- Volume 88, Issue 6, 2007

A C57U nucleotide mutation in a predicted RNA stem structure (nt 11–16/56–61) of the 5′ non-coding region (5′NCR) of dengue 2 (DEN-2) 16681 virus is partially attenuating, but unstable during serial passage of certain candidate DEN-2 PDK-53-based vaccine viruses containing this mutation. Here, 11 different mutations (one or more point substitution and/or deletion) between nt 54 and 70 in the 5′NCR of the pD2/IC-30P-A (16681) infectious clone are described. Four mutants were infectious. Three mutants with single point substitutions replicated well in cell culture and exhibited variable neurovirulence in mice. Constructs containing multiple substitutions or any deletions failed to produce infectious viruses. Unexpectedly, a double C57U+G58C mutant replicated as efficiently as D2/IC-30P-A virus, and was more neurovirulent for newborn ICR mice. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 5′NCR-C57U mutation.

There are two types of feline coronaviruses that can be distinguished by serology and sequence analysis. Type I viruses, which are prevalent in the field but are difficult to isolate and propagate in cell culture, and type II viruses, which are less prevalent but replicate well in cell culture. An important determinant of coronavirus infection, in vivo and in cell culture, is the interaction of the virus surface glycoprotein with a cellular receptor. It is generally accepted that feline aminopeptidase N can act as a receptor for the attachment and entry of type II strains, and it has been proposed that the same molecule acts as a receptor for type I viruses. However, the experimental data are inconclusive. The aim of the studies reported here was to provide evidence for or against the involvement of feline aminopeptidase N as a receptor for type I feline coronaviruses. Our approach was to produce retroviral pseudotypes that bear the type I or type II feline coronavirus surface glycoprotein and to screen a range of feline cell lines for the expression of a functional receptor for attachment and entry. Our results show that type I feline coronavirus surface glycoprotein fails to recognize feline aminopeptidase N as a functional receptor on three continuous feline cell lines. This suggests that feline aminopeptidase N is not a receptor for type I feline coronaviruses. Our results also indicate that it should be possible to use retroviral pseudotypes to identify and characterize the cellular receptor for type I feline coronaviruses.

The genus Orthobunyavirus is composed of segmented, negative-sense RNA viruses that are responsible for mild to severe human diseases. To date, no molecular studies of bunyaviruses in the genus Orthobunyavirus from central Africa have been reported, and their classification relies on serological testing. Four new primer pairs for RT-PCR amplification and sequencing of the complete genomic small (S) RNA segments of 10 orthobunyaviruses isolated from the Central African Republic and pertaining to five different serogroups have been designed and evaluated. Phylogenetic analysis showed that these 10 viruses belong to the Bunyamwera serogroup. The S segment sequences differ from those of the Bunyamwera virus reference strain by 5–15 % at the nucleotide level, and both overlapping reading frames, encoding the nucleocapsid (N) and non-structural (NS) proteins, were evident in sequenced genomes. This study should improve diagnosis and surveillance of African bunyaviruses.

A prototype avian metapneumovirus (AMPV) vaccine (P20) was previously shown to give variable outcomes in experimental trials. Following plaque purification, three of 12 viruses obtained from P20 failed to induce protection against virulent challenge, whilst the remainder retained their protective capacity. The genome sequences of two protective viruses were identical to the P20 consensus, whereas two non-protective viruses differed only in the SH gene transcription termination signal. Northern blotting showed that the alterations in the SH gene-end region of the non-protective viruses led to enhanced levels of dicistronic mRNA produced by transcriptional readthrough. A synthetic minigenome was used to demonstrate that the altered SH gene-end region reduced the level of protein expression from a downstream gene. The genomes of the remaining eight plaque-purified viruses were sequenced in the region where the P20 consensus sequence differed from the virulent progenitor. The seven protective clones were identical, whereas the non-protective virus retained the virulent progenitor sequence at two positions and contained extensive alterations in its attachment (G) protein sequence associated with a reduced or altered expression pattern of G protein on Western blots. The data indicate that the efficacy of a putative protective vaccine strain is affected by mutations altering the balance of G protein expression.

Junín virus (JUNV) entry is conducted by receptor-mediated endocytosis. To explore the cellular entry mechanism of JUNV, inhibitory effects of drugs affecting the main endocytic pathways on JUNV entry into Vero cells were analysed. Compounds that impair clathrin-mediated endocytosis were shown to reduce virus internalization without affecting virion binding. In contrast, drugs that alter lipid-raft microdomains, impairing caveola-mediated endocytosis, were not able to block virus entry. To show direct evidence of JUNV entry, transmission electron microscopy was performed; it showed JUNV particles of about 60–100 nm in membrane depressions that had an electron-dense coating. In addition, JUNV particles were found within invaginations of the plasma membrane and vesicles that resembled those of pits and clathrin-coated vesicles. Taken together, these results demonstrate that clathrin-mediated endocytosis is the main JUNV entry pathway into Vero cells and represent an important contribution to the characterization of the arenavirus multiplication cycle.

Infectious bursal disease virus (IBDV) induces apoptosis and immunosuppression. To understand the molecular mechanisms involved in the pathogenesis of infectious bursal disease (IBD) and the host-directed antiviral responses, cDNA microarrays were used to identify the differentially expressed transcripts in IBDV-infected chicken embryonic fibroblasts. The results suggest a general suppression of surface receptors, including CD40 ligand and SEMA4D. These are related to T- and B-cell activation and differentiation, which may contribute to the immunosuppression of IBD. In addition, activation of genes involved in Toll-like receptor- and interferon (IFN)-mediated antiviral responses was detected. In particular, upregulation of Toll-like receptor 3, a double-stranded (ds) RNA receptor, and MX1, an IFN-inducible antiviral GTPase, may represent the possible host-directed defence responses against the virus and its dsRNA genome. Interestingly, several lines of evidence suggest the modulation of G protein-coupled receptors and receptor tyrosine kinase signalling pathways, especially the possible transactivation of epidermal growth factor receptor by lysophosphatidic acid. Alteration of these may contribute to the previously reported activation of mitogen-activated protein kinases upon IBDV infection, resulting in macrophage activation and inflammatory responses. Additionally, numerous target genes and inducers of nuclear factor kappa B (NF-κB) were upregulated profoundly, implying that IBDV may modulate host-cell survival and apoptosis to support its replication and facilitate viral spread through NF-κB activation. In summary, this investigation of host-gene expression unravelled the candidate physiological pathways involved in host–virus interaction on a molecular level, providing a foundation for researchers to design experiments based on testable hypotheses targeting individual genes.

Analysis of the amino acid sequence of core protein μA of avian reovirus has indicated that it may share similar functions to protein μ2 of mammalian reovirus. Since μ2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant μA ( μA) was designed and used to test these activities. μA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that  μA possessed NTPase activity that enabled the protein to hydrolyse the β–γ phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k cat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of  μA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein  μA. The activity of  μA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal NTPase activity of  μA was achieved between pH 5.5 and 6.0. In addition,  μA enzymic activity increased with temperature up to 40 °C and was almost totally inhibited at temperatures higher than 55 °C. Tests of phosphate release from RNA substrates with  μA or K408A/K412A  μA indicated that  μA, but not K408A/K412A  μA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of  μA might be carried out at the same active site, and that protein μA could play important roles during viral RNA synthesis.

Earlier reports of a human exogenous retrovirus (HMTV) related closely to mouse mammary tumor virus (MMTV) led us to search for these viral sequences in breast cancer tissues and normal tissues. A real-time PCR was developed based on MMTV and published HMTV envelope sequences. The real-time PCR method can detect one to ten copies of MMTV target DNA. Tissue samples were collected prospectively from 18 breast cancer patients and 11 non-malignant control cases, as well as peripheral blood leukocytes from the same women. Despite the high sensitivity of the real-time PCR method used, none of the samples were positive for HMTV DNA or RNA. The absence of HMTV DNA in both breast cancer samples and controls indicates either that the concentration of putative HMTV DNA in the breast cancers was too low for detection or that it did not exist there.

Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of fibroblast growth. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell growth. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all 16 VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence factor that affects the host immune response to infection.

The sheep parapoxvirus orf virus (ORFV) induces acute, pustular skin lesions in humans. ORFV encodes an orthologue of interleukin-10 (IL-10) that, whilst it closely resembles ovine IL-10 (91 % amino acid identity), shows only 75 % amino acid identity to human IL-10 (hIL-10). The anti-inflammatory potential of ORFV IL-10 in human ORFV infection was investigated by examining its immunosuppressive effects on THP-1 monocytes. ORFV IL-10 and hIL-10 were shown to have equivalent inhibitory effects on the synthesis of proinflammatory cytokines in lipopolysaccharide-activated monocytes, but differed in their abilities to inhibit monocyte proliferation. Structural modelling of ORFV IL-10 revealed differences from hIL-10 in residues predicted to interact with IL-10 co-receptor 2 (IL-10R2), whereas there were very few differences in the residues predicted to interact with IL-10R1. These findings suggest that the partial ability of ORFV IL-10 to inhibit THP-1 monocyte proliferation may be due to the absence of critical residues that mediate the interaction with human IL-10R2.

Glycoprotein I (gI) of herpes simplex virus type 1 (HSV-1) contains a tandem repeat (TR) region including the amino acids serine and threonine, residues that can be utilized for O-glycosylation. The length of this TR region was determined for 82 clinical HSV-1 isolates and the results revealed a polymorphic distribution of two to six or eight repeated blocks with a majority harbouring between two and four repeats. Assessment of the O-glycosylation capacity of an acceptor peptide (STPSTTTSTPSTTT), representing two of the gI blocks, showed that the peptide was a universal substrate for O-glycosylation not only for the two most commonly expressed N-acetyl-d-galactosamine (GalNAc)-T1 and -T2 transferases, but also for the GalNAc-T3, -T4 and -T11 transferases. Immunoblotting of virus-infected cells showed that gI was exclusively O-glycosylated with GalNAc monosaccharides (Tn antigen). A polymorphic mucin region has not been described previously for HSV-1 and is a unique finding, as repeated blocks within gI homologues are lacking in other alphaherpesviruses.

The replication and transcriptional activator (Rta), encoded by ORF50 of gammaherpesviruses, initiates the lytic cycle of gene expression; therefore understanding the impact of Rta on viral and cellular gene expression is key to elucidating the transcriptional events governing productive infection and reactivation from latency. To this end, the impact of altering Rta transcription on viral and cellular gene expression was studied in the context of a whole virus infection. Recombinant murine gammaherpesvirus (MHV)-68 engineered to overexpress Rta greatly accelerated expression of specific lytic cycle ORFs, but repressed transcription of the major latency gene, ORF73. Increased expression of Rta accelerated the dysregulation in transcription of specific cellular genes when compared with cells infected with wild-type and revertant viruses. A subset of cellular genes was dysregulated only in cells infected with Rta-overexpressing virus, and never in those infected with non-overexpressing viruses. These data highlight the critical role of Rta abundance in governing viral and cellular gene transcription, and demonstrate the importance of understanding how the relative expression of ORF50 during the virus life cycle impacts on these processes.

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and the plasma-cell variant of multicentric Castleman's disease. Its alternatively spliced K15 gene encodes several membrane proteins with varying numbers of transmembrane domains. Two highly diverged alleles of the K15 gene, termed predominant (P) and minor (M), exist and share only 33 % amino acid identity with one another, but retain conserved putative src homology (SH) 2- and SH3-binding motifs. K15-M is thought to have entered the KSHV genome as the result of recombination with a related γ 2-herpesvirus. The more common K15-P allele has been shown to activate the mitogen-activated protein kinases Erk2 and JNK1 and the nuclear factor κB (NF-κB) pathway. To explore possible functional differences between K15-P and K15-M that might have influenced their spread in the KSHV population, here, the ability of the M form of K15 to activate these pathways was investigated. Similarly to K15-P, K15-M induces the activation of the Erk2 and JNK1 kinases, the NF-κB transcription factor and the expression of a similar range of cellular inflammatory genes, as assessed by gene-expression microarray studies and reporter assays. In epithelial cells, the activation of most K15-M target genes is impaired by mutagenesis of Y490 in its SH2-binding motif Y490EEV, although this motif appears less important in endothelial cells. Therefore, K15-M and K15-P can trigger similar intracellular signalling pathways, despite their extensive sequence divergence.

Many novel vaccine strategies rely on recombinant viral vectors for antigen delivery, and adenovirus vectors have emerged among the most potent of these. In this report, we have compared the immune response induced through priming with adenovirus vector-encoded full-length viral protein to that elicited with an adenovirus-encoded minimal epitope covalently linked to β 2-microglobulin. We demonstrate that the β 2-microglobulin-linked epitope induced an accelerated and augmented CD8+ T-cell response. Furthermore, the immunity conferred by vaccination with β 2-microglobulin-linked lymphocytic choriomeningitis virus (LCMV)-derived epitopes was long-lived and protective. Notably, in contrast to full-length protein, the response elicited with the β 2-microglobulin-linked LCMV-derived epitope was CD4+ T-cell independent. Furthermore, virus-specific CD8+ T cells primed in the absence of CD4+ T-cell help were sustained in the long term and able to expand and control a secondary challenge with LCMV. Our results demonstrate that modifications to the antigen used in adenovirus vaccines may be used to improve the induced T-cell response. Such a strategy for CD4+ T-cell-independent immunity from adenovirus vectors offers prospects for vaccination against opportunistic pathogens in AIDS patients and possibly immunotherapy in chronic virus infections.

Beta interferon (IFN-β) is a cytokine with potent immunomodulatory properties and has been described as a promising therapeutic molecule for the treatment of rheumatoid arthritis (RA). IFN-β was previously overexpressed intra-articularly using an adenoviral vector in rats with adjuvant arthritis (AA) as a model of RA. This effect was powerful, albeit transient due to the vector chosen. Therefore, in the context of pre-clinical development, a delivery vector optimized for intra-articular gene transfer, recombinant adeno-associated virus type 5 (rAAV5), was selected. To exert an optimal effect, protein production should parallel the course of the disease. For this reason, the gene for IFN-β was placed under the control of an inflammation-responsive [nuclear factor (NF)-κB] promoter. After intra-articular injection of the rAAV5 constructs in rats with AA, local transcription of the transgene and production of the IFN-β protein was found, leading to a pronounced and sustained effect on paw swelling when the expression was under the control of the NF-κB-responsive promoter. Additionally, a significant beneficial effect was observed on proteoglycan depletion and erosions. Thus, intra-articular overexpression of IFN-β using a rAAV5 vector exhibits potential as an innovative therapy for the treatment of RA.

Adeno-associated virus (AAV) undergoes preferential Rep-mediated integration into the AAVS1 region of human chromosome 19 during latent infection, at least in highly-selected cell cultures. However, integration at the level of the whole eukaryotic genome in unselected cells has not yet been monitored for AAV as it has been for retro- and lentiviruses. Here we have used ligation-mediated PCR (LMPCR) to monitor the formation of AAV–chromosome junctions within unselected genomic DNA after infection. Our analyses show that, in the absence of selection, the complexity of junction formation is much greater than for selected cells. Sequencing of more than 50 authentic LMPCR clones showed that AAV formed junctions with many different chromosomal sites via DNA micro-homologies that frequently involved GGTC motifs located within the AAV p5 element. One site at position 280 was preferred. Even greater complexity was found when unselected junctions identified by LMPCR were analysed by direct PCR amplification and cloning of genomic DNA. No clones containing AAV–AAVS1 chromosome 19 junctions were identified among the LMPCR clones, although they were readily obtained using chromosomal PCR primers, suggesting that junctions with AAVS1 constituted only a small portion of the total. Thus, we have identified an additional means by which AAV sequences may join to human chromosomes, although the detailed molecular mechanisms remain to be elucidated. These data may have implications for the design of new-generation AAV vectors.

Porcine circovirus type 2 (PCV2) belongs to the family Circoviridae, and is the causative agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. In this study, phylogenetic analyses of three complete PCV2 genomic sequences from Hong Kong suggest that natural recombination happened among different lineages of PCV2. A preliminary investigation of the parental strains of these potential recombinants was carried out using bootscanning. Statistical significance of this recombination event was tested and positions of the potential recombination breakpoints were estimated in a maximum-likelihood framework. The recombinant breakpoints were estimated to be located within the origin of replication (ori) and replicase (rep) gene of PCV2. Interestingly, several GenBank sequences of PCV2 in mainland China were found to have a recombination pattern similar to that of the potential PCV2 recombinants from Hong Kong, implying that this recombinant genotype might already be widespread within mainland China.