- Volume 90, Issue 1, 2009
Volume 90, Issue 1, 2009
- Review
-
-
-
Adenoviruses: update on structure and function
More LessAdenoviruses have been studied intensively for over 50 years as models of virus–cell interactions and latterly as gene vectors. With the advent of more sophisticated structural analysis techniques the disposition of most of the 13 structural proteins have been defined to a reasonable level. This review seeks to describe the functional properties of these proteins and shows that they all have a part to play in deciding the outcome of an infection and act at every level of the virus's path through the host cell. They are primarily involved in the induction of the different arms of the immune system and a better understanding of their overall properties should lead to more effective ways of combating virus infections.
-
-
- Animal
-
- RNA viruses
-
-
Inhibition of mRNA export and dimerization of interferon regulatory factor 3 by Theiler's virus leader protein
Theiler's murine encephalomyelitis virus (TMEV or Theiler's virus) is a neurotropic picornavirus that can persist lifelong in the central nervous system of infected mice, causing a chronic inflammatory demyelinating disease. The leader (L) protein of the virus is an important determinant of viral persistence and has been shown to inhibit transcription of type I interferon (IFN) genes and to cause nucleocytoplasmic redistribution of host proteins. In this study, it was shown that expression of the L protein shuts off synthesis of the reporter proteins green fluorescent protein and firefly luciferase, suggesting that it induces a global shut-off of host protein expression. The L protein did not inhibit transcription or translation of the reporter genes, but blocked cellular mRNA export from the nucleus. This activity correlated with the phosphorylation of nucleoporin 98 (Nup98), an essential component of the nuclear pore complex. In contrast, the data confirmed that the L protein inhibited IFN expression at the transcriptional level, and showed that transcription of other chemokine or cytokine genes was affected by the L protein. This transcriptional inhibition correlated with inhibition of interferon regulatory factor 3 (IRF-3) dimerization. Whether inhibition of IRF-3 dimerization and dysfunction of the nuclear pore complex are related phenomena remains an open question. In vivo, IFN antagonism appears to be an important role of the L protein early in infection, as a virus bearing a mutation in the zinc finger of the L protein replicated as efficiently as the wild-type virus in type I IFN receptor-deficient mice, but had impaired fitness in IFN-competent mice.
-
-
-
Efficient infection of buffalo rat liver-resistant cells by encephalomyocarditis virus requires binding to cell surface sialic acids
More LessIn contrast to the production of virus and cell lysis seen in baby hamster kidney cells (BHK-21) infected with the strain 1086C of encephalomyocarditis virus (EMCV), in buffalo rat liver cells (BRL) neither virus replication nor cytopathic effects were observed. After 29 passages in BRL cells, each alternating with boosts of the recovered virus in BHK-21 cells, the virus acquired the ability to replicate effectively in BRL cells, attaining virus titres comparable to those in BHK-21 cells and producing complete cell destruction. The binding of virus on BRL cells was increased after adaptation and was similar to that observed on BHK-21 cells. Treatment of BRL cells with sialidase resulted in an 87 % reduction in virus binding and inhibition of infection. Sequence analyses revealed three mutations in the VP1 amino acid sequence of the adapted virus at positions 49 (Lys→Glu), 142 (Leu→Phe) and 180 (Ile→Ala). The residue 49 is exposed at the surface of the capsid and is known to be part of a neutralization epitope. These results suggest that the adaptation of EMCV to BRL cells may have occurred through a mutation in a neutralizing site that confers to the virus a capacity to interact with cell surface sialic acid residues. Taken together, these data suggest a link between virus neutralization site, receptor binding and cell permissivity to infection.
-
-
-
Integrin αVβ6 is a high-affinity receptor for coxsackievirus A9
More LessCoxsackievirus A9 (CAV9), a member of the genus Enterovirus in the family Picornaviridae, possesses an integrin-binding arginine-glycine-aspartic acid (RGD) motif in the C terminus of VP1 capsid protein. CAV9 has been shown to utilize integrins αVβ3 and αVβ6 as primary receptors for cell attachment. While CAV9 RGD-mutants (RGE and RGDdel) are capable of infecting rhabdomyosarcoma (RD) cell line, they grow very poorly in an epithelial lung carcinoma cell line (A549). In this study, the relationships between CAV9 infectivity in A549 and RD cells, receptor expression and integrin binding were analysed. A549 cells were shown to express both integrins αVβ3 and αVβ6, whereas αVβ6 expression was not detected on the RD cells. Native CAV9 but not RGE and RGDdel mutants bound efficiently to immobilized αVβ3 and αVβ6. Adhesion of CAV9 but not RGE/RGDdel to A549 cells was also significantly higher than to RD cells. In contrast, no affinity or adhesion of bacterially produced VP1 proteins to the integrins or to the cells was detected. Function-blocking antibodies against αV-integrins blocked CAV9 but not CAV9-RGDdel infectivity, indicating that the viruses use different internalization routes; this may explain the differential infection kinetics of CAV9 and RGDdel. In an affinity assay, soluble αVβ6, but not αVβ3, bound to immobilized CAV9. Similarly, only soluble αVβ6 blocked virus infectivity. These data suggest that CAV9 binding to αVβ6 is a high-affinity interaction, which may indicate its importance in clinical infections; this remains to be determined.
-
-
-
A newly recognized hantavirus in the Asian house rat (Rattus tanezumi) in Indonesia
More LessHantaviral sequences were recovered from the lung tissue of an Asian house rat (Rattus tanezumi) captured in Serang, Indonesia. Phylogenetic analysis of partial L, M and S segment sequences showed that they belonged to a novel hantavirus provisionally named Serang virus (SERV). Notably, SERV is distinct from the hantaviruses associated with rodents of the species Rattus: Seoul virus associated with Rattus norvegicus worldwide and Gou virus isolated from Rattus rattus in China. Instead SERV appeared more closely related to Thailand virus (THAIV) carried by the great bandicoot rat (Bandicota indica). These results suggest the possibility that SERV originated via host-switching, with a possible scenario of (pre)-THAIV ‘jumping’ from (pre)bandicoots to rats and colonizing this new host species.
-
-
-
Crimean-Congo hemorrhagic fever virus entry and replication is clathrin-, pH- and cholesterol-dependent
More LessTo date, the entry pathway and replication mechanisms for members of the family Bunyaviridae, and especially for Crimean-Congo hemorrhagic fever virus (CCHFV), are poorly understood. Considering the severity of disease and the widespread geographical occurrence of CCHFV, investigating viral entry is of great value for development of antivirals. In this study, we have shown that knockdown of clathrin by small interfering RNA significantly reduced CCHFV nucleocapsid protein and viral RNA levels, suggesting that CCHFV utilizes clathrin-dependent endocytosis. In contrast, caveolin-1, an important constituent of caveolae endocytosis, is not important in CCHFV infection. Moreover, treatment with drugs that are known to interfere with the formation of clathrin-coated pits (sucrose and chlorpromazine) or endosome acidification (bafilomycin A1 and NH4Cl) also supported a clathrin-dependent pathway in the entry process of CCHFV. Finally, we demonstrated that cholesterol depletion in the cell plasma membrane significantly inhibited CCHFV infection. In the presence of exogenous cholesterol, this process was reversed, suggesting that cholesterol is important in the life cycle of CCHFV.
-
-
-
Indigenous sources of 2007–2008 H5N1 avian influenza outbreaks in Thailand
Kridsada Chaichoune, Witthawat Wiriyarat, Arunee Thitithanyanont, Rassameepen Phonarknguen, Ladawan Sariya, Sarin Suwanpakdee, Thanom Noimor, Sunisa Chatsurachai, Prapat Suriyaphol, Kumnuan Ungchusak, Parntep Ratanakorn, Robert G. Webster, Mekkla Thompson, Prasert Auewarakul and Pilaipan PuthavathanaOutbreaks of H5N1 avian influenza show strong seasonality. It is not clear where the source of virus originates from in each new outbreak season. This study sought to understand the nature of viral resurgence in recent outbreak seasons in Thailand, where the epidemic is relatively well controlled. In such a situation, indigenous viruses surviving the inter-outbreak season would have to pass through a bottleneck. In order to look for evidence of the bottleneck effect, viral genome sequences from recent outbreaks in the country were analysed. H5N1 avian influenza viruses were isolated from six outbreaks in the rainy season and winter of 2007 through to early 2008. Most of the outbreaks were in the Yom–Nan River basin in the southern part of the northern region of the country. Sequences of these viral isolates were identified as clade 1, genotype Z, similar to viruses from previous years in the central region of the country. The sequences clustered into two groups, one of which was closely related to viruses isolated from the same area in July 2006. These analyses indicated that there was a strong bottleneck effect on the virus population and that only a few lineages remained in the area. In addition, evidence of reassortment among these viruses was found. These indicated re-emergence of viruses from a small pool of indigenous sources that had been silently perpetuated over the dry summer months. Therefore, an approach to eradicate H5N1 avian influenza from the area by eliminating these local reservoirs may be feasible and should be seriously considered.
-
-
-
Genetic characterization of orthobunyavirus Melao, strains BE AR633512 and BE AR8033, and experimental infection in golden hamsters (Mesocricetus auratus)
Melao virus (MELV) strains BE AR8033 and BE AR633512 were isolated from pools of Ochlerotatus scapularis mosquitoes in Belém, Pará State (1955), and Alta Floresta, Rondônia State (2000), Brazil, respectively. The aim of the present study was to molecularly characterize these strains and to describe the histopathological, biochemical and immunological changes in golden hamsters (Mesocricetus auratus) following intraperitoneal injection of MELV strains. Hamsters were susceptible to both of the MELV strains studied. Viraemia was observed 3–6 days post-infection (p.i.) for BE AR633512 and only on the second day p.i. for BE AR8033. Neutralizing antibodies against both strains were detected in blood samples obtained at 5 days p.i. and persisted up to 30 days p.i. Aspartate aminotransferase, alanine aminotransferase and blood urea nitrogen were significantly altered in animals infected with the two MELV strains, while creatinine was only altered in animals inoculated with BE AR633512. Histopathological changes were observed in the central nervous system, liver, kidney and spleen of hamsters, and infection was confirmed by detection of specific MELV antigens by immunohistochemistry. Strain BE AR633512 caused more severe tissue damage than strain BE AR8033, showing increased neurovirulence and pathogenicity. Genetic analysis based on the full-length sequences of the glycoprotein (Gn and Gc) and nucleocapsid protein (N) genes revealed high levels of homology between the MELV strains. Interestingly, the greatest genetic divergence was found for the Gn gene of strain BE AR633512, in which several synonymous and non-synonymous mutations causing changes in RNA secondary structure were observed. Further studies will be necessary to investigate the role of Gn and Gc mutations in the MELV pathogenicity.
-
-
-
Cyanovirin-N potently inhibits human immunodeficiency virus type 1infection in cellular and cervical explant models
More LessIn the absence of a protective vaccine against human immunodeficiency virus (HIV), there is an urgent need for the development of effective topical microbicides to prevent HIV infection. Candidate vaginal microbicides should provide protection against circulating strains, be cheap, stable on storage, safe and easy to use. Here we describe a detailed study of the safety and efficacy of Cyanovirin-N (CV-N) in vitro, and in an ex vivo model of female genital tissue explants. CV-N demonstrated potent activity in the low nanomolar range against laboratory and primary isolates. Activity was related to the affinity of CV-N for binding to whole virions as determined by acoustic resonance. Potent activity was also observed against cell-associated HIV-1, although slightly reduced. CV-N activity in the presence of whole semen was reduced by 7–10-fold, although it remained in the low nanomolar range and was minimally modified by the presence of Candida albicans. Furthermore, CV-N potently inhibited infection of ectocervical explants and virus dissemination by tissue-emigrating cells. In peripheral blood mononuclear cell (PBMC) assays, CV-N was shown to have some mitogenic activity following 3 days exposure to compound, and this was associated with a modest increase in expression of gamma interferon, stromal cell-derived factor 1β and interleukin 4. However, 2 h exposure to CV-N had no effect on cytokine expression in PBMC or tissue explant culture over a 24 h period, suggesting that the potential for inflammation is low. Data presented here indicate that targeting HIV envelope glycoproteins may provide an effective strategy to prevent HIV-1 infection mediated by either cell-free virus or infected cells.
-
- DNA viruses
-
-
Analysis of the subcellular trafficking properties of murine cytomegalovirus M78, a 7 transmembrane receptor homologue
More LessMurine cytomegalovirus (MCMV) M78 is a member of the betaherpesvirus ‘UL78 family’ of seven transmembrane receptor (7TMR) genes. Previous studies of M78 and its counterpart in rat cytomegalovirus (RCMV) have suggested that these genes are required for efficient cell–cell spread of their respective viruses in tissue culture and demonstrated that gene knockout viruses are significantly attenuated for replication in vivo. However, in comparison with other CMV 7TMRs, relatively little is known about the basic biochemical properties and subcellular trafficking of the UL78 family members. We have characterized MCMV M78 in both transiently transfected and MCMV-infected cells to determine whether M78 exhibits features in common with cellular 7TMR. We obtained preliminary evidence that M78 formed dimers, a property that has been reported for several cellular 7TMR. M78 traffics to the cell surface, but was rapidly and constitutively endocytosed. Antibody feeding experiments demonstrated co-localization of M78 with markers for both the clathrin-dependent and lipid raft/caveolae-mediated internalization pathways. In MCMV-infected cells, the subcellular localization of M78 was modified during the course of infection, which may be related to the incorporation of M78 into the virion envelope during the course of virion maturation.
-
-
-
Upstream-binding factor is sequestered into herpes simplex virus type 1 replication compartments
More LessPrevious reports have shown that adenovirus recruits nucleolar protein upstream-binding factor (UBF) into adenovirus DNA replication centres. Here, we report that despite having a different mode of viral DNA replication, herpes simplex virus type 1 (HSV-1) also recruits UBF into viral DNA replication centres. Moreover, as with adenovirus, enhanced green fluorescent protein-tagged fusion proteins of UBF inhibit viral DNA replication. We propose that UBF is recruited to the replication compartments to aid replication of HSV-1 DNA. In addition, this is a further example of the role of nucleolar components in viral life cycles.
-
-
-
Herpes simplex virus infection is sensed by both Toll-like receptors and retinoic acid-inducible gene- like receptors, which synergize to induce type I interferon production
The innate antiviral response is initiated by pattern recognition receptors, which recognize viral pathogen-associated molecular patterns. Here we show that retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) in cooperation with Toll-like receptor (TLR) 9 is required for expression of type I interferons (IFNs) after infection with herpes simplex virus (HSV). Our work also identified RNase L as a critical component in IFN induction. Moreover, we found that TLR9 and RLRs activate distinct, as well as overlapping, intracellular signalling pathways. Thus, RLRs are important for recognition of HSV infection, and cooperate with the Toll pathway to induce an antiviral response.
-
-
-
Cytokine production by human herpesvirus 8-infected dendritic cells
More LessWe have shown previously that human herpesvirus 8 (HHV-8)-infected dendritic cells (DCs) undergo incomplete maturation and have a defective antigen-presenting function. Here, we examined the effects of HHV-8 infection on cytokine production, which is critical to the function of DCs. We detected expression of interleukin (IL)-6, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES and IL-12p40 from 2 to 6 h post-infection, and these peaked by 15–24 h. Expression of these factors decreased 24–48 h post-infection, with the exception of TNF-α which remained high throughout the entire 72 h. Interestingly, while IL-12p40 expression increased post-infection, bioactive IL-12p70 was not detected in the supernatants. These results suggest an intentional skewing of cytokine production in HHV-8-infected DCs towards induction of a Th2 response.
-
-
-
Adenovirus DNA polymerase is recognized by human CD8+ T cells
Donor lymphocytes have potential as a treatment for adenovirus (Ad) disease in haematopoietic stem cell transplant (SCT) recipients, but better understanding of Ad-specific T-cell responses is required. Most healthy adults exhibit memory T-cell responses to hexon, a capsid protein synthesized late after infection. However, since the Ad E3-19k downregulates major histocompatibility complex (MHC) class I molecules, cytotoxic T cells (CTLs) targeted to early viral proteins may be more effective in eliminating Ad-infected cells in vivo. Here we show that Ad-specific CTLs recognize the early region 2 proteins DNA polymerase (Pol) and DNA-binding protein (DBP). Firstly, memory Ad-specific CD8+ T cells were amplified from healthy donors by in vitro stimulation with Ad-infected dendritic cells and found to exhibit MHC-restricted cytotoxicity to targets expressing Pol and DBP. Secondly, gamma interferon responses to HLA A2-binding motif peptides from Pol and DBP were directly detected in peripheral blood mononuclear cells (PBMCs) from a recently infected normal donor. Peptide-specific CTLs generated to Pol and DBP epitopes were confirmed to exhibit HLA A2-restricted killing of targets expressing Pol or DBP. Lastly, Pol-epitope-specific T cells were detected at similar or higher frequencies than hexon and DBP in three of three SCT recipients recovering from invasive Ad disease. Pol epitopes were well conserved among different Ad serotypes. Therefore, Pol is a promising target for immunotherapy of Ad disease.
-
-
-
Adenovirus type 5 E4 Orf3 protein targets promyelocytic leukaemia (PML) protein nuclear domains for disruption via a sequence in PML isoform II that is predicted as a protein interaction site by bioinformatic analysis
More LessHuman adenovirus type 5 infection causes the disruption of structures in the cell nucleus termed promyelocytic leukaemia (PML) protein nuclear domains or ND10, which contain the PML protein as a critical component. This disruption is achieved through the action of the viral E4 Orf3 protein, which forms track-like nuclear structures that associate with the PML protein. This association is mediated by a direct interaction of Orf3 with a specific PML isoform, PMLII. We show here that the Orf3 interaction properties of PMLII are conferred by a 40 aa residue segment of the unique C-terminal domain of the protein. This segment was sufficient to confer interaction on a heterologous protein. The analysis was informed by prior application of a bioinformatic tool for the prediction of potential protein interaction sites within unstructured protein sequences (predictors of naturally disordered region analysis; PONDR). This tool predicted three potential molecular recognition elements (MoRE) within the C-terminal domain of PMLII, one of which was found to form the core of the Orf3 interaction site, thus demonstrating the utility of this approach. The sequence of the mapped Orf3-binding site on PML protein was found to be relatively poorly conserved across other species; however, the overall organization of MoREs within unstructured sequence was retained, suggesting the potential for conservation of functional interactions.
-
-
-
Yemen and Vietnam capripoxviruses demonstrate a distinct host preference for goats compared with sheep
Sheeppox and goatpox are caused by viruses that are members of the genus Capripoxvirus, and globally result in significant production losses. To improve the understanding of disease pathogenesis and evaluate host species preferences, sheep and goats were inoculated either with a capripoxvirus isolate from Yemen or from a recent outbreak in Vietnam. Blood, swabs and tissues were collected at various time points following experimental challenge and assessed for viral DNA content using real-time PCR and infectivity using virus isolation. The Yemen isolate was considerably more pathogenic in goats with 100 % mortality and morbidity compared with sheep with 0 % mortality and 100 % morbidity. The Vietnam isolate was also more pathogenic in goats with 100 % morbidity and an estimated 33 % mortality rate compared with mild morbidity and a 0 % mortality rate in sheep. Higher viral titres were observed in nasal, oral and conjunctival swabs from goats inoculated with either the Yemen or Vietnam isolate compared with those collected from sheep. Although the highest viral titres were detected in primary and secondary skin lesions in sheep and goats, the severity of clinical disease observed in each species varied according to the inoculum used. Whereas both the Yemen and Vietnam isolates clearly caused more severe disease in goats, the Yemen isolate was also moderately pathogenic in sheep. The Vietnam isolate, in contrast, caused only very mild disease in sheep. Limited DNA sequencing revealed ORF 074 of the Vietnam isolate to be identical to that of several goatpox virus isolates from China, suggesting a possible Chinese origin.
-
-
-
Hepatitis B virus (HBV)-specific short hairpin RNA is capable of reducing the formation of HBV covalently closed circular (CCC) DNA but has no effect on established CCC DNA in vitro
More LessHepatitis B virus (HBV) covalently closed circular (CCC) DNA is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA, thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA)-expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication, and the levels of HBV RNA, HBV surface antigens (HBsAg) and replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced until day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have a therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA following antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication.
-
-
-
Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells
More LessThe recently described hepatic cell line HepaRG is the sole hepatoma cell line susceptible to hepatitis B virus (HBV) infection. It provides a unique tool for investigating some unresolved issues of the virus' biology, particularly the formation of the viral mini-chromosome believed to be responsible for the persistence of infection. In this study, we characterized the main features of HBV infection: it is restricted to a subpopulation of differentiated hepatocyte-like cells that express albumin as a functional marker and represents around 10 % of all differentiated HepaRG cells. Infection may persist for more than 100 days in cells maintained at the differentiated state. Even though infected cells continued to produce infectious viral particles, very limited or no spreading of infection was observed. Low genetic variation was also observed in the viral DNA from viruses found in the supernatant of infected cells, although this cannot explain the lack of reinfection. HBV infection of HepaRG cells appears to be a very slow process: viral replication starts at around day 8 post-infection and reaches a maximum at day 13. Analysis of viral DNA showed slow and inefficient conversion of the input relaxed circular DNA into covalently closed circular (CCC) DNA, but no further amplification. Continuous lamivudine treatment inhibited viral replication, but neither prevented viral infection nor initial formation of CCC DNA. In conclusion, HBV infection in differentiated HepaRG cells is characterized by long-term persistence without a key feature of hepadnaviruses, the so-called ‘CCC DNA amplification’ described in the duck hepatitis B model.
-
-
-
Serological relationship between cutaneous human papillomavirus types 5, 8 and 92
Evidence of a possible association of cutaneous human papillomavirus (HPV) types, especially members of the genus Betapapillomavirus, and the development of non-melanoma skin cancer (NMSC) is accumulating. Vaccination with virus-like particles (VLPs) consisting of self-assembled L1, the major capsid protein, has been introduced to control anogenital HPV infection. This study examined the serological relationship between betapapillomavirus (β-PV) types 5 and 8 and the new type HPV-92, which has recently been isolated from a basal cell carcinoma containing a high number of viral genomes. Following expression by recombinant baculoviruses, the L1 protein of HPV-92 self-assembled into VLPs that elicited high-titre antibodies after immunization, similar to VLPs from HPV-5 and -8. Haemagglutination inhibition (HAI) assays were used as a surrogate method for the detection of virion-neutralizing antibodies, which correlates with protection from infection. Antisera raised against HPV-5 and -8 VLPs displayed HAI activity not only against the homologous type, but also against heterologous HPV types 5, 8 and 92, whereas HAI activity of antisera against HPV-92 VLP was restricted to the homologous type. The results of neutralization assays using HPV-5 pseudovirions were consistent with those from HAI assays. Cross-neutralizing immune responses by VLP vaccination against heterologous HPV types may provide broader protection against the multiplicity of HPV types detected in NMSC. If a close link to HPV infection can be conclusively established, these results may provide a basis for further evaluation of VLPs of β-PVs as candidates for a prophylactic skin-type HPV vaccine, aimed at reducing the incidence of NMSC.
-
-
-
Distribution patterns of BK polyomavirus (BKV) subtypes and subgroups in American, European and Asian populations suggest co-migration of BKV and the human race
BK polyomavirus (BKV) is ubiquitous in the human population, infecting children asymptomatically and then persisting in the kidney. Based on serological and genotyping methods, BKV isolates worldwide are classified into four subtypes (I–IV), with subtype I prevalent throughout the world, subtype IV prevalent in Asia and part of Europe, and subtypes II and III rare throughout the world. Phylogenetic analyses of complete genome sequences have identified several geographically distinct subgroups of subtypes I and IV. To explain how the geographical distribution patterns of BKV subtypes and subgroups were formed, this study hypothesized that BKV co-migrated with human populations (the co-migration hypothesis), and examined this hypothesis by comparing the BKV subtype and subgroup profiles among two American populations in North-east USA and southern California, two European populations in Finland and Ireland/England, and two Asian populations in Japan and China (both American populations were composed mainly of European Americans). The frequency of subtype I was always the highest throughout the populations, but that of subtype IV was variable among populations. A subgroup of subtype I (I/b-2) was detected primarily in all of the European and American populations, whereas subgroup I/c was predominant in the Asian populations (the observed difference was statistically significant). Additionally, all of the five fully sequenced subtype IV isolates from the American and European populations belonged to subgroup IV/c-2, whereas all subtype IV isolates from the Asian populations belonged to the other subgroups. Collectively, the current findings provide support for the co-migration hypothesis.
-
-
-
Variants of open reading frame Bm126 in wild-type Bombyx mori nucleopolyhedrovirus isolates exhibit functional differences
The open reading frame (ORF) 126 (Bm126) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homologue of Ac150 and belongs to the baculovirus 11K protein family. Bm126 was amplified from BmNPVs isolated from five different regions of China. Sequence analysis showed that the isolates had two different subtypes of Bm126, Bm126-SX and Bm126-GD, and both were different from that of the BmNPV T3 isolate. All of the BM126 ORFs contained a hydrophobic N terminus and a C6 motif at their C terminus, but the sequence between the N terminus and C6 motif varied in each isolate. The function of Bm126 was studied using bacmid BmBacJS13 derived from a BmNPV containing Bm126-SX. A 3′ rapid amplification of cDNA ends showed that the transcript of Bm126 was first detected at 6 h post-infection. A Bm126-knockout bacmid was constructed in which the majority of the coding region of Bm126 was deleted. Subsequently, the gene was repaired with Bm126-SX or Bm126-GD and tested for infectivity. The deletion of Bm126 had no obvious effect on the budded virus growth curve and the mean lethal dose of the occlusion bodies (OBs); however, the mean survival time of the larvae infected with Bm126-null virus was significantly delayed compared with that of the control virus. The delay was rescued by repairing the deletion with Bm126-SX but not with Bm126-GD. In addition, the virus repaired with Bm126-GD showed a significant increase in OB yield, both in vitro and in vivo.
-
-
-
Bombyx mori nucleopolyhedrovirus ORF9 is a gene involved in the budded virus production and infectivity
The ORF9 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm9) is conserved in all completely sequenced lepidopteran nucleopolyhedroviruses. RT-PCR analysis demonstrated that Bm9 is an early and late transcribed gene that is initiated at 3 h post-infection, and immunofluorescence microscopy showed that Bm9 is localized mainly in the cytoplasm of infected cells. To determine the role of Bm9 during virus infection, Bm9 was knocked out by recombination in a BmNPV genome propagated as a bacmid in Escherichia coli. The budded virus (BV) production of Bm9-deleted bacmids was reduced more than 10-fold compared with wild-type (wt) bacmid; however, the kinetics of viral DNA replication were unaffected. The defect in BV production was recovered by the Bm9 rescue bacmid. In addition, electron microscope observations revealed that polyhedra formation was not affected by the deletion of Bm9. Bioassays showed that the Bm9-deleted bacmid took approximately 14–22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD50 was about 15 times higher than that of the wt bacmid. In conclusion, Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro.
-
-
-
N-linked glycans located in the pro-region of Bombyx mori nucleopolyhedrovirus V-CATH are essential for the proper folding of V-CATH and V-CHIA
More LessPost-mortem host degradation by infection of Bombyx mori nucleopolyhedrovirus (BmNPV) requires the synergistic activation of two virus-encoded genes, cathepsin (v-cath) and chitinase (v-chiA). Previous studies have suggested that V-CHIA is essential for the proper folding of the nascent V-CATH polypeptide in the endoplasmic reticulum, and that the putative V-CHIA–V-CATH interaction might be mediated by N-linked glycans of V-CATH. Sequence analysis shows that BmNPV V-CATH includes three consensus N-linked glycosylation sites (asparagine 38, 65 and 158). To clarify the role of N-linked glycans of V-CATH in its biological activity, we generated three recombinant BmNPVs expressing mutant V-CATHs, and found that the two residues, asparagine 38 and 65, which are localized in the pro-region of V-CATH, are the glycosylation sites of BmNPV V-CATH. Western blot analysis also showed that removal of N-linked glycans from BmNPV V-CATH resulted in production of the insoluble forms of V-CATH and V-CHIA. These results demonstrate that N-linked glycans located in the pro-region of BmNPV V-CATH are essential for the proper folding of V-CATH and V-CHIA.
-
- Plant
-
-
-
The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants
More LessThe prevalence of necrotic potato virus Y (PVY) in natural populations could reflect increased fitness of necrotic isolates. In this paper, the effects of the acquisition of molecular determinants (A/G2213 and A/C2271) involved in necrosis capacity on both the number of progeny produced and the competitiveness of PVY were characterized. The relationship between necrosis and fitness was tested using (i) Nicotiana tabacum cv. Xanthi and Nicotiana clevelandii, (ii) necrotic PVYN-605 and non-necrotic PVYO-139 isolates, (iii) single-mutated (PVYKR and PVYED) and double-mutated (PVYKRED) versions of PVYN-605 and (iv) three quantitative PCR assays specific for nt A2213, G2213 and A2271 of the PVY genome. The data demonstrated effects of both the genetic background and nt 2213 and 2271 on the fitness of PVY. Quantification of PVY RNA in singly infected plants revealed that both the PVYN-605 genetic background and the acquisition of necrotic capacity resulted in a decrease in the number of progeny produced. Competition experiments revealed that the genetic background of PVYN had a positive impact on competitiveness. In contrast, nucleotides involved in necrotic properties were associated with decreased fitness. Finally, in the host that did not respond to infection with necrosis, the benefit associated with the PVYN-605 genetic background was higher than the cost associated with the acquisition of molecular determinants involved in necrosis capacity. The opposite result was obtained in the host responding to the infection with necrosis. These results indicate that the emergence of necrotic isolates from a non-necrotic population is unlikely in tobacco.
-
-
-
-
Identification of sequence elements regulating promoter activity and replication of a monopartite begomovirus-associated DNA β satellite
More LessDNA β is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA β contains a single gene, βC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the β-glucuronidase reporter gene driven by a βC1 promoter-deletion series of the DNA β associated with cotton leaf curl Multan virus identified a 68 nt region (between −139 and −207) which is important for βC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the βC1 start codon. Mutation of the G-box resulted in a significant reduction in βC1 promoter activity and DNA β replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA β.
-
- Other Agents
-
-
-
Alteration of the biological and biochemical characteristics of bovine spongiform encephalopathy prions during interspecies transmission in transgenic mice models
More LessIn the interspecies transmission of prions, the species barrier influences the susceptibility of the host. Bovine spongiform encephalopathy (BSE) prions affect a wide range of host species but do not affect hamsters. In order to study this species barrier, this study analysed the transmissibility of BSE prions to several lines of transgenic (Tg) mice, including those expressing mouse and hamster chimeric prion proteins (MH2M and MHM2 mice). BSE prions were transmitted to tga20, MHM2 and ICR mice, and the incubation period was approximately 400 days. Thus, these mice were classified as ‘susceptible mice’. However, BSE prions were not transmitted to MH2M and TgHaNSE mice, and these mice were classified as ‘resistant mice’. After the BSE prions were passaged once in wild-type mice, they could be transmitted to resistant mice. The characteristics of the accumulated abnormal isoform of PrP (PrPSc) in susceptible and resistant mice were determined using Western blotting. A BSE-like glycoform pattern of PrPSc was detected in all of the susceptible mice using two different antibodies that recognized either the N- or the C-terminal end of the 27–30 kDa protease-resistant fragment of PrP (PrP27–30) as the epitope. In contrast, proteinase digestion followed by deglycosylation analysis showed that, in addition to PrP27–30, truncated PrPSc fragments existed in resistant mice. These mixed PrPSc fragments may have resulted from the adaptation of resistant mice to BSE prions.
-
-
-
-
Microinjection of lentiviral vectors expressing small interfering RNAs directed against laminin receptor precursor mRNA prolongs the pre-clinical phase in scrapie-infected mice
We examined therapeutic in vitro and in vivo approaches using lentivirus-based packaging of small interfering RNAs (siRNAs) targeting the non-integrin laminin receptor mRNA for treatment and prevention of prion disorders. Transfection of N2aSc+ cells with recombinant plasmids expressing three different siRNAs, significantly reduced both the LRP (laminin receptor precursor) and PrPSc levels by approximately 40–60 %. Stereotactic intracerebral microinjection of recombinant lentiviral vectors LVsiRNA-LRP 7 and 9 into the cortex of C57BL/6 wild-type mice resulted in a significant reduction of the LR levels in the cortex 15 days post-injection by 62 and 82 %, respectively. Intracerebral RML inoculation of C57BL/6 mice after microinjection with recombinant lentiviral vector LVsiRNA-LRP 7 into the hippocampus resulted in a significant reduction of both LRP and PrPSc levels by 36 and 41 %, respectively, concomitant with a significant prolongation of the pre-clinical phase. Lentiviral vectors expressing siRNAs targeting LRP mRNA represent a novel delivery system for the treatment of transmissible spongiform encephalopathies.
-
-
-
Pathogenic prion protein is degraded by a manganese oxide mineral found in soils
Prions, the aetiological agents of transmissible spongiform encephalopathies, exhibit extreme resistance to degradation. Soil can retain prion infectivity in the environment for years. Reactive soil components may, however, contribute to the inactivation of prions in soil. Members of the birnessite family of manganese oxides (MnO2) rank among the strongest natural oxidants in soils. Here, we report the abiotic degradation of pathogenic prion protein (PrPTSE) by a synthetic analogue of naturally occurring birnessite minerals. Aqueous MnO2 suspensions degraded the PrPTSE as evidenced by decreased immunoreactivity and diminished ability to seed protein misfolding cyclic amplification reactions. Birnessite-mediated PrPTSE degradation increased as a solution's pH decreased, consistent with the pH-dependence of the redox potential of MnO2. Exposure to 5.6 mg MnO2 ml−1 (PrPTSE : MnO2=1 : 110) decreased PrPTSE levels by ≥4 orders of magnitude. Manganese oxides may contribute to prion degradation in soil environments rich in these minerals.
-
- Jgv Direct
-
-
-
In vivo imaging of murid herpesvirus-4 infection
Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine herpesvirus-4 (MuHV-4). The recombinant virus strongly expressed luciferase in lytically infected cells without significant attenuation. We used it to compare different routes of virus inoculation. After intranasal infection of anaesthetized mice, luciferase was expressed in the nose and lungs for 7–10 days and in lymphoid tissue, most consistently the superficial cervical lymph nodes, for up to 30 days. Gastrointestinal infection was not observed. Intraperitoneal infection was very different to intranasal, with strong luciferase expression in the liver, kidneys, intestines, reproductive tract and spleen, but none in the nose or lungs. The nose has not previously been identified as a site of MuHV-4 infection. After intranasal infection of non-anaesthetized mice, it was the only site of non-lymphoid luciferase expression. Nevertheless, lymphoid colonization and persistence were still established, even at low inoculation doses. In contrast, virus delivered orally was very poorly infectious. Inoculation route therefore had a major impact on pathogenesis. Low dose intranasal infection without anaesthesia seems most likely to mimic natural transmission, and may therefore be particularly informative about normal viral gene functions.
-
-
-
-
Murine cytomegalovirus displays selective infection of cells within hours after systemic administration
More LessA distinctive feature of the cytomegaloviruses is their wide tissue tropism, demonstrated by the infection of many organs and cell types in an active infection. However, in experimental models of systemic infection, the earliest stages of infection are not well characterized, and it is unclear whether only certain cells are initially infected. Using a recombinant murine cytomegalovirus (MCMV) expressing green fluorescent protein (GFP), we tracked viral infection after systemic administration via intraperitoneal injection and showed that specific cells are infected within the first hours. We provide evidence that MCMV traffics as free virus from the peritoneal cavity into the mediastinal lymphatics, providing access to the bloodstream. We demonstrate that MCMV productively infected CD169+ subcapsular sinus macrophages in the mediastinal lymph nodes, ER-TR7+ CD29+ reticular fibroblasts in the spleen and hepatocytes. Infection in the spleen followed a distinctive pattern, beginning in the marginal zone at 6 h and spreading into the red pulp by 17 h. By 48 h after infection, there was widespread infection in the spleen and liver with degeneration of infected cells. In addition, infected dendritic cells appeared in the white pulp of the spleen at 48 h post-infection. On the other hand, cowpox virus showed a different pattern of infectivity in the spleen and liver. Thus, early MCMV infection produces a distinct pattern of infection of selective cells.
-
-
-
Partial molecular characterization of alphaherpesviruses isolated from tropical bats
Herpesviruses have previously been isolated from African and South-American bats. Recently, herpesviruses detected from European insectivorous bats (family Vespertilionidae) were classified molecularly as betaherpesviruses and gammaherpesviruses. In the current study, we performed PCR analyses targeting the UL30 catalytic subunit region of the DNA polymerase gene of the African and South American herpesviruses and new Malagasy and Cambodian herpesviruses isolated from bats, especially frugivorous bats from the families Pteropodidae and Phyllostomidae. The sequences obtained from the amplified products indicated that these isolates belonged to the genus Simplexvirus of the subfamily Alphaherpesvirinae. These results extend the taxonomic range of bat herpesviruses with the description of four members in the subfamily Alphaherpesvirinae. Furthermore, these data confirm and extend the geographical distribution of herpesvirus in bats to three more continents (Africa, South America and Asia) and indicate the presence of these viruses in frugivorous bats of the families Pteropodidae and Phyllostomidae.
-
-
-
CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells
Hepatitis C virus (HCV) infects cells by the direct uptake of cell-free virus following virus engagement with specific cell receptors such as CD81. Recent data have shown that HCV is also capable of direct cell-to-cell transmission, although the role of CD81 in this process is disputed. Here, we generated cell culture infectious strain JFH1 HCV (HCVcc) genomes carrying an alanine substitution of E2 residues W529 or D535 that are critical for binding to CD81 and infectivity. Co-cultivation of these cells with naïve cells expressing enhanced green fluorescent protein (EGFP) resulted in a small number of cells co-expressing both EGFP and HCV NS5A, showing that the HCVcc mutants are capable of cell-to-cell spread. In contrast, no cell-to-cell transmission from JFH1ΔE1E2-transfected cells occurred, indicating that the HCV glycoproteins are essential for this process. The frequency of cell-to-cell transmission of JFH1W529A was unaffected by the presence of neutralizing antibodies that inhibit E2–CD81 interactions. By using cell lines that expressed little or no CD81 and that were refractive to infection with cell-free virus, we showed that the occurrence of viral cell-to-cell transmission is not influenced by the levels of CD81 on either donor or recipient cells. Thus, our results show that CD81 plays no role in the cell-to-cell spread of HCVcc and that this mode of transmission is shielded from neutralizing antibodies. These data suggest that therapeutic interventions targeting the entry of cell-free HCV may not be sufficient in controlling an ongoing chronic infection, but need to be complemented by additional strategies aimed at disrupting direct cell-to-cell viral transmission.
-
Volumes and issues
-
Volume 105 (2024)
-
Volume 104 (2023)
-
Volume 103 (2022)
-
Volume 102 (2021)
-
Volume 101 (2020)
-
Volume 100 (2019)
-
Volume 99 (2018)
-
Volume 98 (2017)
-
Volume 97 (2016)
-
Volume 96 (2015)
-
Volume 95 (2014)
-
Volume 94 (2013)
-
Volume 93 (2012)
-
Volume 92 (2011)
-
Volume 91 (2010)
-
Volume 90 (2009)
-
Volume 89 (2008)
-
Volume 88 (2007)
-
Volume 87 (2006)
-
Volume 86 (2005)
-
Volume 85 (2004)
-
Volume 84 (2003)
-
Volume 83 (2002)
-
Volume 82 (2001)
-
Volume 81 (2000)
-
Volume 80 (1999)
-
Volume 79 (1998)
-
Volume 78 (1997)
-
Volume 77 (1996)
-
Volume 76 (1995)
-
Volume 75 (1994)
-
Volume 74 (1993)
-
Volume 73 (1992)
-
Volume 72 (1991)
-
Volume 71 (1990)
-
Volume 70 (1989)
-
Volume 69 (1988)
-
Volume 68 (1987)
-
Volume 67 (1986)
-
Volume 66 (1985)
-
Volume 65 (1984)
-
Volume 64 (1983)
-
Volume 63 (1982)
-
Volume 62 (1982)
-
Volume 61 (1982)
-
Volume 60 (1982)
-
Volume 59 (1982)
-
Volume 58 (1982)
-
Volume 57 (1981)
-
Volume 56 (1981)
-
Volume 55 (1981)
-
Volume 54 (1981)
-
Volume 53 (1981)
-
Volume 52 (1981)
-
Volume 51 (1980)
-
Volume 50 (1980)
-
Volume 49 (1980)
-
Volume 48 (1980)
-
Volume 47 (1980)
-
Volume 46 (1980)
-
Volume 45 (1979)
-
Volume 44 (1979)
-
Volume 43 (1979)
-
Volume 42 (1979)
-
Volume 41 (1978)
-
Volume 40 (1978)
-
Volume 39 (1978)
-
Volume 38 (1978)
-
Volume 37 (1977)
-
Volume 36 (1977)
-
Volume 35 (1977)
-
Volume 34 (1977)
-
Volume 33 (1976)
-
Volume 32 (1976)
-
Volume 31 (1976)
-
Volume 30 (1976)
-
Volume 29 (1975)
-
Volume 28 (1975)
-
Volume 27 (1975)
-
Volume 26 (1975)
-
Volume 25 (1974)
-
Volume 24 (1974)
-
Volume 23 (1974)
-
Volume 22 (1974)
-
Volume 21 (1973)
-
Volume 20 (1973)
-
Volume 19 (1973)
-
Volume 18 (1973)
-
Volume 17 (1972)
-
Volume 16 (1972)
-
Volume 15 (1972)
-
Volume 14 (1972)
-
Volume 13 (1971)
-
Volume 12 (1971)
-
Volume 11 (1971)
-
Volume 10 (1971)
-
Volume 9 (1970)
-
Volume 8 (1970)
-
Volume 7 (1970)
-
Volume 6 (1970)
-
Volume 5 (1969)
-
Volume 4 (1969)
-
Volume 3 (1968)
-
Volume 2 (1968)
-
Volume 1 (1967)