- Volume 87, Issue 2, 2006

Alfuy virus (ALFV) is classified as a subtype of the flavivirus Murray Valley encephalitis virus (MVEV); however, despite preliminary reports of antigenic and ecological similarities with MVEV, ALFV has not been associated with human disease. Here, it was shown that ALFV is at least 104-fold less neuroinvasive than MVEV after peripheral inoculation of 3-week-old Swiss outbred mice, but ALFV demonstrates similar neurovirulence. In addition, it was shown that ALFV is partially attenuated in mice that are deficient in α/β interferon responses, in contrast to MVEV which is uniformly lethal in these mice. To assess the antigenic relationship between these viruses, a panel of monoclonal antibodies was tested for the ability to bind to ALFV and MVEV in ELISA. Although the majority of monoclonal antibodies recognized both viruses, confirming their antigenic similarity, several discriminating antibodies were identified. Finally, the entire genome of the prototype strain of ALFV (MRM3929) was sequenced and phylogenetically analysed. Nucleotide (73 %) and amino acid sequence (83 %) identity between ALFV and MVEV confirmed previous reports of their close relationship. Several nucleotide and amino acid deletions and/or substitutions with putative functional significance were identified in ALFV, including the abolition of a conserved glycosylation site in the envelope protein and the deletion of the terminal dinucleotide 5′-CUOH-3′ found in all other members of the genus. These findings confirm previous reports that ALFV is closely related to MVEV, but also highlights significant antigenic, genetic and phenotypic divergence from MVEV. Accordingly, the data suggest that ALFV is a distinct species within the serogroup Japanese encephalitis virus.

Chimeric (D2/WN) viruses containing the pre-membrane (prM) and envelope (E) proteins of West Nile virus (WN virus) and the capsid (C) and non-structural proteins of dengue 2 (DEN2) virus were used to evaluate the protective immunity elicited by either the flaviviral E protein or non-structural proteins. AG129 interferon-deficient mice, previously shown to be protected against lethal DEN1 or DEN2 viral infection after vaccination with a wild-type or candidate vaccine strain of DEN1 or DEN2 virus, respectively, were immunized with chimeric D2/WN virus and then challenged with DEN2 virus. D2/WN chimeric viruses were non-pathogenic in AG129 mice. These viruses elicited little anti-DEN E antibody, high levels of anti-DEN NS1 antibody and no or very low levels of DEN2 virus-neutralizing antibodies. Only 15 % of D2/WN-immunized mice survived challenge with DEN2 virus. However, their mean survival time increased by 11–14 days over non-immunized controls. These results suggest that, whilst the non-structural proteins were able to enhance mean survival times of AG129 mice, this protection was not as effective as protection mediated by the E protein.

To investigate RNA-dependent RNA polymerase (RdRp) further, mutational analysis of the N-terminal domain of the NS5B protein of Classical swine fever virus was performed. Results show that the N-terminal domain (positions 1–300) of the protein might be divided artificially into four different regions, N1–N4. The N1 region (positions 1–61) contained neither conserved lysine nor conserved arginine residues. NS5B protein with deletion of the N1 region has the capacity for elongative RNA synthesis, but not for de novo RNA synthesis on natural templates. All substitutions of the conserved lysines and arginines in the N2 region (positions 63–216) destroyed RdRp activity completely. Substitutions of the conserved arginines in the N3 region (positions 217–280) seriously reduced RdRp activity. However, all substitutions of the conserved lysines in this region enhanced RNA synthesis and made the mutants synthesize RNA on any template. Substitutions of the conserved arginines in the N4 region (positions 281–300) reduced elongative synthesis and destroyed de novo RNA synthesis. In contrast, substitutions of lysines in this region did not affect RdRp activity significantly. These data indicate that the N3 region might be related to the enzymic specificity for templates, and the conserved lysines and arginines in different regions have different effects on RdRp activity. In combination with the published crystal structure of bovine viral diarrhea virus NS5B, these results define the important role of the N-terminal domain of NS5B for template recognition and de novo RNA synthesis.

Feline calicivirus (FCV) belongs to the family Caliciviridae and is an important pathogen of the upper respiratory tract of cats. Recent studies have shown that cells infected with FCV undergo apoptosis, as evidenced by caspase activation, chromatin condensation and cleavage of poly(ADP-ribose) polymerase. Here, the upstream events were investigated in order to define the molecular mechanism of apoptosis in FCV-infected cells. It was shown that FCV induced translocation of phosphatidylserine to the cell outer membrane and release of cytochrome c from mitochondria at about 6–8 h post-infection. These events were preceded by the loss of mitochondrial membrane potential and Bax translocation from the cytosol to mitochondria between 4 and 6 h after infection. Release of cytochrome c from mitochondria triggered the activation of caspase-9 and the subsequent activation of the executioner caspase, caspase-3. These results suggest that the mitochondrial pathway of apoptosis is triggered during FCV infection.

Caliciviruses are a major cause of gastroenteritis in humans and cause a wide variety of other diseases in animals. Here, the characterization of protein–protein interactions between the individual proteins of Feline calicivirus (FCV), a model system for other members of the family Caliciviridae, is reported. Using the yeast two-hybrid system combined with a number of other approaches, it is demonstrated that the p32 protein (the picornavirus 2B analogue) of FCV interacts with p39 (2C), p30 (3A) and p76 (3CD). The FCV protease/RNA polymerase (ProPol) p76 was found to form homo-oligomers, as well as to interact with VPg and ORF2, the region encoding the major capsid protein VP1. A weak interaction was also observed between p76 and the minor capsid protein encoded by ORF3 (VP2). ORF2 protein was found to interact with VPg, p76 and VP2. The potential roles of the interactions in calicivirus replication are discussed.

The 3a protein of severe acute respiratory syndrome (SARS)-associated coronavirus is expressed and transported to the plasma membrane in tissue cells of infected patients. Its short N-terminal ectodomain was found to elicit strong humoral responses in half of the patients who had recovered from SARS. The ectodomain-specific antibodies from the convalescent-phase plasma readily recognized and induced destruction of 3a-expressing cells in the presence of the human complement system, demonstrating their potential ability to provide immune protection by recognizing and eliminating SARS coronavirus-infected cells that express the target protein. In addition, when coupled to a carrier protein, the ectodomain peptide elicited 3a-specific antibodies in mice and rabbit at high titres. These results showed that the N terminus of the 3a protein is highly immunogenic and elicits potentially protective humoral responses in infected patients. Therefore, the short extracellular domain may be a valuable immunogen in the development of a vaccine for infectious SARS.

Bovine enteroviruses are currently classified into two serotypes within the species Bovine enterovirus (BEV). Comparison of the sequences of six American and eleven German BEV isolates with published BEV sequences revealed the necessity to revise the taxonomy of these viruses. Molecular data indicate that the bovine enteroviruses are composed of two clusters (designated BEV-A and -B) each with two and three geno-/serotypes, respectively. Whereas low amino acid identity of the capsid proteins 1C (VP3) and 1D (VP1) is the main criterion for the discrimination of geno-/serotypes, the BEV clusters, presumably representing species, differ in sequence identity of all viral proteins. In addition, characteristic lengths of (i) the capsid proteins 1B, 1C and 1D, (ii) the 2C protein, and (iii) the 3′-non-translated region are observed. The BEVs can be distinguished from the other enteroviruses by sequence identity and unique features of the 5′-non-translated region, i.e. a conserved second cloverleaf and characteristic RNA structures of the internal ribosome entry site. Phylogenetically, the closest relatives of the bovine enteroviruses are the porcine enteroviruses. Incongruent phylogenies of the 5′-non-translated region, the capsid proteins and the 3D polymerase indicate frequent intraserotypic and interserotypic recombination within the non-capsid and the capsid region of the BEV genome.

Genomic characterization of Heliothis armigera cypovirus (HaCPV) isolated from China showed that insects were co-infected with several cypoviruses (CPVs). One of the CPVs (HaCPV-5) could be separated from the others by changing the rearing conditions of the Heliothis armigera larvae. This finding was further confirmed by nucleotide sequencing analysis. Genomic sequences of segments S10–S7 from HaCPV-14, S10 and S7 from HaCPV-5, and S10 from Heliothis assulta CPV-14 were compared. Results from database searches showed that the nucleotide sequences and deduced amino acid sequences of the newly identified CPVs had high levels of identity with those of reported CPVs of the same type, but not with CPVs of different types. Putative amino acid sequences of HaCPV-5 S7 were similar to that of the protein from Rice ragged stunt virus (genus Oryzavirus, family Reoviridae), suggesting that CPVs and oryzaviruses are related more closely than other genera of the family Reoviridae. Conserved motifs were also identified at the ends of each RNA segment of the same virus type: type 14, 5′-AGAAUUU…CAGCU-3′; and type 5, 5′-AGUU…UUGC-3′. Our results are consistent with classification of CPV types based on the electrophoretic patterns of CPV double-stranded RNA.

The cytoplasmic domains of the fusion proteins encoded by several viruses play a role in cell fusion and contain sites for palmitoylation associated with viral protein trafficking and virus assembly. The fusion (F) protein of Human respiratory syncytial virus (HRSV) has a predicted cytoplasmic domain of 26 residues containing a single palmitoylated cysteine residue that is conserved in bovine RSV F protein, but not in the F proteins of other pneumoviruses such as pneumonia virus of mice, human metapneumovirus and avian pneumovirus. The cytoplasmic domains in other paramyxovirus fusion proteins such as Newcastle disease virus F protein play a role in fusion. In this study, it was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue (C550S) of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion.

In this study, the design and preclinical development of a multigene human immunodeficiency virus type 1 (HIV-1) subtype C DNA vaccine are described, developed as part of the South African AIDS Vaccine Initiative (SAAVI). Genetic variation remains a major obstacle in the development of an HIV-1 vaccine and recent strategies have focused on constructing vaccines based on the subtypes dominant in the developing world, where the epidemic is most severe. The vaccine, SAAVI DNA-C, contains an equimolar mixture of two plasmids, pTHr.grttnC and pTHr.gp150CT, which express a polyprotein derived from Gag, reverse transcriptase (RT), Tat and Nef, and a truncated Env, respectively. Genes included in the vaccine were obtained from individuals within 3 months of infection and selection was based on closeness to a South African subtype C consensus sequence. All genes were codon-optimized for increased expression in humans. The genes have been modified for safety, stability and immunogenicity. Tat was inactivated through shuffling of gene fragments, whilst maintaining all potential epitopes; the active site of RT was mutated; 124 aa were removed from the cytoplasmic tail of gp160; and Nef and Gag myristylation sites were inactivated. Following vaccination of BALB/c mice, high levels of cytotoxic T lymphocytes were induced against multiple epitopes and the vaccine stimulated strong CD8+ gamma interferon responses. In addition, high titres of antibodies to gp120 were induced in guinea pigs. This vaccine is the first component of a prime–boost regimen that is scheduled for clinical trials in humans in the USA and South Africa.

This study compares the replication of primary isolates of human immunodeficiency virus type 2 (HIV-2) and type 1 (HIV-1) in monocyte-derived macrophages (MDMs). Eleven HIV-2 and five HIV-1 primary isolates that use CCR5, CXCR4 or both coreceptors to enter cells were included. Regardless of coreceptor preference, 10 of 11 HIV-2 viruses could enter, reverse transcribe and produce fully infectious virus in MDMs with efficiency equal to that in peripheral blood mononuclear cells. However, the kinetics of replication of HIV-2 compared with HIV-1 over time were distinct. HIV-2 had a burst of virus replication 2 days after infection that resolved into an apparent ‘latent state’ at day 3. HIV-1, however, continued to produce infectious virions at a lower, but steady, rate throughout the course of infection. These results may have implications for the lower pathogenesis and viral-load characteristics of HIV-2 infection.

The fingers subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a hotspot for nucleoside analogue resistance mutations. Some multi-nucleoside analogue-resistant variants contain a T69S substitution along with dipeptide insertions between residues 69 and 70. This set of mutations usually co-exists with classic zidovudine-resistance mutations (e.g. M41L and T215Y) or an A62V mutation and confers resistance to multiple nucleoside analogue inhibitors. As insertions lie in the vicinity of the dNTP-binding pocket, their influence on RT fidelity was investigated. Commonly occurring insertion mutations were selected, i.e. T69S-AG, T69S-SG and T69S-SS alone, in combination with 3′-azido-2′,3′-deoxythymidine-resistance mutations M41L, L210W, R211K, L214F, T215Y (LAGAZ and LSGAZ) or with an alternate set where A62V substitution replaces M41L (VAGAZ, VSGAZ and VSSAZ). Using a lacZα gapped duplex substrate, the forward mutation frequencies of recombinant wild-type and mutant RTs bearing each of the above sets of mutations were measured. All of the mutants displayed significant decreases in mutation frequencies. Whereas the dipeptide insertions alone showed the least decrease (4·0- to 7·5-fold), the VAG series showed an intermediate reduction (5·0- to 11·4-fold) and the LAG set showed the largest reduction in mutation frequencies (15·3- and 16·3-fold for LAGAZ and LSGAZ, respectively). Single dNTP exclusion assays for mutants LSGAZ and LAGAZ confirmed their large reduction in misincorporation efficiencies. The increased in vitro fidelity was not due to excision of the incorrect nucleotide via ATP-dependent removal. There was also no direct correlation between increased fidelity and template–primer affinity, suggesting a change in the active site that is conducive to better discrimination during dNTP insertion.

Cercopithecine herpesvirus 1 (monkey B virus; BV) produces extremely severe and usually fatal infections when transmitted from macaque monkeys to humans. Cercopithecine herpesvirus 16 (herpesvirus papio 2; HVP2) is very closely related to BV, yet cases of human HVP2 infection are unknown. However, following intramuscular inoculation of mice, HVP2 rapidly invades the peripheral nervous system and ascends the central nervous system (CNS) resulting in death, very much like human BV infections. In this study, the neurovirulence of HVP2 in mice was further evaluated as a potential model system for human BV infections. HVP2 was consistently neurovirulent when administered by epidermal scarification, intracranial inoculation and an eye splash. Quantitative real-time PCR, histopathology and immunohistochemistry were used to follow the temporal spread of virus following skin scarification and to compare the pathogenesis of neurovirulent and apathogenic isolates of HVP2. Apathogenic isolates were found to be capable of reaching the CNS but were extremely inefficient at replicating within the CNS. It is concluded that neurovirulent strains of HVP2 exhibit a pathogenesis in mice that parallels that observed in human BV infections and that this model system may prove useful in dissecting the viral determinants underlying the extreme severity of zoonotic BV infections.

In this study, the role of cholesterol in the envelope of human herpesvirus 6 (HHV-6) was examined by using methyl-β-cyclodextrin (MβCD) depletion. When cholesterol was removed from HHV-6 virions with MβCD, infectivity was abolished, but it could be rescued by the addition of exogenous cholesterol. HHV-6 binding was affected slightly by MβCD treatment. In contrast, envelope cholesterol depletion markedly affected HHV-6 infectivity and HHV-6-induced cell fusion. These results suggest that the cholesterol present in the HHV-6 envelope plays a prominent role in the fusion process and is a key component in viral entry.

Survivin has recently been identified as a novel member of the inhibitor of apoptosis (IAP) gene family. The product of this gene not only suppresses apoptosis but also controls cell division. Survivin is undetectable in most terminally differentiated normal tissues but is expressed in embryonic and fetal organs and is present in most malignant tumours. Human papillomaviruses (HPV) are thought to play an important role in the development of cervical cancer. By interfering in the cell cycle, the viral oncoproteins (E6 and E7) can induce the immortalization of the host cell. The transcriptional effects of the HPV-16 E6 and E7 proteins on the survivin promoter in transiently transfected cell lines using luciferase tests were examined. HPV-16 E6, but not E7, was found to significantly transactivate the survivin promoter. Experiments performed in different cancer cell lines and with different E6 mutants indicated that the effect of E6 on the survivin promoter is largely dependent on p53 status. In accordance with this, the p53 tumour suppressor protein downregulated the expression of survivin. As E6 is able to interact with p53 and induces its ubiquitin-dependent degradation, it appears that the transactivation effect of E6 on survivin is mediated by the p53 degradation pathway. Transduction of HPV-16 E6 and E7 into human embryonic fibroblast cells showed that the HPV oncoproteins can upregulate endogenous survivin mRNA. Importantly, cell cycle synchronization experiments showed that the effect of HPV-16 E6 on survivin transcription is independent of the cell cycle.

In order to evaluate the genetic variability of Porcine parvovirus (PPV), the complete capsid protein sequences (VP1/VP2) from seven recent field isolates from Germany, one isolate from the UK and one German vaccine strain were sequenced and analysed, along with two American (NADL-2 and Kresse), three Asian and 22 Brazilian partial PPV sequences retrieved from GenBank. The analysis revealed a high degree of diversity: 1·2–2·6 % at the nucleotide level and 1·2–6·8 % at the amino acid level. Phylogenetic analysis defined two German clusters: one formed by four German isolates and the English, Asian and American sequences; and the second, distinct cluster formed by the other three of the seven German isolates examined. The latter cluster was still observed when the 22 partial sequences (853 nt of the 3′ terminus of the VP2 gene) from the Brazilian isolates were included in the analyses, indicating that the VP2 sequence determines the phylogeny.

To clarify the stability of the BK polyomavirus (BKPyV) genome in renal transplant (RT) recipients, three to five complete BKPyV genomes from each of six RT recipients with surviving renal allografts were molecularly cloned. The complete sequences of these clones were determined and compared in each patient. No nucleotide difference was detected among clones in two patients, and a few nucleotide variations were found among those in four patients. In each of these patients a parental sequence (usually the major sequence), from which variant sequences (usually minor sequences) with nucleotide substitutions would have been generated, were identified. A comparison between the parental and variant sequences in each patient identified a single nucleotide substitution in each variant sequence. From these findings, it was concluded that the genome of BKPyV is stable in RT recipients without nephropathy, with only minor nucleotide substitutions in the coding region.

Chronic carriers of hepatitis B infection often harbour virus strains with mutations in the precore region. These mutations are temporally associated with the development of HBeAg loss and seroconversion to anti-HBe. The most common precore mutation is a stop codon at position 1896, but other mutations leading to abolished HBeAg secretion have been described. Here, a novel precore mutation introducing a lysine in the precore position 28, a sequence shared by non-human primates but not by other human isolates, is described. However, the insertion causes a frame-shift preventing the expression of HBeAg by introducing a stop codon 5 aa downstream of the mutation. Analysis of the predicted RNA secondary structure indicates that the insertion could occur without fatally affecting the stability of the stem–loop encapsidation signal.

Glyptapanteles indiensis (Braconidae, Hymenoptera) is an endoparasitoid of Lymantria dispar, the gypsy moth. Expression of G. indiensis polydnavirus (GiBV)-encoded genes within the pest host results in inhibition of immune response and development and alteration of physiology, enabling successful development of the parasitoid. Here, GiBV genome segment F (segF), an 18·6 kb segment shown to encode nine protein tyrosine phosphatase (PTP) genes and a single ankyrin repeat gene (ank), is analysed. PTPs have presumed function as regulators of signal transduction, while ankyrin repeat genes are hypothesized to function in inhibition of NF-κB signalling in the parasitized host. In this study, transcription of each gene was mapped by 5′- and 3′-RACE (rapid amplification of cDNA ends) and temporal and tissue-specific expression was examined in the parasitized host. For polydnavirus gene prediction in the parasitized host, no available gene prediction parameters were entirely precise. The mRNAs for each GiBV segF gene initiated between 30 and 112 bp upstream of the translation initiation codon. All were encoded in single open reading frames (ORFs), with the exception of PTP9, which was transcribed as a bicistronic message with the adjacent ank gene. RT-PCR indicated that all GiBV segF PTPs were expressed early in parasitization and, for most, expression was sustained over the course of at least 7 days after parasitization, suggesting importance in both early and sustained virus-induced immunosuppression and alteration of physiology. Tissue-specific patterns of PTP expression of GiBV segF genes were variable, suggesting differing roles in facilitating parasitism.