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.
Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-β and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-β transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-β inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development.IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.
The performance of a commercial immunochromatography test for rapid detection of dengue NS1 antigen present in serum or plasma of patients was evaluated against a commercial dengue NS1 antigen-capture ELISA. The rapid immunochromatography test gave an overall sensitivity of 90.4% with a specificity of 99.5%. The sensitivity was highest for serum samples from which virus was isolated (96.3%) and lowest for those from which virus was not isolated and RT-PCR was negative (76.4%). The sensitivity was significantly higher for serum samples from patients with acute primary dengue (92.3%) than those from patients with acute secondary dengue (79.1%). The positive predictive value and negative predictive value of this commercial immunochromatography test were 99.6% and 87.9% respectively.
One of the critical stages of the T-cell immune response is the dimerization of the intramembrane domains of T-cell receptors (TCR). Structural similarities between the immunosuppressive domains of viral proteins and the transmembrane domains of TCR have led several authors to hypothesize the mechanism of immune response suppression by highly pathogenic viruses: viral proteins embed themselves in the membrane and act on the intramembrane domain of the TCRalpha subunit, hindering its functional oligomerization. It has also been suggested that this mechanism is used by influenza A virus in NS1-mediated immunosuppression. We have shown that the peptide corresponding to the primary structure of the potential immunosuppressive domain of NS1 protein (G51) can reduce concanavalin A-induced proliferation of PBMC cells, as well as in vitro, G51 can affect the oligomerization of the core peptide corresponding to the intramembrane domain of TCR, using AFM and small-angle neutron scattering. The results obtained using in cellulo and in vitro model systems suggest the presence of functional interaction between the NS1 fragment and the intramembrane domain of the TCR alpha subunit. We have proposed a possible scheme for such interaction obtained by computer modeling. This suggests the existence of another NS1-mediated mechanism of immunosuppression in influenza.
The pathogenic dengue virus (DV) is a growing global threat, particularly in South East Asia, for which there is no specific treatment available. The virus possesses a two-component (NS2B/NS3) serine protease that cleaves the viral precursor proteins. Here, we performed molecular dynamics simulations of the NS2B/NS3 protease complexes with six peptide substrates (capsid, intNS3, 2A/2B, 4B/5, 3/4A and 2B/3 containing the proteolytic site between P(1) and P(1)' subsites) of DV type 2 to compare the specificity of the protein-substrate binding recognition. Although all substrates were in the active conformation for cleavage reaction by NS2B/NS3 protease, their binding strength was somewhat different. The simulated results of intermolecular hydrogen bonds and decomposition energies suggested that among the ten substrate residues (P(5)-P(5)') the P(1) and P(2) subsites play a major role in the binding with the focused protease. The arginine residue at these two subsites was found to be specific preferential binding at the active site with a stabilization energy of intNS3>2A/2B>4B/5>3/4A>2B/3 in a relative correspondence with previous experimentally derived values.
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.
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.
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.
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.
Early definitive diagnosis of dengue virus infection may help in the timely management of dengue virus infection. We evaluated the Standard Diagnostics (SD, South Korea) dengue virus nonstructural protein NS1 antigen enzyme-linked immunosorbent assay (SD dengue NS1 Ag ELISA) for the detection of dengue virus NS1 antigen in patients' sera, using a total of 399 serum samples in a comparison with real-time reverse transcription (RT)-PCR, an in-house IgM capture (MAC)-ELISA, and a hemagglutination inhibition (HI) assay. Of the 320 dengue sera, 205 (64%) tested positive for NS1 antigen compared to 300 (93.75%) by either MAC-ELISA or RT-PCR, 161 (50.31%) by RT-PCR, and 226 (70.36%) by MAC-ELISA only. The assay was able to detect NS1 antigen in convalescent-phase sera until day 14 of infection. The NS1 detection rate is inversely proportional while the IgM detection rate is directly proportional to the presence of IgG antibodies. The overall sensitivity and specificity of the SD dengue NS1 Ag ELISA in the detection of "confirmed dengue virus" sera are 76.76% and 98.31%, respectively. This suggests that the SD kit is highly specific and sensitive for the detection of NS1 antigen. However, caution is needed when the kit is used as a single assay, as detection in samples that contained the virus was only about 81.97%. Combining this assay with an IgM and/or IgG assay will increase the sensitivity of detection, especially in areas with a higher prevalence of secondary dengue virus infections.
The infectious salmon anaemia virus (ISAV) is a piscine virus, a member of Orthomyxoviridae family. It encodes at least 10 proteins from eight negative-strand RNA segments. Since ISAV belongs to the same virus family as Influenza A virus, with similarities in protein functions, they may hence be characterised by analogy. Like NS1 protein of Influenza A virus, s8ORF2 of ISAV is implicated in interferon antagonism and RNA-binding functions. In this study, we investigated the role of s8ORF2 in RNAi suppression in a well-established Agrobacterium transient suppression assay in stably silenced transgenic Nicotiana xanthi. In addition, s8ORF2 was identified as a novel interactor with SsMov10, a key molecule responsible for RISC assembly and maturation in the RNAi pathway. This study thus sheds light on a novel route undertaken by viral proteins in promoting viral growth, using the host RNAi machinery.
Dengue infection is a major public health problem affecting millions of people living in tropical countries. With no suitable vaccines and specific antiviral drugs, treatment for dengue is usually symptomatic and supportive. Early diagnosis and recognition of severe disease is therefore crucial for better management of the patient. Two-dimension electrophoresis was used to identify disease-associated proteins that can be used for diagnosis and as drug targets for treatment. Two markers, identified by mass spectrometry analysis as alpha1-antitrypsin and NS1 proteins were found to be upregulated in dengue fever (DF; n=10) and dengue haemorrhagic fever (DHF; n=10) patients compared with healthy individuals (n=8). Both alpha1-antitrypsin and NS1 proteins were overexpressed two-fold in DHF patients compared with DF patients. Our study suggests that alpha1-antitrypsin and NS1 protein could be used as biomarkers as early indicators of DHF risk among patients with suspected dengue infection.
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.
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.
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.
Dengue is the most prevalent mosquito borne infection worldwide. Vertical transmissions after maternal dengue infection to the fetus and pregnancy losses in relation to dengue illness have been reported. The relationship of dengue to miscarriage is not known.
Zika virus (ZIKV) is an arthropod-borne flavivirus that causes newborn microcephaly and Guillian-Barré syndrome in adults. No therapeutics are available to treat ZIKV infection or other flaviviruses. In this study, we explored the inhibitory effect of glycosaminoglycans and analogues against ZIKV infection. Highly sulfated heparin, dextran sulfate and suramin significantly inhibited ZIKV infection in Vero cells. De-sulfated heparin analogues lose inhibitory effect, implying that sulfonate groups are critical for viral inhibition. Suramin, an FDA-approved anti-parasitic drug, inhibits ZIKV infection with 3-5 log10 PFU viral reduction with IC50value of ∼2.5-5 μg/ml (1.93 μM-3.85 μM). A time-of-drug-addition study revealed that suramin remains potent even when administrated at 1-24 hpi. Suramin inhibits ZIKV infection by preventing viral adsorption, entry and replication. Molecular dynamics simulation revealed stronger interaction of suramin with ZIKV NS3 helicase than with the envelope protein. Suramin warrants further investigation as a potential antiviral candidate for ZIKV infection. Heparan sulfate (HS) is a cellular attachment receptor for multiple flaviviruses. However, no direct ZIKV-heparin interaction was observed in heparin-binding analysis, and downregulate or removal of cellular HS with sodium chlorate or heparinase I/III did not inhibit ZIKV infection. This indicates that cell surface HS is not utilized by ZIKV as an attachment receptor.
Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010.
Severe dengue can be lethal caused by manifestations such as severe bleeding, fluid accumulation and organ impairment. This study aimed to investigate the role of dengue non-structural 1 (NS1) protein and host factors contributing to severe dengue. Electrical cell-substrate impedance sensing system was used to investigate the changes in barrier function of microvascular endothelial cells treated NS1 protein and serum samples from patients with different disease severity. Cytokines and metabolites profiles were assessed using a multiplex cytokine assay and liquid chromatography mass spectrometry respectively. The findings showed that NS1 was able to induce the loss of barrier function in microvascular endothelium in a dose dependent manner, however, the level of NS1 in serum samples did not correlate with the extent of vascular leakage induced. Further assessment of host factors revealed that cytokines such as CCL2, CCL5, CCL20 and CXCL1, as well as adhesion molecule ICAM-1, that are involved in leukocytes infiltration were expressed higher in dengue patients in comparison to healthy individuals. In addition, metabolomics study revealed the presence of deregulated metabolites involved in the phospholipid metabolism pathway in patients with severe manifestations. In conclusion, disease severity in dengue virus infection did not correlate directly with NS1 level, but instead with host factors that are involved in the regulation of junctional integrity and phospholipid metabolism. However, as the studied population was relatively small in this study, these exploratory findings should be confirmed by expanding the sample size using an independent cohort to further establish the significance of this study.
The NS2B/NS3 protease is crucial for the pathogenesis of the DENV. Therefore, the inhibition of this protease is considered to be the key strategy for the development of new antiviral drugs. In the present study, malabaricones C (3) and E (4), acylphenols from the fruits of Myristica cinnamomea King, have been respectively identified as moderate (27.33 ± 5.45 μM) and potent (7.55 ± 1.64 μM) DENV-2 NS2B/NS3 protease inhibitors, thus making this the first report on the DENV-2 NS2B/NS3 protease inhibitory activity of acylphenols. Based on the molecular docking studies, compounds 3 and 4 both have π-π interactions with Tyr161. While compound 3 has hydrogen bonding interactions with Gly151, Gly153 and Tyr161, compound 4 however, forms hydrogen bonds with Ser135, Asp129, Phe130 and Ile86 instead. The results from the present study suggests that malabaricones C (3) and E (4) could be employed as lead compounds for the development of new dengue antivirals from natural origin.