Displaying publications 1 - 20 of 30 in total

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  1. Sequences of E/NS1 gene junction from four dengue-2 viruses of northeastern Thailand and their evolutionary relationships with other dengue-2 viruses
    Zin K, Morita K, Igarashi A
    Microbiol. Immunol., 1995;39(8):581-90.
    PMID: 7494497
    We determined the 240-nucleotide sequences of the E/NS1 gene junction of four dengue-2 viruses by the primer extension dideoxy chain termination method. These viruses were isolated from dengue patients with different clinical severities in Nakhon Phanom, Northeastern Thailand in 1993. The results were compared with the 52 published dengue-2 sequences of the same gene region. Sequence divergence of four new isolates varied from 4.17% to 5.42% compared with dengue-2 prototype New Guinea C strain whereas it varied from 5.42% to 6.67% and from 6.67% to 7.09% when compared with Jamaica 1409 strain and PR159/S1 strain, respectively. All nucleotide substitutions were found at the 3rd position of the codons which were silent mutations. All 56 isolates studied were classified into five genotypic groups by constructing the dendrogram. The results indicated that four new isolates from Northeastern Thailand belong to genotype II of dengue virus serotype 2, and were most closely related to prototype New Guinea C strain. We also observed the variation in nucleotide and amino acid sequences among clusters of isolates (Thailand-1980, Malaysia-1989 and Thailand-1993) which were obtained from the dengue patients with different clinical severities. The significance of these genetic differences have been discussed in terms of the possible correlation between genetic variability and virulence.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  2. Fulltext Isolation and molecular detection of dengue and chikungunya virus from field-collected adult mosquitoes in Kelantan, Malaysia
    Dissanayake DS, Wijekoon CD, Wegiriya H
    J Vector Borne Dis, 2024 Jan 01;61(1):564-573.
    PMID: 38648407 DOI: 10.4103/0972-9062.392269
    BACKGROUND OBJECTIVES: Dengue and chikungunya infections are one of the major health problems that have plagued the human population globally. All dengue virus (DENV) serotypes circulate within Malaysia with particular serotypes dominating in different years/outbreaks. In the state of Kelantan, an increasing number of DENV and chikungunya virus (CHIKV) new cases have been reported, including several deaths. This study aimed to isolate and detect these arboviruses from adult mosquitoes in Kelantan.

    METHODS: Adult mo squito samples were collected from January to August 2019 and were identified according to gender, species and locality. The isolation of the virus was done in C6/36 cells. Dengue NS1 antigen was carried out using direct mosquito lysate and mosquito culture supernatant. Detection and serotyping of the DENV was performed using multiplex RT-PCR and CHIKV detection using a one-step RT-PCR assay.

    RESULTS: Of 91 mosquito pools, four were positive for NS1 antigen comprising two pools (2.2%) of male Ae. albopictus (Pulau Melaka and Kubang Siput) and two pools (2.2%) of Ae. aegypti (Kampung Demit Sungai). DENV 1 was detected in one pool (0.9%) of female Ae. albopictus among 114 tested Aedes pools. Two pools of 114 pools (1.7%) from both male Aedes species were positive with double serotypes, DENV 1 and DENV 2 (Pulau Melaka). However, no pool was positive for CHIKV.

    INTERPRETATION CONCLUSION: The presence of DENV and the main vectors of arboviruses in Kelantan are pertinent indicators of the need to improve vector controls to reduce arbovirus infections among people in the localities.

    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  3. Fulltext Clinical diagnosis of early dengue infection by novel one-step multiplex real-time RT-PCR targeting NS1 gene
    Kim JH, Chong CK, Sinniah M, Sinnadurai J, Song HO, Park H
    J Clin Virol, 2015 Apr;65:11-9.
    PMID: 25766980 DOI: 10.1016/j.jcv.2015.01.018
    BACKGROUND: Dengue is a mosquito-borne disease that causes a public health problem in tropical and subtropical countries. Current immunological diagnostics based on IgM and/or nonstructural protein 1 (NS1) antigen are limited for acute dengue infection due to low sensitivity and accuracy.
    OBJECTIVES: This study aimed to develop a one-step multiplex real-time RT-PCR assay showing higher sensitivity and accuracy than previous approaches.
    STUDY DESIGN: Serotype-specific primers and probes were designed through the multiple alignment of NS1 gene. The linearity and limit of detection (LOD) of the assay were determined. The assay was clinically validated with an evaluation panel that was immunologically tested by WHO and Malaysian specimens.
    RESULTS: The LOD of the assay was 3.0 log10 RNA copies for DENV-1, 2.0 for DENV-3, and 1.0 for DENV-2 and DENV-4. The assay showed 95.2% sensitivity (20/21) in an evaluation panel, whereas NS1 antigen- and anti-dengue IgM-based immunological assays exhibited 0% and 23.8-47.6% sensitivities, respectively. The assay showed 100% sensitivity both in NS1 antigen- and anti-dengue IgM-positive Malaysian specimens (26/26). The assay provided the information of viral loads and serotype with discrimination of heterotypic mixed infection.
    CONCLUSIONS: The assay could be clinically applied to early dengue diagnosis, especially during the first 5 days of illness and approximately 14 days after infection showing an anti-dengue IgM-positive response.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  4. An amino acid change in nsP4 of chikungunya virus confers fitness advantage in human cell lines rather than in Aedes albopictus
    Fu JYL, Chua CL, Vythilingam I, Sulaiman WYW, Wong HV, Chan YF, et al.
    J Gen Virol, 2019 11;100(11):1541-1553.
    PMID: 31613205 DOI: 10.1099/jgv.0.001338
    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.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  5. Fulltext Sequence analysis of E/NS1 gene junction of dengue virus type 2 isolated in Brunei
    Osman O, Fong MY, Devi S
    Southeast Asian J Trop Med Public Health, 2008 Jan;39(1):62-78.
    PMID: 18567445
    A preliminary study of dengue infection in Brunei between 2005 and 2006 showed that dengue 2 was the predominant serotype. A total of five DEN-2 isolates were isolated and maintained in the mosquito cell-line, albopictus C6/36. The sequence spanning the envelope and non-structural protein 1 (E/NS1) junction (positions 2311 to 2550) of the isolates were determined and analysed at the amino acid and nucleotide levels. Alignment of the 240 nucleotide sequences among the five isolates showed changes occurring at 7 positions (2.9%) of the region. All but one nucleotide substitution (position 2319, amino acid 742 V --> F) were found at the 3rd position of the codons and were silent mutations. Amino acid homology ranged from 98% to 100%. Sequence divergence of the Brunei isolates varied from 5% to 6.6% compared with dengue-2 prototype New Guinea C strain. Comparison of the Brunei DEN-2 isolates with sixty-five other strains placed them in a cluster containing Indonesian strains isolated in 1973, 1978 and 2004 and Malaysian strains isolated in 1996, 1998 and 1999 in genotype group IV.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  6. Fulltext The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen
    Teoh BT, Sam SS, Tan KK, Johari J, Abd-Jamil J, Hooi PS, et al.
    Sci Rep, 2016 06 09;6:27663.
    PMID: 27278716 DOI: 10.1038/srep27663
    Timely and accurate dengue diagnosis is important for differential diagnosis and immediate implementation of appropriate disease control measures. In this study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n = 375). The NS1 Ag Strip and qRT-PCR showed a fair concordance (κ = 0.207, p = 0.001). While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2-94.8%) than in those from primary dengue (21.7-64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  7. Comparative analysis of NS3 sequences of temporally separated dengue 3 virus strains isolated from southeast Asia
    Chow VT, Seah CL, Chan YC
    Intervirology, 1994;37(5):252-8.
    PMID: 7698880
    By a combination of PCR and direct-cycle sequencing using consensus primers, we analyzed approximately 400-bp fragments within the NS3 genes of twenty-one dengue virus type 3 strains isolated from five neighboring Southeast Asian countries at different time intervals from 1956 to 1992. The majority of base disparities were silent mutations, with few predicted amino acid substitutions, thus emphasizing the strict conservation of the NS3 gene. Phylogenetic trees constructed on the basis of these nucleotide differences revealed distinct but related clusters of strains from the Philippines, Indonesia, and strains from Singapore and Malaysia of the 1970s and early 1980s, while the Thai cluster was relatively more distant. This genetic relationship was compatible with that proposed by other workers who have studied other dengue 3 virus genes such as E, M and prM. However, we observed that the more recent, epidemic-associated dengue 3 strains from Singapore and Malaysia of the late 1980s and early 1990s were more closely related to the Thai cluster, implying their evolution from the latter, and emphasizing the importance of viral spread via increasing travel within the Southeast Asian area and elsewhere. Nucleotide sequence analysis of the NS3 genes of dengue viruses can serve to advance the understanding of the epidemiology and evolution of these viruses.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  8. Molecular epidemiology of Malaysian dengue 2 viruses isolated over twenty-five years (1968-1993)
    Fong MY, Koh CL, Lam SK
    Res. Virol., 1998 Nov-Dec;149(6):457-64.
    PMID: 9923022
    The limited sequencing approach was used to study the molecular epidemiology of 24 Malaysian dengue 2 viruses which were isolated between 1968 and 1993. The sequences of a 240-nucleotide-long region across the envelope/non-structural 1 protein (E/NS1) gene junction of the isolates were determined and analysed. Alignment and comparison of the nucleotide and deduced amino acid sequences of the isolates revealed that nucleotide changes occurred mostly at the third position of a particular codon and were of the transition (AG, CU) type. Five nucleotide changes resulted in amino acid substitutions. Pairwise comparisons of the nucleotide sequences gave divergence values ranging from 0 to 9.2%. At the amino acid level, the divergence ranged between 0 and 3.8%. Based on the 6% divergence as the cut-off point for genotypic classification, the isolates were grouped into two genotypes, I and II. Comparison of the nucleotide sequences of the Malaysian dengue isolates with those of the dengue viruses of other regions of the world revealed that members of genotypes I and II were closely related to viruses from the Indian Ocean and Western Pacific regions, respectively.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  9. Expression of surface-bound nonstructural 1 (NS1) protein of influenza virus A H5N1 on Lactobacillus casei strain C1
    Tan TS, Syed Hassan S, Yap WB
    Lett Appl Microbiol, 2017 Jun;64(6):446-451.
    PMID: 28370088 DOI: 10.1111/lam.12738
    The study aimed to construct a recombinant Lactobacillus casei expressing the nonstructural (NS) 1 protein of influenza A virus H5N1 on its cell wall. The NS1 gene was first amplified and fused to the pSGANC332 expression plasmid. The NS1 protein expression was carried out by Lact. casei strain C1. PCR screening and DNA sequencing confirmed the presence of recombinant pSG-NS1-ANC332 plasmid in Lact. casei. The plasmid was stably maintained (98·94 ± 1·65%) by the bacterium within the first 20 generations without selective pressure. The NS1 was expressed as a 49-kDa protein in association with the anchoring peptide. The yield was 1·325 ± 0·065 μg mg(-1) of bacterial cells. Lactobacillus casei expressing the NS1 on its cell wall was red-fluorescently stained, but the staining was not observed on Lact. casei carrying the empty pSGANC332. The results implied that Lact. casei strain C1 is a promising host for the expression of surface-bound NS1 protein using the pSGANC332 expression plasmid.

    SIGNIFICANCE AND IMPACT OF THE STUDY: The study has demonstrated, for the first time, the expression of nonstructural 1 (NS1) protein of influenza A virus H5N1 on the cell wall of Lactobacillus casei using the pSGANC332 expression plasmid. Display of NS1 protein on the bacterial cell wall was evident under an immunofluorescence microscopic observation. Lactobacillus casei carrying the NS1 protein could be developed into a universal oral influenza vaccine since the NS1 is highly conserved among influenza viruses.

    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  10. Admission Clinicopathological Factors Associated with Prolonged Hospital Stay Among Hospitalized Patients with Dengue Viral Infections
    Willeam Peter SS, Hassan SS, Khei Tan VP, Ngim CF, Azreen Adnan NA, Pong LY, et al.
    Vector Borne Zoonotic Dis, 2019 07;19(7):549-552.
    PMID: 30668248 DOI: 10.1089/vbz.2018.2379
    Background:
    There is an escalation of frequency and magnitude of dengue epidemics in Malaysia, with a concomitant increase in patient hospitalization. Prolonged hospitalization (PH) due to dengue virus (DENV) infections causes considerable socioeconomic burden. Early identification of patients needing PH could optimize resource consumption and reduce health care costs. This study aims to identify clinicopathological factors present on admission that are associated with PH among patients with DENV infections.
    Methods:
    This study was conducted in a tertiary referral hospital in Southern Malaysia. Relevant clinical and laboratory data upon admission were retrieved from medical records of 253 consecutive DENV nonstructural protein 1 (NS1) antigen and PCR-positive hospitalized patients. The DENV serotype present in each patient was determined. Patients were stratified based on duration of hospital stay (<4 vs. ≥4 days). Data were analyzed using IBM® SPSS® 25.0. Multivariate logistic regression was performed to examine the association between PH and admission parameters.
    Results:
    Of 253 DENV hospitalized patients, 95 (37.5%) had PH (≥4 days). The mean duration of hospital stay was 3.43 ± 2.085 days (median = 3 days, interquartile range = 7 days). Diabetes mellitus (adjusted odds ratio [AOR] = 6.261, 95% confidence interval [CI] = 2.130-18.406, p = 0.001), DENV-2 serotype (AOR = 2.581, 95% CI = 1.179-5.650, p = 0.018), duration of fever ≤4 days (AOR = 2.423, 95% CI = 0.872-6.734, p = 0.09), and a shorter preadmission fever duration (AOR = 0.679, 95% CI = 0.481-0.957, p = 0.027) were independently associated with PH. However, PH was not found to be associated with symptoms on admission, secondary DENV infections or platelet count, hematocrit, or liver enzyme levels on admission.
    Conclusions:
    Early identification of these factors at presentation may alert clinicians to anticipate and recognize challenges in treating such patients, leading to more focused management plans that may shorten the duration of hospitalization.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  11. Fulltext A new paradigm for Aedes spp. surveillance using gravid ovipositing sticky trap and NS1 antigen test kit
    Lau SM, Chua TH, Sulaiman WY, Joanne S, Lim YA, Sekaran SD, et al.
    Parasit Vectors, 2017 Mar 21;10(1):151.
    PMID: 28327173 DOI: 10.1186/s13071-017-2091-y
    BACKGROUND: Dengue remains a serious public health problem in Southeast Asia and has increased 37-fold in Malaysia compared to decades ago. New strategies are urgently needed for early detection and control of dengue epidemics.

    METHODS: We conducted a two year study in a high human density dengue-endemic urban area in Selangor, where Gravid Ovipositing Sticky (GOS) traps were set up to capture adult Aedes spp. mosquitoes. All Aedes mosquitoes were tested using the NS1 dengue antigen test kit. All dengue cases from the study site notified to the State Health Department were recorded. Weekly microclimatic temperature, relative humidity (RH) and rainfall were monitored.

    RESULTS: Aedes aegypti was the predominant mosquito (95.6%) caught in GOS traps and 23% (43/187 pools of 5 mosquitoes each) were found to be positive for dengue using the NS1 antigen kit. Confirmed cases of dengue were observed with a lag of one week after positive Ae. aegypti were detected. Aedes aegypti density as analysed by distributed lag non-linear models, will increase lag of 2-3 weeks for temperature increase from 28 to 30 °C; and lag of three weeks for increased rainfall.

    CONCLUSION: Proactive strategy is needed for dengue vector surveillance programme. One method would be to use the GOS trap which is simple to setup, cost effective (below USD 1 per trap) and environmental friendly (i.e. use recyclable plastic materials) to capture Ae. aegypti followed by a rapid method of detecting of dengue virus using the NS1 dengue antigen kit. Control measures should be initiated when positive mosquitoes are detected.

    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  12. Human-porcine reassortant rotavirus generated by multiple reassortment events in a Sri Lankan child with diarrhea
    Yahiro T, Takaki M, Chandrasena TGAN, Rajindrajith S, Iha H, Ahmed K
    Infect Genet Evol, 2018 11;65:170-186.
    PMID: 30055329 DOI: 10.1016/j.meegid.2018.07.014
    A human-porcine reassortant rotavirus, strain R1207, was identified from 74 group A rotaviruses detected in 197 (37.6%) stool samples collected from patients who attended a tertiary care hospital in Ragama, Sri Lanka. This is the first report of a human-porcine reassortant rotavirus in Sri Lanka. The patient was a 12-month-old boy who had been hospitalized with fever and acute diarrhea with a duration of 6 days. The family had pigs at home before the birth of this boy. However, the neighbors still practice pig farming. The genotype constellation of R1207 was G4-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. This is based on the assignment of all the eleven gene segments a full genome-based genotyping system. R1207 showed a 4-2-3-2 genomic electrophoretic migration pattern, which is characteristic of group A rotaviruses. Our analyses revealed that five (NSP2, NSP4, VP1, VP2, and VP7) of the 11 genes were closely related to the respective genes of porcine strains. Although the remaining six genes (NSP1, NSP3, NSP5, VP3, VP4, and VP6) were related to human strains, with the exception of the gene sequence of NSP1, all of these human strains were human-porcine reassortants. With a genogroup 1 genetic backbone, this strain was possibly formed via multiple genetic reassortments. We do not know whether this strain is circulating in pigs, as no data are available on porcine rotaviruses in Sri Lanka. Surveillance should be strengthened to determine the epidemiology of this genotype of rotavirus in Sri Lanka and to assess whether the infection was limited or sustained by ongoing human-to-human transmission.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  13. Fulltext Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients
    Aw-Yong KL, Sam IC, Koh MT, Chan YF
    PLoS One, 2016;11(11):e0165659.
    PMID: 27806091 DOI: 10.1371/journal.pone.0165659
    Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142-156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41-55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  14. Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR
    Kong YY, Thay CH, Tin TC, Devi S
    J Virol Methods, 2006 Dec;138(1-2):123-30.
    PMID: 17000012 DOI: 10.1016/j.jviromet.2006.08.003
    The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  15. Fulltext Comparison of sequences of E/NS1 gene junction of dengue type 3 virus following culture subpassage in C6/36 cells to study the possible occurrence of mutations
    Thayan R, Morita K, Vijayamalar B, Zainah S, Chew TK, Oda K, et al.
    Southeast Asian J Trop Med Public Health, 1997 Jun;28(2):380-6.
    PMID: 9444025
    The aim of this study was to determine whether mutations could occur in the dengue virus genome following three subpassages of the virus in a mosquito cell line. This was done because sources of virus isolates used for sequencing studies are usually maintained in cell lines rather than in patients' sera. Therefore it must be assured that no mutation occurred during the passaging. For this purpose, sequencing was carried out using the polymerase chain reaction (PCR) products of the envelope/non-structural protein 1 junction region (280 nucleotides) of dengue type 3 virus. Sequence data were compared between the virus from a patient's serum against the virus subpassaged three times in the C6/36 cell line. We found that the sequence data of the virus from serum was identical to the virus that was subpassaged three times in C6/36 cell line.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  16. Antibody neutralization and viral virulence in recurring dengue virus type 2 outbreaks
    Wong SS, Abd-Jamil J, Abubakar S
    Viral Immunol, 2007 Sep;20(3):359-68.
    PMID: 17931106
    Outbreaks involving dengue viruses (DENV) of the same genotype occur in a cyclical pattern in Malaysia. Two cycles of outbreaks involving dengue virus type 2 (DENV-2) of the same genotype occurred in the 1990s in the Klang Valley, Malaysia. Sera of patients from the first outbreak and sera of mice inoculated with virus from the same outbreak had poorer neutralization activity against virus of the second outbreak. Conversely, patient sera from the second outbreak showed higher neutralization titer against virus of the early outbreak. At subneutralizing concentrations, sera of mice immunized with second outbreak virus did not significantly enhance infection with viruses from the earlier outbreak. Amino acid substitution from valine to isoleucine at position 129 of the envelope protein (E), as well as threonine to alanine at position 117 and lysine to arginine at position 272 of the NS1 protein, differentiated viruses of the two outbreaks. These findings highlight the potential influence of specific intragenotypic variations in eliciting varied host immune responses against the different DENV subgenotypes. This could be an important contributing factor in the recurring homogenotypic dengue virus outbreaks seen in dengue-endemic regions.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  17. Genomic analysis of some Japanese isolates of Getah virus
    Wekesa SN, Inoshima Y, Murakami K, Sentsui H
    Vet Microbiol, 2001 Nov 08;83(2):137-46.
    PMID: 11557154
    Using the reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing, capsid protein and non-structural protein 1 (nsP1) regions of Sagiyama virus and eight Getah virus strains were analysed. The viruses were isolated from Malaysia and various areas of Japan over a period of 30 years. Based on the available published sequence data, oligonucleotide primers were designed for RT-PCR and the sequences were determined. Our findings showed that though there were differences in the nucleotide sequences in the nsP1 region, there was 100% amino acid homology. On the other hand, in the capsid region, the nucleotide differences caused a major difference in the amino acid sequence. Therefore, the difference in the capsid region is one of the useful markers in the genetic classification between Sagiyama virus and strains of Getah virus, and might be responsible for the serological difference in complement fixation test. The genomic differences among the Getah virus strains are due to time factor rather than geographical distribution.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics*
  18. Genome structure of Sagiyama virus and its relatedness to other alphaviruses
    Shirako Y, Yamaguchi Y
    J Gen Virol, 2000 May;81(Pt 5):1353-60.
    PMID: 10769079
    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.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  19. Fulltext Viral Fitness Landscapes in Diverse Host Species Reveal Multiple Evolutionary Lines for the NS1 Gene of Influenza A Viruses
    Muñoz-Moreno R, Martínez-Romero C, Blanco-Melo D, Forst CV, Nachbagauer R, Benitez AA, et al.
    Cell Rep, 2019 12 17;29(12):3997-4009.e5.
    PMID: 31851929 DOI: 10.1016/j.celrep.2019.11.070
    Influenza A viruses (IAVs) have a remarkable tropism in their ability to circulate in both mammalian and avian species. The IAV NS1 protein is a multifunctional virulence factor that inhibits the type I interferon host response through a myriad of mechanisms. How NS1 has evolved to enable this remarkable property across species and its specific impact in the overall replication, pathogenicity, and host preference remain unknown. Here we analyze the NS1 evolutionary landscape and host tropism using a barcoded library of recombinant IAVs. Results show a surprisingly great variety of NS1 phenotypes according to their ability to replicate in different hosts. The IAV NS1 genes appear to have taken diverse and random evolutionary pathways within their multiple phylogenetic lineages. In summary, the high evolutionary plasticity of this viral protein underscores the ability of IAVs to adapt to multiple hosts and aids in our understanding of its global prevalence.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics
  20. Genetic diversity of bluetongue viruses in south east Asia
    Pritchard LI, Sendow I, Lunt R, Hassan SH, Kattenbelt J, Gould AR, et al.
    Virus Res, 2004 May;101(2):193-201.
    PMID: 15041187
    Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.
    Matched MeSH terms: Viral Nonstructural Proteins/genetics
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