells cleared infected cells. This novel approach demonstrates that CD8+ cells are a major determinant of HTLV-I proviral load. This assay is applicable to quantifying the CD8+ cell response to other viruses and malignancies and may be of particular importance in assessing vaccines.

Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses, constituting major sites for latency/reactivation events. Here, the in vitro kinetics of productive infection of the swine alphaherpesvirus pseudorabies virus (PRV) and resulting cell death in primary porcine TG neurons were determined, and these were compared with similar kinetics in many other porcine cell types. Confocal microscopy showed that all TG neurons expressed late genes such as viral glycoproteins, and that these glycoproteins were processed through the Golgi and reached the cell surface as in other cell types, albeit with a delay of ±2–6 h. However, TG neurons were much more resistant towards PRV-induced cell death compared with all other porcine cell types tested (non-neuronal TG cells, superior cervical ganglion neurons, epithelial kidney cells, arterial endothelial cells, dermal fibroblasts and cells derived from a porcine swine kidney cell line). About half of the TG neurons survived up to 96 h post-inoculation (end of experiment), whereas all other cell types almost completely succumbed within 2 days post-inoculation. In addition, infection with a strongly pro-apoptotic PRV strain that misses the anti-apoptotic US3 protein did not lead to substantial apoptosis in TG neurons, even at 72 h post-inoculation. Thus, primary porcine TG neurons can be infected with PRV in vitro, and are remarkably more resistant to PRV-induced cell death compared with other porcine cell types, suggesting a cell type-specific resistance to alphaherpesvirus-induced cell death that may have important implications for different aspects of the virus life cycle, including latency/reactivation events.