Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced 'medium-sized' EVs (CVB1i-mEVs). These mEVs (100-300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection.
Chikungunya virus (CHIKV) has caused large-scale epidemics of fever, rash and arthritis since 2004. This unprecedented re-emergence has been associated with mutations in genes encoding structural envelope proteins, providing increased fitness in the secondary vector Aedes albopictus. In the 2008-2013 CHIKV outbreaks across Southeast Asia, an R82S mutation in non-structural protein 4 (nsP4) emerged early in Malaysia or Singapore and quickly became predominant. To determine whether this nsP4-R82S mutation provides a selective advantage in host cells, which may have contributed to the epidemic, the fitness of infectious clone-derived CHIKV with wild-type nsP4-82R and mutant nsP4-82S were compared in Ae. albopictus and human cell lines. Viral infectivity, dissemination and transmission in Ae. albopictus were not affected by the mutation when the two variants were tested separately. In competition, the nsP4-82R variant showed an advantage over nsP4-82S in dissemination to the salivary glands, but only in late infection (10 days). In human rhabdomyosarcoma (RD) and embryonic kidney (HEK-293T) cell lines coinfected at a 1 : 1 ratio, wild-type nsP4-82R virus was rapidly outcompeted by nsP4-82S virus as early as one passage (3 days). In conclusion, the nsP4-R82S mutation provides a greater selective advantage in human cells than in Ae. albopictus, which may explain its apparent natural selection during CHIKV spread in Southeast Asia. This is an unusual example of a naturally occurring mutation in a non-structural protein, which may have facilitated epidemic transmission of CHIKV.
Tembusu virus (TMUV, genus Flavivirus, family Flaviviridae) was first isolated in 1955 from Culex tritaeniorhynchus mosquitoes in Kuala Lumpur, Malaysia. In April 2010, duck TMUV was first identified as the causative agent of egg-drop syndrome, characterized by a substantial decrease in egg laying and depression, growth retardation and neurological signs or death in infected egg-laying and breeder ducks, in the People's Republic of China. Since 2010, duck TMUV has spread to most of the duck-producing regions in China, including many of the coastal provinces, neighbouring regions and certain Southeast Asia areas (i.e. Thailand and Malaysia). This review describes the current understanding of the genome characteristics, host range, transmission, epidemiology, phylogenetic and immune evasion of avian-origin TMUV and the innate immune response of the host.
The ability of passage in HeLa cells to attenuate flaviviruses was investigated for three different strains of the mosquito-borne West Nile (WN) virus and two tick-borne viruses, louping-ill and Langat. One strain of WN virus, Sarawak, was attenuated 4000-fold for adult mice by intraperitoneal or intranasal challenge after six HeLa passages. The HeLa-passaged virus was also found to be antigenically different and temperature-sensitive in its growth characteristics compared with the parent. After six HeLa cell passages the Egypt 101 and Smithburn strains of WN virus lost their ability to infect monkey kidney cells and no longer killed adult mice, although inoculated animals became sick for several days. In contrast, two tick-borne flaviviruses remained as virulent for mice after six HeLa passages as the parent non-HeLa-passaged virus. Neither of the tick-borne viruses exhibited characteristics associated with temperature sensitivity. The results, therefore, indicate that the mosquito-borne, but not tick-borne, flaviviruses can be attenuated by very few passages in HeLa cells. This observation may provide a model system with which to analyse the molecular basis of attenuation and/or virulence of mosquito-borne flaviviruses.
Broadly neutralizing antibodies (bnAbs) have been considered to be potent therapeutic tools and potential vaccine candidates to enable protection against various clades of human immunodeficiency virus (HIV). The generation of bnAbs has been associated with enhanced exposure to antigen, high viral load and low CD4+ T cell counts, among other factors. However, only limited data are available on the generation of bnAbs in viraemic non-progressors that demonstrate moderate to high viraemia. Further, since HIV-1 subtype C viruses account for more than 50 % of global HIV infections, the identification of bnAbs with novel specificities is crucial to enable the development of potent tools to aid in HIV therapy and prevention. In the present study, we analysed and compared the neutralization potential of responses in 70 plasma samples isolated from ART-naïve HIV-1 subtype C-infected individuals with various disease progression profiles against a panel of 30 pseudoviruses. Among the seven samples that exhibited a neutralization breadth of ≥70 %, four were identified as 'elite neutralizers', and three of these were from viraemic non-progressors while the fourth was from a typical progressor. Analysis of the neutralization specificities revealed that none of the four elite neutralizers were reactive to epitopes in the membrane proximal external region (MPER), CD4-binding site and V1V2 or V3 glycan. However, two of the four elite neutralizers exhibited enhanced sensitivity towards viruses lacking N332 glycan, indicating high neutralization potency. Overall, our findings indicate that the identification of potent neutralization responses with distinct epitope specificities is possible from the as yet unexplored Indian population, which has a high prevalence of HIV-1 subtype C infection.
Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.
Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.
The complete nucleotide sequences are reported of two strains of echovirus 7, the prototype Wallace strain (Eo7-Wallace) and a recent Malaysian strain isolated from the cerebrospinal fluid of a child with fatal encephalomyelitis (Eo7-UMMC strain). The molecular findings corroborate the serological placement of the UMMC strain as echovirus 7. Both Eo7-Wallace and Eo7-UMMC belong to the species human enterovirus B and are most closely related to echovirus 11. Eo7-UMMC has undergone significant genetic drift from the prototype strain in the 47 years that separate the isolation of the two viruses. Phylogenetic analysis revealed that Eo7-UMMC did not arise from recombination with another enterovirus serotype. The molecular basis for the severely neurovirulent phenotype of Eo7-UMMC remains unknown. However, it is shown that mutations in the nucleotide sequence of the 5' untranslated region (UTR) of Eo7-UMMC result in changes to the putative structure of the 5' UTR. It is possible that these changes contribute to the neurovirulence of Eo7-UMMC.
We have completely sequenced the genomes of two Nipah virus (NiV) isolates, one from the throat secretion and the other from the cerebrospinal fluid (CSF) of the sole surviving encephalitic patient with positive CSF virus isolation in Malaysia. The two genomes have 18246 nucleotides each and differ by only 4 nucleotides. The NiV genome is 12 nucleotides longer than the Hendra virus (HeV) genome and both genomes have identical leader and trailer sequence lengths and hexamer-phasing positions for all their genes. Both NiV and HeV are also very closely related with respect to their genomic end sequences, gene start and stop signals, P gene-editing signals and deduced amino acid sequences of nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, glycoprotein and RNA polymerase. The existing evidence demonstrates a clear need for the creation of a new genus within the subfamily Paramyxovirinae to accommodate the close similarities between NiV and HeV and their significant differences from other members of the subfamily.
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 (alpha and beta). However, these two strains did not differ in their response to type II IFN (gamma). Although unable to suppress IFN signalling, TSV01 infection caused a weaker IFN-beta 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.
Apoptosis was detected in Vero cell cultures expressing transfected dengue virus type 2 (DENV-2) genes. Approximately 17.5 and 51.5 % of cells expressing NS3 serine protease and NS2B-NS3(185) serine protease precursor protein [NS2B-NS3(185)(pro)] genes, respectively, were apoptotic. The percentage of apoptotic cells was significantly higher in cell cultures expressing NS2B-NS3(185)(pro). NS2B-NS3(185)(pro) was detected as NS2B-NS3(185)(pro)-EGFP fusion protein in cytoplasmic vesicular structures in the apoptotic cells. Site-directed mutagenesis which replaced His(51) with Ala within the protease catalytic triad significantly reduced the ability of the expressed NS3 and NS2B-NS3(185)(pro) to induce apoptosis. Results from the present study showed that DENV-2-encoded NS3 serine protease induces apoptosis, which is enhanced in cells expressing its precursor, NS2B-NS3(185)(pro). These findings suggest the importance of NS2B as a cofactor to NS3 protease-induced apoptosis.
Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model.
Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130-DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130-DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130-DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130-DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130-DREAM complex.
Phylogenetic analyses of the envelope (E) gene sequence of five recently isolated dengue virus type 4 (DENV-4) suggested the emergence of a distinct geographical and temporal DENV-4 subgenotype IIA in Malaysia. Four of the isolates had direct ancestral lineage with DENV-4 Indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated DENV-4, MY01-22713. The E gene of isolate MY01-22713 had strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and genotype I Malaysia 1969. These results suggest that intra-serotypic recombination amongst DENV-4 from independent ancestral lineages may have contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.
The relationship between Epstein-Barr virus (EBV) and the germinal centre (GC) of the asymptomatic host remains an enigma. The occasional appearance of EBV-positive germinal centres in some patients, particularly those with a history of immunosuppression, suggests that EBV numbers in the GC are subject to immune control. The relationship, if any, between lymphoid hyperplasia with EBV-positive germinal centres and subsequent or concurrent lymphomagenesis remains to be clarified. As far as the development of EBV-associated Hodgkin's lymphoma is concerned, the suppression of virus replication, mediated by LMP1 on the one hand, and the loss of B-cell receptor signalling on the other, appears to be an important pathogenic mechanism. A further important emerging concept is that alterations in the microenvironment of the EBV-infected B-cell may be important for lymphomagenesis.
We previously described three new Malaysian orthoreoviruses designated Pulau virus, Melaka virus and Kampar virus. Melaka and Kampar viruses were shown to cause respiratory disease in humans. These viruses, together with Nelson Bay virus, isolated from Australian bats, are tentatively classified as different strains within the species Pteropine orthoreovirus (PRV), formerly known as Nelson Bay orthoreovirus, based on the small (S) genome segments. Here we report the sequences of the large (L) and medium (M) segments, thus completing the whole-genome characterization of the four PRVs. All L and M segments were highly conserved in size and sequence. Conserved functional motifs previously identified in other orthoreovirus gene products were also found in the deduced proteins encoded by the cognate segments of these viruses. Detailed sequence analysis identified two genetic lineages divided into the Australian and Malaysian PRVs, and potential genetic reassortment among the M and S segments of the three Malaysian viruses.
Japanese encephalitis virus (JEV), the prototype member of the JEV serocomplex, genus Flavivirus, family Flaviviridae, is the most significant arthropod-borne encephalitis worldwide in terms of morbidity and mortality. At least four genotypes (GI-GIV) of the virus have been identified; however, to date, the genomic nucleotide sequence of only one GII virus has been determined (FU strain, Australia, 1995). This study sequenced three additional GII strains of JEV isolated between 1951 and 1978 in Korea, Malaysia and Indonesia, respectively, and compared them with the FU strain, as well as with virus strains representing the other three genotypes. Based on nucleotide and amino acid composition, the genotype II strains were the most similar to GI strains; however, these two genotypes are epidemiologically distinct. Selection analyses revealed that the strains utilized in this study are under predominantly purifying selection, and evidence of positive selection was detected at aa 24 of the NS4B protein, a protein that functions as an alpha/beta interferon signalling inhibitor.
The full-length genomes of two DENV-1 viruses isolated during the 2005-2006 dengue incidents in Brunei were sequenced. Twenty five primer sets were designed to amplify contiguous overlapping fragments of approximately 500-600 base pairs spanning the entire sequence of the genome. The amplified PCR products were sent to a commercial laboratory for sequencing and the nucleotides and the deduced amino acids were determined. Sequence analysis of the envelope gene at the nucleotide and amino acid levels between the two isolates showed 92 and 96 % identity, respectively. Comparison of the envelope gene sequences with 68 other DENV-1 viruses of known genotypes placed the two isolates into two different genotypic groups. Isolate DS06/210505 belongs to genotype V together with some of the recent isolates from India (2003) and older isolates from Singapore (1990) and Burma (1976), while isolate DS212/110306 was clustered in genotype IV with the prototype Nauru strain (1974) and with some of the recent isolates from Indonesia (2004) and the Philippines (2002, 2001). In the full-length genome analysis at the nucleotide level, isolate DS06/210505 showed 94 % identity to the French Guyana strain (1989) in genotype V while isolate DS212/110306 had 96 % identity to the Nauru Island strain (1974) in genotype IV. This work constitutes the first complete genetic characterization of not only Brunei DENV-1 virus isolates, but also the first strain from Borneo Island. This study was the first to report the isolation of dengue virus in the country.
Forty-six strains of Japanese encephalitis (JE) virus from a variety of geographic areas in Asia were examined by primer-extension sequencing of the RNA template. A 240 nucleotide sequence from the pre-M gene region was selected for study because it provided sufficient information for determining genetic relationships among the virus isolates. Using 12% divergence as a cutoff point for virus relationships, the 46 isolates fell into three distinct genotypic groups. One genotypic group consisted of JE virus isolates from northern Thailand and Cambodia. A second group was composed of isolates from southern Thailand, Malaysia, Sarawak and Indonesia. The remainder of the isolates, from Japan, China, Taiwan, the Philippines, Sri Lanka, India and Nepal, made up a third group. The implications of these findings in relation to the epidemiology of JE are discussed. Results of this study demonstrate that the comparison of short nucleotide sequences can provide insight into JE virus evolution, transmission and, possibly, pathogenesis.
Sagiyama virus (SAG) is a member of the genus Alphavirus in the family Togaviridae, isolated in Japan from mosquitoes in 1956. We determined the complete nucleotide sequence of the SAG genomic RNA from the original stock virus which formed a mixture of plaques with different sizes, and that from a full-length cDNA clone, pSAG2, infectious RNA transcripts from which formed uniform large plaques on BHK-21 cells. The SAG genome was 11698 nt in length exclusive of the 3' poly(A) tail. Between the complete nucleotide sequences of the full-length cDNA clone, pSAG2, and the consensus sequence from the original stock virus, there were nine amino acid differences; two each in nsP1, nsP2 and E1, and three in E2, some of which may be responsible for plaque phenotypic variants in the original virus stock. SAG was most closely related to Ross River virus among other alphaviruses fully sequenced, with amino acid sequence identities of 86% in the nonstructural proteins and of 83% in the structural proteins. The 3' terminal 280 nt region of SAG was 82% identical to that of Barmah Forest virus, which was otherwise not closely related to SAG. Comparison of the nucleotide sequence of SAG with partial nucleotide sequences of Getah virus (GET), which was originally isolated in Malaysia in 1955 and is closely related to SAG in serology and in biology, showed near identity between the two viruses, suggesting that SAG is a strain of GET.