- Volume 89, Issue 7, 2008

DNAzyme (Dz) molecules have been shown to be highly efficient inhibitors of virus replication. Hepatitis C virus RNA translation is mediated by an internal ribosome entry site (IRES) element located mostly in the 5′ untranslated region (UTR), the mechanism of which is fundamentally different from cap-dependent translation of cellular mRNAs, and thus an attractive target for designing antiviral drugs. Inhibition of HCV IRES-mediated translation has drastic consequences for the replication of viral RNA as well. We have designed several Dzs, targeting different regions of HCV IRES specific for 1b and also sequences conserved across genotypes. The RNA cleavage and translation inhibitory activities of these molecules were tested in a cell-free system and in cell culture using transient transfections. The majority of Dzs efficiently inhibited HCV IRES-mediated translation. However, these Dz molecules did not show significant inhibition of coxsackievirus B3 IRES-mediated translation or cap-dependent translation of reporter gene, showing high level of specificity towards target RNA. Also, Northern blot hybridization analysis showed significant cleavage of HCV IRES by the Dz molecules in Huh7 cells transiently transfected with the HCV–FLuc monocistronic construct. Interestingly, one of the Dzs was more effective against genotype1b, whereas the other showed significant inhibition of viral RNA replication in Huh7 cells harbouring a HCV 2a monocistronic replicon. As expected, mutant-Dz failed to cleave RNA and inhibit HCV RNA translation, showing the specificity of inhibition. Taken together, these findings suggest that the Dz molecule can be used as selective and effective inhibitor of HCV RNA replication, which can be explored further for development of a potent therapeutic agent against HCV infection.

While hepatocytes are the major site of hepatitis C virus (HCV) infection, a number of studies have suggested that HCV can replicate in lymphocytes. However, in vitro culture systems to investigate replication of HCV in lymphocytic cells are severely limited. Robust HCV culture systems have been established using the HCV JFH-1 strain and Huh-7 cells. To gain more insights into the tissue tropism of HCV, we investigated the infection, replication, internal ribosome entry site (IRES)-dependent translation and polyprotein processing of the HCV JFH-1 strain in nine lymphocytic cell lines. HCV JFH-1 failed to infect lymphocytes and replicate, but exhibited efficient polyprotein processing and IRES-dependent translation in lymphocytes as well as in Huh-7 cells. Our results suggest that lymphocytic cells can support HCV JFH-1 translation and polyprotein processing, but may lack some host factors essential for HCV JFH-1 infection and replication.

Hepatitis A virus (HAV) antagonizes the innate immune response by inhibition of retinoic acid-inducible gene I-mediated and melanoma differentiation-associated gene 5-mediated beta interferon (IFN-β) gene expression. This study showed that this is due to an interaction of HAV with mitochondrial antiviral signalling protein (MAVS)-dependent signalling, in which the viral non-structural protein 2B and the protein intermediate 3ABC recently suggested in this context seem to be involved, cooperatively affecting the activities of MAVS and the kinases TANK-binding kinase 1 (TBK1) and the inhibitor of NF-κB kinase ϵ (IKKϵ). In consequence, interferon regulatory factor 3 (IRF-3) is not activated. As IRF-3 is necessary for IFN-β transcription, inhibition of this factor results in efficient suppression of IFN-β synthesis. This ability might be of vital importance for HAV, which is an exceptionally slow growing virus sensitive to IFN-β, as it allows the virus to establish infection and maintain virus replication for a longer period of time.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes an acute febrile disease in humans, characterized by musculoskeletal pain, headache, rash and leukopenia. The cause of myalgia during DENV infection is still unknown. To determine whether DENV can infect primary muscle cells, human muscle satellite cells were exposed to DENV in vitro. The results demonstrated for the first time high-efficiency infection and replication of DENV in human primary muscle satellite cells. Changes in global gene expression were also examined in these cells following DENV infection using Affymetrix GeneChip analysis. The differentially regulated genes belonged to two main functional categories: cell growth and development, and antiviral type I interferon (IFN) response genes. Increased expression of the type I IFN response genes for tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), melanoma-derived antigen 5 (MDA-5), IFN-γ-inducible protein 10 (IP-10), galectin 3 soluble binding protein (LGals3BP) and IFN response factor 7 (IRF7) was confirmed by quantitative RT-PCR. Furthermore, higher levels of cell-surface-bound intracellular adhesion molecule-1 (ICAM-1) and soluble ICAM-1 in the cell-culture medium were detected following DENV infection. However, DENV infection impaired the ability of the infected cells in the culture medium to upregulate cell-surface expression of MHC I molecules, suggesting a possible mechanism of immune evasion by DENV. The findings of this study warrant further clinical research to identify whether muscle cells are targets for DENV infection during the acute stage of the disease in vivo.

The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393–401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101–109 sequence containing 105-C by G substitution and the 393–401 sequence are good candidates for diagnostic assays and cross-protection experiments.

A mouse-adapted strain of human enterovirus 71 (HEV71) was selected by serial passage of a HEV71 clinical isolate (HEV71-26M) in Chinese hamster ovary (CHO) cells (CHO-26M) and in newborn BALB/c mice (MP-26M). Despite improved growth in CHO cells, CHO-26M did not show increased virulence in newborn BALB/c mice compared with HEV71-26M. By contrast, infection of newborn mice with MP-26M resulted in severe disease of high mortality. Skeletal muscle was the primary site of replication in mice for both viruses. However, MP-26M infection induced severe necrotizing myositis, whereas CHO-26M infection caused only mild inflammation. MP-26M was also isolated from whole blood, heart, liver, spleen and brain of infected mice. CHO-26M harboured a single mutation within the open reading frame (ORF), resulting in an amino acid substitution of K149→I in the VP2 capsid protein; two further ORF mutations that resulted in amino acid substitutions were identified in MP-26M, located within the VP1 capsid protein (G145→E) and the 2C protein (K216→R). Infectious cDNA clone-derived mutant virus populations containing the mutations identified in CHO-26M and MP-26M were generated in order to study the molecular basis of CHO cell and mouse adaptation. The VP2 (K149→I) change was responsible only for improved growth in CHO cells and did not lead to increased virulence in mice. Of the two amino acid substitutions identified in MP-26M, the VP1 (G145→E) mutation alone was sufficient to increase virulence in mice to the level observed in MP-26M-infected mice.

West Nile virus (WNV), a mosquito-borne flavivirus, has significantly expanded its geographical and host range since its 1999 introduction into North America. The underlying mechanisms of evolution of WNV and other arboviruses are still poorly understood. Studies evaluating virus adaptation and fitness in relevant in vivo systems are largely lacking. In order to evaluate the capacity for host-specific adaptation and the genetic correlates of adaptation in vivo, this study measured phenotypic and genotypic changes in WNV resulting from passage in Culex pipiens mosquitoes. An increase in replicative ability of WNV in C. pipiens was attained for the two lineages of WNV tested. This adaptation for replication in mosquitoes did not result in a replicative cost in chickens, but did decrease cell-to-cell spread of virus in vertebrate cell culture. Genetic analyses of one mosquito-adapted lineage revealed a total of nine consensus nucleotide substitutions with no accumulation of a significant mutant spectrum. These results differed significantly from previous in vitro studies. When St Louis encephalitis virus (SLEV), a closely related flavivirus, was passaged in C. pipiens, moderately attenuated growth in C. pipiens was observed for two lineages tested. These results suggest that significant differences in the capacity for mosquito adaptation may exist between WNV and SLEV, and demonstrate that further comparative studies in relevant in vivo systems will help elucidate the still largely unknown mechanisms of arboviral adaptation in ecologically relevant hosts.

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.

The genetic diversity of Puumala hantavirus (PUUV) was studied in a local population of its natural host, the bank vole (Myodes glareolus). The trapping area (2.5×2.5 km) at Konnevesi, Central Finland, included 14 trapping sites, at least 500 m apart; altogether, 147 voles were captured during May and October 2005. Partial sequences of the S, M and L viral genome segments were recovered from 40 animals. Seven, 12 and 17 variants were detected for the S, M and L sequences, respectively; these represent new wild-type PUUV strains that belong to the Finnish genetic lineage. The genetic diversity of PUUV strains from Konnevesi was 0.2–4.9 % for the S segment, 0.2–4.8 % for the M segment and 0.2–9.7 % for the L segment. Most nucleotide substitutions were synonymous and most deduced amino acid substitutions were conservative, probably due to strong stabilizing selection operating at the protein level. Based on both sequence markers and phylogenetic clustering, the S, M and L sequences could be assigned to two groups, ‘A’ and ‘B’. Notably, not all bank voles carried S, M and L sequences belonging to the same group, i.e. SAMALA or SBMBLB. A substantial proportion (8/40, 20 %) of the newly characterized PUUV strains possessed reassortant genomes such as SBMALA, SAMBLB or SBMALB. These results suggest that at least some of the PUUV reassortants are viable and can survive in the presence of their parental strains.

The CD4+ T-cell reduction characteristic of human immunodeficiency virus type 1 (HIV-1) infection is thought to result, in addition to infected T-cell death, mainly from uninfected bystander T-cell apoptosis. Nevertheless, the immunological and virological mechanisms leading to T-cell death during HIV-1 infection are not yet fully understood. In the present study, we analysed the individual implication of the p38 mitogen-activated protein kinase (MAPK) isoforms (p38α, p38β, p38γ and p38δ) during apoptosis induced by HIV-1, taking into account that HIV-1 replication is known to be blocked by p38 inhibitors. For this purpose, we used the SupT1 cell line, where death induced by HIV-1 mainly occurs by uninfected bystander cell apoptosis. A variety of SupT1-based cell lines were constructed constitutively expressing, under the control of cytomegalovirus promoter (PCMV), each dominant-negative (dn) p38 isoform and each wild-type p38 isoform as a control. An enhanced green fluorescent protein marker gene, under the control of the HIV-1 promoter, was inserted in all of them. These cell lines were infected with HIV-1 and analysed by flow cytometry. We found that survival in SupT1-based cell lines infected by HIV-1 was increased by the p38αdn, p38γdn and p38δdn isoforms, but not by the p38βdn isoform. HIV-1 replication was delayed most by p38δdn and to a lesser extent by p38αdn and p38γdn. Moreover, these three isoforms, p38αdn, p38γdn and p38δdn, reduced apoptosis induced by HIV-1. These results suggest that, in SupT1-based cell lines, p38α, p38γ and p38δ, but not p38β, are implicated in both HIV-1 induced replication and apoptosis in infected and uninfected bystander cells.

Emergence of antiviral drug resistance is a major challenge to human immunodeficiency virus (HIV) therapy. The archetypal example of this problem is loss of antiviral activity of the nucleoside analogue 3′-azido-3′-deoxythymidine (AZT), caused by mutations in reverse transcriptase (RT), the viral polymerase. AZT resistance results from an imbalance between rates of AZT-induced proviral DNA chain termination and RT-induced excision of the chain-terminating nucleotide. Conversion of the AZT prodrug from its monophosphorylated to diphosphorylated form by human thymidylate kinase (TMPK) is inefficient, resulting in accumulation of the monophosphorylated AZT metabolite (AZT-MP) and a low concentration of the active triphosphorylated metabolite (AZT-TP). We reasoned that introduction of an engineered, highly active TMPK into T cells would overcome this functional bottleneck in AZT activation and thereby shift the balance of AZT activity sufficiently to block replication of formerly AZT-resistant HIV. Molecular engineering was used to link highly active, engineered TMPKs to the protein transduction domain of Tat for direct cell delivery. Combined treatment of HIV-infected T cells with AZT and these cell-permeable, engineered TMPKs restored AZT-induced repression of viral production. These results provide an experimental basis for the development of new strategies to therapeutically increase the intracellular concentrations of active nucleoside analogue metabolites as a means to overcome emerging drug resistance.

To analyse the potential karyophilic activity of prototype foamy viruses (PFVs), we expressed the PFV integrase (IN) and its mutants as fusion proteins with enhanced green fluorescence protein. The subcellular localization of the fusion proteins was investigated by fluorescence microscopy. The PFV IN was found to be karyophilic and targeted the fusion protein to the nucleus. Mutational analyses demonstrated that the PFV IN contains a potent but non-transferable nuclear localization signal (NLS) in its C-terminal domain and contains five arginine and lysine residues between amino acids 308 and 329 that are critical for its NLS function.

Deformed wing virus (DWV) induces wing deformation when bees are infected during their pupal development. Field observations and laboratory experiments suggest that the mite Varroa destructor is a vector of the virus. Moreover, it has been stated that DWV replicates within this mite. In order to understand the role of V. destructor in the transmission of DWV, the objective of this work was to locate the sites of retention and/or replication of DWV within the mite by immunohistochemistry. There was no evidence that DWV was replicating in the mite as no tissues showed specific antibody binding to DWV. Also, there were no specific structures that could be suggested as retention sites. DWV was found only in the midgut lumen of V. destructor in structures resembling large, dense spheres, which were presumably faecal pellets.

A surveillance of human, porcine and bovine rotaviruses was carried out in Slovenia in 2004 and 2005. Stool samples were collected from a total of 406 pigs (373 from asymptomatic animals), 132 cattle (126 from asymptomatic animals) and 241 humans (all with diarrhoea), tested for group A rotaviruses using RT-PCR and analysed by sequencing. The aims of the study were to determine the incidence of asymptomatic rotavirus infection in animals, to look for evidence of zoonotic transmission and to detect reassortment among rotaviruses. The rates of asymptomatic shedding of rotaviruses in pigs and cattle were 18.0 % (67/373) and 4.0 % (5/126), respectively. Evidence for zoonotic transmission was detected in one human rotavirus strain, SI-MB6, with the G3P[6] genotype combination, as the nucleotide and predicted amino acid sequences of the VP6, VP7, VP8* and NSP4 genes of strain SI-MB6 and of porcine strains showed high nucleotide and amino acid sequence identity. Two porcine rotavirus strains carried VP7 of probable human origin, suggesting an interspecies reassortment event in the past.

Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamily Gammaherpesvirinae, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virus in vitro has made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2 from nasal secretions collected from naturally infected sheep during shedding episodes results in infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by nebulization resulted in infection in all lambs, but no infection was established in any lambs after intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.), increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels (∼7500 copies) at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2 open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs. In addition, selected immune response genes were also highly expressed in the lung at 5 and 7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial infection in sheep and suggest that viral replication is promptly controlled by a host defence mechanism.

The UL15, UL28 and UL33 proteins of herpes simplex virus type 1 (HSV-1) are thought to comprise a terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. Immunofluorescence studies confirmed that shortly after infection with wild-type HSV-1 these three proteins localize to viral DNA replication compartments within the nucleus, identified by the presence of the single-stranded DNA-binding protein, ICP8. In cells infected with either UL28- or UL33-null mutants, the other two terminase proteins also co-localized with ICP8. In contrast, neither UL28 nor UL33 was detectable in replication compartments following infection with a UL15-null mutant, although Western blot analysis showed they were present in normal amounts in the infected cells. Provision of UL15 in a complementing cell line restored the ability of all three proteins to localize to replication compartments. These data indicate that UL15 plays a key role in localizing the terminase complex to DNA replication compartments, and that it can interact independently with UL28 and UL33.

Integrated human papillomavirus type 16 (HPV-16) viral loads are currently estimated by quantification with real-time PCR of HPV-16 E6 (RT-E6 and HPV-16 PG) and E2 (RT-E2-1) DNA. We assessed the influence of HPV-16 E2 polymorphism on quantification of integrated HPV-16 DNA in anogenital specimens. HPV-16 E2 was sequenced from 135 isolates (123 from European and 12 from non-European lineages). An assay targeting conserved HPV-16 E2 sequences (RT-E2-2) was optimized and applied with RT-E6 and RT-E2-1 on 139 HPV-16-positive cervicovaginal lavages collected from 74 women [58 human immunodeficiency virus (HIV)-seropositive and 16 HIV-seronegative]. Ratios of HPV-16 copies measured with RT-E2-2 and RT-E2-1 obtained with African 2 (median=3.23, range=1.92–3.49) or Asian–American (median=3.78, range=1.47–37) isolates were greater than those obtained with European isolates (median=1.02, range=0.64–1.80; P<0.02 for each comparison). The distribution of HPV-16 E2 copies measured in 139 samples with RT-E2-2 (median=6150) and RT-E2-1 (median=8960) were different (P<0.0001). The risk of high-grade cervical intraepithelial neoplasia (CIN-2,3) compared with women without CIN was increased with higher HPV-16 total [odds ratio (OR)=2.17, 95 % confidence interval (CI)=1.11–4.23], episomal (OR=2.14, 95 % CI=1.09–4.19), but not for HPV-16 integrated viral load (OR=1.71, 95 % CI=0.90–3.26), after controlling for age, race, CD4 count, HIV and HPV-16 polymorphism. The proportion of samples with an E6/E2 ratio >2 in women without squamous intraepithelial lesion (7 of 35) was similar to that of women with CIN-2,3 (5 of 11, P=0.24) or CIN-1 (5 of 14, P=0.50). HPV-16 E2 polymorphism was a significant factor that influenced measures of HPV-16 integrated viral load.

Recombination and segment reassortment are important contributors to the standing genetic variation of RNA viruses and are often involved in the genesis of new, emerging viruses. This study explored the role played by these two processes in the evolutionary radiation of the plant virus family Bromoviridae. The evolutionary history of this family has been explored previously using standard molecular phylogenetic methods, but incongruences have been found among the trees inferred from different gene sequences. This would not be surprising if RNA exchange was a common event, as it is well known that recombination and reassortment of genomes are poorly described by standard phylogenetic methods. In an attempt to reconcile these discrepancies, this study first explored the extent of segment reassortment and found that it was common at the origin of the bromoviruses and cucumoviruses and at least at the origin of alfalfa mosaic virus, American plum line pattern virus and citrus leaf rugose virus. Secondly, recombination analyses were performed on each of the three genomic RNAs and it was found that recombination was very common in members of the genera Bromovirus, Cucumovirus and Ilarvirus. Several cases of recombination involving species from different genera were also identified. Finally, a phylogenetic network was constructed reflecting these genetic exchanges. The network confirmed the taxonomic status of the different genera within the family, despite the phylogenetic noise introduced by genetic exchange.