- Volume 88, Issue 5, 2007

Five hepatitis C virus (HCV) complete genome sequences (Th846, 537796, QC227, QC216 and QC99) from a blood donor in Thailand and three Asian immigrants and one Caucasian in North America were determined. Phylogenetically, they represent the first complete genomes for subtypes 6c, 6l, 6o, 6p and 6q, respectively. Similarity analysis showed no evidence of inter- or intrasubtype recombination. Further analysis in conjunction with partial sequences from the Los Alamos HCV database led to the identification of other closely related isolates from south-eastern Asia or immigrants from that region. However, Th846 did not cluster with any reference sequence and is the sole isolate of subtype 6c reported so far. This study completes the full genome sequencing of all 17 assigned HCV genotype 6 subtypes (6a–6q). The utility of this panel of complete sequences for accurate detection and classification of infection, and for estimating the origin of this genotype of HCV, is discussed.

Egypt has one of the world’s highest prevalences of hepatitis C virus (HCV) infection, with a majority of genotype 4 infections. To explore the genetic diversity of HCV in Egypt, sera from 131 Egyptians [56 from community studies, 37 chronic hepatitis patients, 28 hepatocellular carcinoma (HCC) patients and 10 patients with non-Hodgkin’s lymphoma] were genotyped by restriction fragment-length polymorphism and phylogenetic analyses of sequences from the mid-core and non-structural 5B regions. The different genotyping methods showed good agreement. The majority of the viruses (83 of 131; 63 %) were of subtype 4a, but five other subtypes within genotype 4 were also observed, as well as three genotype 1b, five genotype 1g and one genotype 3a samples. Interestingly, subtype 4o, which was easily identifiable in all three genomic regions, showed an association with HCC (P=0.017), which merits further investigation.

Avian hepatitis E virus (HEV) is genetically and antigenically related to human HEV. Vertical transmission of HEV has been reported in humans, but not in other animals. In this study, we showed that avian HEV could be detected in chicken egg-white samples. Subsequently, avian HEV in egg white was found to be infectious, as evidenced by the appearance of viraemia, faecal virus shedding and seroconversion in chickens inoculated with avian HEV-positive egg white, but not in chickens inoculated with HEV-negative egg white. To further assess the possibility of vertical transmission of avian HEV, batches of embryonated eggs from infected hens were hatched, and hatched chicks were monitored for evidence of avian HEV infection. However, no virus was detected in samples collected from the hatched chicks throughout this study, suggesting that avian HEV could not complete the vertical transmission cycle. The possible implications of our findings are also discussed.

Avian hepatitis E virus (HEV) was identified from chickens with hepatitis–splenomegaly syndrome. In this study, the complete genomic sequence of an apparently avirulent strain of avian HEV was determined to be 6649 nt in length, excluding the poly(A) tail, which is 5 nt shorter than the prototype avian HEV. Sequence analyses revealed that the ORF1 has 89.6 % nucleotide sequence identity, with numerous non-silent mutations and deletions, compared to the prototype strain. The ORF2 capsid gene showed 90.7 % sequence identity with six non-silent mutations, and ORF3 had four non-silent mutations with 97 % sequence identity. Overall, the apparently avirulent strain shares only 90.1 % nucleotide sequence identity with the prototype strain. The identification of significant non-silent mutations in the capsid gene and other regions suggests that these mutations may play a role in HEV attenuation. This is the first report of the full-length sequence of an apparently avirulent strain of HEV.

Mosquito midgut epithelial cells (MEC) play a major role in determining whether an arbovirus can successfully infect and be transmitted by mosquitoes. The Sindbis virus (SINV) strain TR339 efficiently infects Aedes aegypti MEC but the SINV strain TE/5′2J poorly infects MEC. SINV determinants for MEC infection have been localized to the E2 glycoprotein. The E2 amino acid sequences of TR339 and TE/5′2J differ at two sites, E2-55 and E2-70. We have altered the TE/5′2J virus genome by site-directed mutagenesis to contain two TR339 residues, E2-55 H→Q (histidine to glutamine) and E2-70 K→E (lysine to glutamic acid). We have characterized the growth patterns of derived viruses in cell culture and determined the midgut infection rate (MIR) in A. aegypti mosquitoes. Our results clearly show that the E2-55 H→Q and the E2-70 K→E mutations in the TE/5′2J virus increase MIR both independently and in combination. TE/5′2J virus containing both TR339 E2 residues had MIRs similar to the parental TR339 virus. In addition, SINV propagated in a mammalian cell line had a significantly lower A. aegypti midgut 50 % infectious dose than virus propagated in a mosquito cell line.

Paramyxovirus genomes contain a linear array of five to ten genes sequentially transcribed by the viral RNA polymerase. mRNA synthesis initiates at a nucleotide signal (gs1) within the genomic promoter located at the genome 3′ end. To gain information about the mechanism involved in transcription initiation, a search was carried out for upstream nucleotides required for gs1 and the effects of the gs1 nucleocapsid protein (N) phase context on transcription regulation were determined. For both purposes, tandem promoter mini-genomes carrying a transcription signal ectopically positioned downstream of a replication-only signal were used. The requirement for hygromycin resistance gene expression was used in an attempt to select essential nucleotides within randomized stretches of nucleotides. Nucleotide insertions or deletions were also made on either side of the transcription signal to change its original N phase context in the five remaining possibilities and GFP expression from these modified signals was assessed. Cell cultures resistant to hygromycin treatment were readily obtained following amplification of mini-genomes harbouring randomized sequences. However, selected nucleotides upstream of gs1 could not be identified under conditions where nucleotides within gs1 were selected. In contrast, it was observed that changing the gs1 N phase context progressively decreased transcription by five- to tenfold. These results are discussed in relation to two different mechanisms of transcription initiation.

Measles virus (MeV) research is largely dependent on the B95a cell line, which is derived from marmoset B lymphocytes. As this cell line is persistently infected with Epstein–Barr virus (EBV), a novel cell line, COBL-a, was established from human umbilical cord blood. COBL-a cells have a significant advantage over B95a cells because they are of human origin, are free from EBV and have higher sensitivity to wild-type MeV. Thus, COBL-a cells should prove very valuable for both epidemiological and basic studies of MeV.

The pathogenesis of Lassa fever is poorly understood. As the liver is a major target organ of Lassa virus, gene expression in Lassa virus-infected HuH-7 cells, a differentiated human hepatoma cell line, was studied. Cellular mRNA levels were measured at the late phase of acute infection, when virtually all cells expressed large amounts of nucleoprotein, and virus RNA concentration had reached >108 copies (ml supernatant)−1. Two types of transcription array were used: cDNA-based macroarrays with a set of 3500 genes (Atlas Human 1.2 arrays; Clontech) and oligonucleotide-based microarrays covering 18 400 transcripts (Human Genome U133A array; Affymetrix). Data analysis was based on statistical frameworks controlling the false-discovery rate. Atlas array data were considered relevant if they could be verified by U133A array or real-time RT-PCR. According to these criteria, there was no evidence for true changes in gene expression. Considering the precision of the U133A array and the number of replicates tested, potential expression changes due to Lassa virus infection are probably smaller than twofold. To substantiate the array data, beta interferon (IFN-β) gene expression was studied longitudinally in Lassa virus-infected HuH-7 and FRhK-4 cells by using real-time RT-PCR. IFN-β mRNA levels increased only twofold upon Lassa virus infection, although there was no evidence that the virus inhibited poly(I : C)-induced IFN-β gene expression. In conclusion, Lassa virus interferes only minimally with gene expression in HuH-7 cells and poorly induces IFN-β gene transcription.

The Tetraviridae are a family of small, non-enveloped, insect RNA viruses consisting of one or two single-stranded, positive-sense genomic RNAs encapsidated in an icosahedral capsid with T=4 symmetry. Tetravirus procapsids undergo maturation when exposed to a low pH environment in vitro. While the structural biology of the conformational changes that mediate acid-dependent maturation is well understood, little is known about the significance of acid-dependent maturation in vivo. To address this question, the capsid-coding sequence of the tetravirus Helicoverpa armigera stunt virus was expressed in Saccharomyces cerevisiae cells. Virus-like particles were shown to assemble as procapsids that matured spontaneously in vivo as the cells began to age. Growth in the presence of hydrogen peroxide or acetic acid, which induced apoptosis or programmed cell death in the yeast cells, resulted in virus-like particle maturation. The results demonstrate that assembly-dependent maturation of tetravirus procapsids in vivo is linked to the onset of apoptosis in yeast cells. We propose that the reduction in pH required for tetraviral maturation may be the result of cytosolic acidification, which is associated with the early onset of programmed cell death in infected cells.

Rhopalosiphum padi virus (RhPV) is a member of the family Dicistroviridae. The genomes of viruses in this family contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The RhPV 5′ IRES is functional in mammalian, insect and plant translation systems and can form 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and eIF1. Large regions of the 5′ untranslated region (UTR) can be deleted without affecting initiation-complex formation. The minimal sequences required for directing internal initiation in mammalian (rabbit reticulocyte lysate), plant (wheatgerm extract) and insect (Sf21 cells) translation systems have now been defined. A fragment (nt 426–579) from the 3′ portion of the 5′ UTR can direct translation in each of these translation systems. In addition, a distinct region (nt 300–429) is also active. Thus, unstructured regions within the 5′ UTR seem to be critical for IRES function.

CD4+ T cells are involved in several immune response pathways used to control viral infections. In this study, a group of genetically defined goats was immunized with a synthetic peptide known to encompass an immunodominant helper T-cell epitope of caprine arthritis encephalitis virus (CAEV). Fifty-five days after challenge with the molecularly cloned CAEV strain CO, the vaccinated animals had a higher proviral load than the controls. The measurement of gamma interferon and interleukin-4 gene expression showed that these cytokines were reliable markers of an ongoing immune response but their balance did not account for more or less efficient control of CAEV replication. In contrast, granulocyte–macrophage colony-stimulating factor appeared to be a key cytokine that might support virus replication in the early phase of infection. The observation of a potential T-cell-mediated enhancement of virus replication supports other recent findings showing that lentivirus-specific T cells can be detrimental to the host, suggesting caution in designing vaccine candidates.

Two important issues regarding a virus mutant that is resistant to an antiviral drug are its ability to replicate in animal hosts (in vivo fitness) relative to other genetic variants, including wild type, and its ability to cause disease. These issues have been investigated for a herpes simplex virus 1 mutant that is resistant to thiourea compounds, which inhibit encapsidation of viral DNA. Following corneal inoculation of mice, the mutant virus replicated very similarly to its wild-type parent in the eye, trigeminal ganglion and brain. The mutant virus was as lethal to mice as its wild-type parent following this route of inoculation. Indeed, it exhibited increased virulence. Thus, unlike most drug-resistant virus mutants, this mutant retained in vivo fitness and virulence.

CD46 (membrane cofactor protein; MCP) is a molecule that functions as either a complement-regulatory protein (CRP) or a receptor for some pathogens, including human herpesvirus 6. DNA microarray analysis suggested that the expression of CD46 was upregulated in T cells infected with human herpesvirus 7 (HHV-7). Northen and Western blot analyses supported this result at both the transcriptional and translational levels. Flow-cytometric analysis revealed that upregulation of CD46 occurred at a late stage of infection in both SupT1 cells and primary CD4+ T cells, and also that expression of another CRP, CD59, was increased at a late stage of infection. Whether these CRPs actually function in HHV-7-infected cells was addressed and it was found that HHV-7-infected cells were more resistant to complement-dependent cytotoxicity than were uninfected cells. This study is the first report demonstrating that HHV-7 infection causes elevation of the CRPs CD46 and CD59, which may be a possible mechanism for HHV-7 to evade humoral immunity via complement.

Human herpesvirus 7 (HHV-7) is a member of the subfamily Betaherpesvirinae that exhibits a restricted cell tropism, preferentially infecting CD4+ T cells in vitro. HHV-7 encodes two functional chemokine receptors, U12 and U51. The human chemokines that act as ligands for these receptors have been identified as CCL22 (the natural ligand for CCR4) and CCL19 (the natural ligand for CCR7). It was found that murine L1.2 cells co-expressing CCR4 or CCR7 and U12 responded to both CCL22 and CCL19 in calcium-mobilization assays, but migrated in response only to the appropriate ligand for the expressed cellular receptor. Similar results were obtained with L1.2 cells co-expressing CCR4 or CCR7 with U51. These results suggest that the HHV-7 U12 and U51 receptors can function in concert with CCR4 and CCR7 in host-cell signalling pathways.

Immune control of human cytomegalovirus (HCMV) infection can be mediated by CD8+ cytolytic T lymphocytes (CTL). Adoptive transfer of antiviral CTL confers protection against HCMV reactivation and disease. The tegument protein pp65 and the immediate-early 1 protein (IE1) are recognized to be major CTL targets, even though during productive infection the viral immunoevasion proteins gpUS2–11 act to suppress major histocompatibility complex (MHC) class I-restricted antigen presentation. Thus it was not clear how infected cells could be labelled with antigenic peptides in the face of immunoevasion. We show here that the immunodominant peptide pp65NLV was presented by MHC class I in cells infected with a gpUS2–11-competent virus. Presentation of pp65NLV was still detectable at 96 h post-infection, although at low levels. Partial suppression of pp65NLV presentation was dependent on the ability of the infecting strain to express gpUS2–11. MHC class I-restricted antigen presentation in HCMV-infected cells (encoding gpUS2–11) exhibited specificity for pp65-derived peptides, as infected fibroblasts did not present the IE1-derived nonapeptide IE1TMY. Remarkably, infected cells could restore pp65NLV peptide presentation after acid removal of MHC class I despite gpUS2–11 expression. This recovery was shown to be dependent on proteasome functionality. In contrast to IE1, pp65 peptides are loaded on MHC class I molecules to be transported to the cell surface at early and late times after infection in the face of gpUS2–11-mediated immunoevasion. pp65 is therefore the first example of an HCMV protein only incompletely subjected to gpUS2–11-mediated immunoevasion.

Following acute infection, murine cytomegalovirus (MCMV) replicates persistently in the salivary glands, despite the vigorous response of activated CD8 T cells that infiltrate this gland. Virus-specific CD8 T lymphocytes isolated from this organ were found to express the inhibitory CD94/NKG2A receptor that, in some virus models, confers an inhibitory response to cytotoxic T lymphocytes (CTLs). In response to MCMV infection, expression of the CD94/NKG2A ligand, Qa-1b, increased dramatically in the submandibular gland (SMG) prior to upregulation of H-2Dd. However, there was no net negative impact on virus-specific T-cell function, as virus titres were similar in CD94− and CD94+ mice. CD94/NKG2A expression, also known to inhibit apoptosis, did not influence the numbers of accumulated T, NK and NK T cells. These data indicate that expression of inhibitory CD94/NKG2A receptors does not account for the failure of MCMV-specific CTLs to clear the SMG of infection.

The objective of this work was to identify novel viral ‘evasion’ genes without homology in the database through functional assays. Using this approach, the ‘unassigned’, conserved murine gammaherpesvirus ORF20 gene was shown to localize in the nucleus and to induce cell-cycle arrest followed by apoptosis in both mouse and human cells. Such growth-arrested cells did not express phospho-histone H3, demonstrating that the virus protein caused arrest at the G2 stage of the cell cycle. To characterize the mechanism further, Western blots of ORF20-recombinant lentivirus-infected cells were developed with antibodies to cyclin B1, Cdc2 and phospho-Tyr-15-Cdc2. This analysis revealed a relative increase in cyclin B and phospho-Tyr-15-Cdc2, from 24 to 72 h after infection with recombinant lentivirus. The demonstration that Cdc2 is in its inactive phosphorylated form and the clearly increased levels of cyclin B indicated that the virus gene blocks the progression of cells into mitosis by acting at the level of the Cdc2–cyclin B complex. To confirm this result, the Cdc2–cyclin B complex in ORF20-expressing cells was shown to be essentially without kinase activity. As the ORF20 gene is conserved in all herpesvirus, it may be presumed to have evolved to fulfil an important, as yet undefined, biological role in host-cell modification.

Epstein–Barr virus (EBV) latent membrane protein 2A (LMP2A) blocks B-cell receptor (BCR) signalling after BCR cross-linking to inhibit activation of lytic EBV, and ectopically expressed LMP2B negatively regulates LMP2A. Here, it is demonstrated that silencing of LMP2B in EBV-harbouring Burkitt's lymphoma Akata cells results in reduced expression of EBV immediate-early lytic BZLF1 gene mRNA and late lytic gp350/220 protein upon BCR cross-linking. Similarly, reduction of lytic EBV activation was observed in Akata cells overexpressing LMP2A. In contrast, silencing of LMP2A expression resulted in higher lytic EBV mRNA and protein expression in BCR cross-linked Akata cells. These observations indicate a role for LMP2B distinct from that of LMP2A in regulation of lytic EBV activation in the host cell and support the hypothesis that LMP2B exhibits a negative-regulatory effect on the ability of LMP2A to maintain EBV latency by preventing the switch to lytic replication.

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17 % total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11 % of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means.