- Volume 89, Issue 12, 2008

Outbreaks of dengue disease are constant threats to tropical and subtropical populations but range widely in severity, from mild to haemorrhagic fevers, for reasons that are still elusive. We investigated the interferon (IFN) response in infected human cell lines A549 and HepG2, using two strains (NGC and TSV01) of dengue serotype 2 (DEN2) and found that the two viruses exhibited a marked difference in inducing type I IFN response. While TSV01 infection led to activation of type I antiviral genes such as EIF2AK2 (PKR), OAS, ADAR and MX, these responses were absent in NGC-infected cells. Biochemical analysis revealed that NGC but not TSV01 suppressed STAT-1 and STAT-2 activation in response to type I IFN (α and β). However, these two strains did not differ in their response to type II IFN (γ). Although unable to suppress IFN signalling, TSV01 infection caused a weaker IFN-β induction compared with NGC, suggesting an alternative mechanism of innate immune escape. We extended our study to clinical isolates of various serotypes and found that while MY10245 (DEN2) and MY22713 (DEN4) could suppress the IFN response in a similar fashion to NGC, three other strains of dengue [EDEN167 (DEN1), MY02569 (DEN1) and MY10340 (DEN2)] were unable to suppress the IFN response, suggesting that this difference is strain-dependent but not serotype-specific. Our report indicates the existence of a strain-specific virulence factor that may impact on disease severity.

Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147–165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N-linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain.

Hamsters experimentally infected with the neuroinvasive West Nile virus (WNV) strain NY385-99 frequently develop persistent renal infection and viruria. Viruses recovered from the urine of such animals no longer cause neurological disease when inoculated into naïve hamsters. To examine if this phenotypic change is stable, and if additional nucleotide changes occur during further passages, a urine isolate from a persistently infected hamster (WNV 9317B) was serially passaged in hamsters, and representative isolates from each passage were analysed for pathogenesis in hamsters and by nucleotide sequencing. The progeny viruses tested all resulted in asymptomatic infection when inoculated into hamsters and caused no mortality. Most of the original nucleotide changes were retained in these serial WNV isolates. Changes were distributed throughout the genome at 116 sites, ranging from 0.082 to 0.262 %, compared with the parent strain NY385-99, and they were mostly in coding regions. Our findings indicate that WNV underwent additional genetic changes during serial passage in hamsters, but there was no reversion to neurotropism and virulence.

Rabbit hemorrhagic disease virus (RHDV), a member of the family Caliciviridae comprising positive-stranded RNA viruses, is a highly virulent pathogen of rabbits. Until recently, studies into the molecular mechanisms of RHDV replication and pathogenesis have been hindered by the lack of an in vitro culture system and reverse genetics. This study describes the generation of a DNA-based reverse genetics system for RHDV and the subsequent investigation of the biological role of the RHDV VP2 protein. The full-length RHDV genome was assembled as a single cDNA clone and placed under the control of the eukaryotic human cytomegalovirus promoter. Transfection of cells with the DNA clone resulted in a clear cytopathic effect and the generation of infectious progeny virions. The reconstituted virus was stable and grew to titres similar to that of the parental virus. Although previous reports have suggested that the minor structural protein (VP2) of other caliciviruses is essential for the production of infectious virions, using the DNA-launch-based RHDV reverse genetics system described here it was demonstrated that VP2 is not essential for RHDV infectivity. Transfection of cells with a cDNA clone of RHDV lacking VP2 resulted in the generation of infectious virions. These studies indicate that the presence of VP2 could reduce the replication of RHDV, suggesting that it may play a regulatory role in the life cycle of RHDV.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major problem in the pork industry worldwide. The limitations of current PRRSV vaccines require the development of a new generation of vaccines. One of the key steps in future vaccine development is to include markers for diagnostic differentiation of vaccinated animals from those naturally infected with wild-type virus. Using a cDNA infectious clone of type 1 PRRSV, this study constructed a recombinant green fluorescent protein (GFP)-tagged PRRSV containing a deletion of an immunogenic epitope, ES4, in the nsp2 region. In a nursery pig disease model, the recombinant virus was attenuated with a lower level of viraemia in comparison with that of the parental virus. To complement the marker identification, GFP and ES4 epitope-based ELISAs were developed. Pigs immunized with the recombinant virus lacked antibodies directed against the corresponding deleted epitope, but generated a high-level antibody response to GFP by 14 days post-infection. These results demonstrated that this recombinant marker virus, in conjunction with the diagnostic tests, enables serological differentiation between marker virus-infected animals and those infected with the wild-type virus. This rationally designed marker virus will provide a basis for further development of PRRSV marker vaccines to assist with the control of PRRS.

Influenza A virus genome RNA segment 7 encodes three known mRNAs, two of which, M2 mRNA and M mRNA3, are derived by alternative splicing of the primary collinear mRNA transcript using alternative 5′ splice sites. The function of M mRNA3 is currently unknown, therefore we attempted to determine whether it is essential for virus replication. Recombinant viruses unable to produce M mRNA3 and/or M2 mRNA were created by mutating the shared 3′ splice site. Growth of the mutant viruses in M2-expressing MDCK cells was not significantly affected by the lack of M mRNA3. During the course of a wild-type virus infection, levels of M mRNA3 began to decrease while those of M2 mRNA increased, which may indicate a potential mechanism of alternative splicing control. These data suggest that neither M mRNA3 nor any potential protein product are essential for influenza virus replication in tissue culture.

In the current study, we characterized H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation during a 5 year period (1998–2002). Phylogenetic analysis of the 8 genes of 11 representative viruses showed that they all shared high similarity to that of the first isolate, A/Chicken/Shanghai/F/1998 (Ck/SH/F/98), and clustered to the same lineages. Furthermore, all 11 viruses had a 9 nt deletion between positions 206 and 214 of the neuraminidase gene. These genetic characteristics strongly suggest that these viruses are descendants of the first isolate. In addition, our study also showed that the H9N2 viruses circulating in the operation during this 5 year period were evolving, as shown by antigenic variations between viruses manifested by reactivity with polyclonal antisera and monoclonal antibodies, by haemagglutination with erythrocytes from different animals, by amino acid differences in haemagglutinin and neuraminidase proteins, and by variation in their ability to replicate in the respiratory and intestinal tract and to be transmitted by aerosol. Phylogenetic analysis revealed that the internal genes from some H5N1 viruses of duck origin clustered together with those from H9N2 virus and that the RNP genes of these H5N1 viruses isolated after 2001 are more closely related to the genes of the Ck/SH/F/98-like H9N2 viruses, indicating more recent reassortment events between these two subtypes of viruses. Continuous surveillance of influenza virus in poultry and waterfowl is critical for monitoring the genesis and emergence of potentially pandemic strains in this region.

Human metapneumovirus (hMPV) is genetically related to respiratory syncytial virus (RSV); both cause respiratory tract illnesses ranging from a mild cough to bronchiolitis and pneumonia. The F protein-directed monoclonal antibody (mAb) palivizumab has been shown to prevent severe lower respiratory tract RSV infection in animals and humans. We have previously reported on a panel of mAbs against the hMPV F protein that neutralize hMPV in vitro and, in two cases, in vivo. Here we describe the generation of hMPV mAb-resistant mutants (MARMs) to these neutralizing antibodies. Sequencing the F proteins of the hMPV MARMs identified several neutralizing epitopes. Interestingly, some of the epitopes mapped on the hMPV F protein coincide with homologous regions mapped previously on the RSV F protein, including the site against which the broadly protective mAb palivizumab is directed. This suggests that these homologous regions play important, conserved functions in both viruses.

Recombination is recognized as a primary force in human immunodeficiency virus type 1 (HIV-1) evolution, increasing viral diversity through reshuffling of genomic portions. The strand-switching activity of reverse transcriptase is required to complete HIV-1 replication and can occur randomly throughout the genome, leading to viral recombination. Some recombination hotspots have been identified and found to correlate with RNA structure or sequence features. The aim of this study was to evaluate the presence of recombination hotspots in the pol gene of HIV-1 and to assess their correlation with the underlying RNA structure. Analysis of the recombination pattern and breakpoint distribution in a group of unique recombinant forms (URFs) detected two recombination hotspots in the pol region. Two stable and conserved hairpins were consistently predicted corresponding to the identified hotspots using six different RNA-folding algorithms on the URF parental strains. These findings suggest that such hairpins may play a role in the higher recombination rates detected at these positions.

Various G protein-coupled receptors (GPCRs) have the potential to work as co-receptors for human and simian immunodeficiency virus (HIV/SIV). HIV/SIV co-receptors have several tyrosines in their extracellular N-terminal region (NTR) as a common feature. However, the domain structure of the NTR that is critical for GPCRs to have co-receptor activity has not been identified. Comparative studies of different HIV/SIV co-receptors are an effective way to clarify the domain. These studies have been carried out only for the major co-receptors, CCR5 and CXCR4. A chemokine receptor, D6, has been shown to mediate infection of astrocytes with HIV-1. Recently, it was also found that an orphan GPCR, GPR1, and a formyl peptide receptor, FPRL1, work as potent HIV/SIV co-receptors in addition to CCR5 and CXCR4. To elucidate more about the domain of the NTR critical for HIV/SIV co-receptor activity, this study analysed the effects of mutations in the NTR on the co-receptor activity of D6, FPRL1 and GPR1 in addition to CCR5. The results identified a number of tyrosines that are indispensable for the activity of these co-receptors. The number and positions of those tyrosines varied among co-receptors and among HIV-1 strains. Moreover, it was found that a small domain of a few amino acids containing a tyrosine is critical for the co-receptor activity of GPR1. These findings will be useful in elucidating the mechanism that allows GPCRs to have the potential to act as HIV/SIV co-receptors.

There have been various attempts to redirect the cell entry receptor tropism of the murine leukemia virus vectors. We have recently reported the successful retargeting of the ecotropic Moloney murine leukemia virus vector. This vector (S3-D84K) contains a viral envelope (Env) protein into which a full-length (68 aa) stromal cell-derived factor-1alpha (SDF-1α) was inserted at Pro-79. The S3-D84K vector transduces a certain human cell line through the CXC chemokine receptor 4 (CXCR4) at a titre of about 104 c.f.u. ml−1. Here, the S3-D84K vector was found to transduce another human cell line through CXCR4 with a titre close to 106 c.f.u. ml−1. The SDF-1α ligand of the S3-D84K Env protein was modified in different ways. In one, C-terminal truncations (by 3–51 aa) with or without a Cys-to-Gly change were performed, and in the other, Cys-to-Ala changes of the disulfide-forming cysteines without truncation were made. Seven truncation and three alanine mutant chimeric Env proteins were examined for virion incorporation, and the retroviral vectors displaying the mutant protein were examined for CXCR4 binding and retargeted transduction. Two mutant vectors showed transduction through CXCR4 with titres not higher than those of the S3-D84K vector, while the other mutant vectors minimally transduced cells through CXCR4 either due to a defect in virion incorporation of the chimeric Env protein or an inability to bind to CXCR4. These results suggest that a full-length sequence that may fold into a distinct domain within the chimeric Env protein is preferable as a targeting ligand.

The matrix domain (MA) of human immunodeficiency virus type 1 Pr55Gag is covalently modified with a myristoyl group that mediates efficient viral production. However, the role of myristoylation, particularly in the viral entry process, remains uninvestigated. This study replaced the myristoylation signal of MA with a well-studied phosphatidylinositol 4,5-biphosphate-binding plasma membrane (PM) targeting motif, the phospholipase C-δ1 pleckstrin homology (PH) domain. PH–Gag–Pol PM targeting and viral production efficiencies were improved compared with Gag–Pol, consistent with the estimated increases in Gag–PM affinity. Both virions were recovered in similar sucrose density-gradient fractions and had similar mature virion morphologies. Importantly, PH–Gag–Pol and Gag–Pol pseudovirions had almost identical infectivity, suggesting a dispensable role for myristoylation in the virus life cycle. PH–Gag–Pol might be useful in separating the myristoylation-dependent processes from the myristoylation-independent processes. This the first report demonstrating infectious pseudovirion production without myristoylated Pr55Gag.

Comparisons of the relative activities of 11 intergenic region (IGR) internal ribosome entry site (IRES) elements of insect dicistrovirus with 5′ IRES elements of the hepatitis C and encephalomyocarditis viruses were performed in insect and mammalian cells. Dual luciferase assays were performed to determine the most effective dicistrovirus IGR IRES in the lepidopteran cell lines Sf9 (Spodoptera frugiperda) and BmN (Bombyx mori), and the dipteran cell lines S2 (Drosophila melanogaster) and ATC-10 (Aedes aegypti). Evaluation of dual luciferase expression from DNA plasmids and in vitro-transcribed RNA revealed apparent splicing with certain IRES elements. Though IRES activity depended upon the cell line examined, the black queen cell and Drosophila C dicistrovirus intergenic IRES elements were most effective for coupled gene expression in the diverse insect cell lines examined.

Birnaviruses are bi-segmented double-stranded RNA (dsRNA) viruses infecting insects, avian species and a wide range of aquatic species. Although homologous recombination is a common phenomenon in positive-sense RNA viruses, recombination in dsRNA viruses is rarely reported. Here we performed a comprehensive survey on homologous recombination in all available sequences (>1800) of the family Birnaviridae based on phylogenetic incongruence. Although inter-species recombination was not evident, potential intra-species recombination events were detected in aquabirnaviruses and infectious bursal disease virus (IBDV). Eight potential recombination events were identified and the possibility that these events were non-naturally occurring was assessed case by case. Five of the eight events were identified in IBDVs and all of these five events involved live attenuated vaccine strains. This finding suggests that homologous recombination between vaccine and wild-type IBDV strains may have occurred; the potential risk of mass vaccination using live vaccines is discussed. This is the first report of evidence for homologous recombination within the family Birnaviridae.

Vaccinia virus (VACV) has been associated with several bovine vaccinia outbreaks in Brazil, affecting cattle and humans. There are no available data about VACV environmental circulation or the role of wildlife in the emergence of an outbreak. Since VACV was isolated from rodents in Brazil, we investigated shedding and transmission of VACV strains in mice. The VACV excretion profile was assessed by PCR and chicken chorioallantoic membrane infection, revealing viral DNA and infectious virus in the faeces and urine of intranasally infected mice. Horizontal transmission was assessed by exposure of sentinel mice to wood shavings contaminated with excrement, to mimic a natural infection. Sentinel mice showed orthopoxvirus antibodies, and VACV DNA and infectious virus were detected in their faeces and intestines, even after six rounds of natural transmission. Together, these data suggest that murine excrement could play a relevant role in VACV spread and transmission, perhaps helping to explain how these viruses circulate between their natural hosts.

The antibody responses elicited by immunization of humans with vaccinia virus (VACV) strains Lister, Dryvax and NYVAC have been determined and compared. Neutralizing antibodies against intracellular mature virus (IMV) and extracellular enveloped virus (EEV), and binding antibody titres (ELISA) against the EEV protein B5, the IMV proteins A27 and H3, and VACV-infected cell lysate were measured. Lister and Dryvax induced broadly similar antibody titres, consistent with the fact that these vaccines each protected against smallpox. In contrast, antibody titres induced by NYVAC were significantly lower than those induced by both Lister and Dryvax. Moreover, there were qualitative differences with NYVAC-immunized subjects failing to induce A27-specific antibodies. These observations suggest that although NYVAC is a safer VACV strain, it does not induce an optimal VACV-specific antibody response. However, NYVAC strains engineered to express antigens from other pathogens remain promising candidate vaccines for immunization against other diseases.

Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an oestrogen receptor-enhanced cell line when treated with oestrogen and the promoter-enhancer binds unidentified proteins that recognize a consensus oestrogen response element (ERE). Co-transfection assays with the CAV promoter and the nuclear receptor chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) showed that expression of EGFP was decreased by 50 to 60 % in DF-1 and LMH cells. The CAV promoter that included sequences at and downstream of the transcription start point had less expression than a short promoter construct. Mutation of a putative E box at this site restored expression levels. Electromobility shift assays showed that the transcription regulator delta-EF1 (δEF1) binds to this E box region. These findings indicate that the CAV promoter activity can be affected directly or indirectly by COUP-TF1 and δEF1.

Enteropathogens are known to disrupt apical actin filaments and/or tight-junction barriers of intestinal epithelial cells to promote infection. In this study, we show that a controlled, cytochalasin-D (Cyto-D)-mediated disruption of actin filaments and tight junctions enhanced the apical delivery of the gene-therapy vector recombinant adeno-associated virus serotype 2 (rAAV2). This increase in transduction efficiency can be attributed to the enhanced delivery of rAAV2 across the Cyto-D disrupted tight junctions, allowing basolateral entry of rAAV2. Previously, we have shown that MG101 and doxorubicin are capable of overcoming proteasome-mediated transduction barriers of rAAV2 in enterocytes. In this study, when Cyto-D was combined with MG101 and doxorubicin in apical delivery of rAAV2 to transduce the differentiated Caco-2 enterocytes, a synergistic >2300-fold increase in transgene expression was achieved. We conclude that Cyto-D is capable of permeating the polarized enterocytes for rAAV2 transduction, which may potentially be a useful device to facilitate intestinal gene transfer via the gut lumen.

Hepatitis B virus (HBV) genotype G (HBV/G) is an unusual variant, and little is known about its epidemiology and natural history, particularly the requirement for a co-infecting HBV genotype and their relationship during infection. This study investigated the quasispecies nature of co-infecting genotypes in 39 samples collected over a 6 year period from 13 HBV/G-infected patients. HBV/G infections were found to occur predominantly in males (92 %) and were primarily associated with male homosexual sex (67 %). All patients were infected with HBV/G and HBV/A, or a recombinant HBV/A/G strain. Co-infecting genotypic prevalence was often observed to fluctuate over time, with periods of HBV/G monoinfection in some patients. The average sequence divergence among Canadian HBV/G strains was 1.57±0.62 %. Thus, all HBV/G infections in Canada occur in the context of co-infection or recombination with HBV/A, and strains display increased sequence divergence compared with all known HBV/G sequences described to date.