Structure and its transformation of elliptical nege-like virus Tanay virus.

Negeviruses that infect insects are recently identified virus species that are phylogenetically related to several plant viruses. They exhibit a unique virion structure, an elliptical core with a short projection. Negeviruses encode two structural proteins, a glycoprotein that forms a short projection, and an envelope protein that forms an elliptical core. The glycoprotein has been reported only in the negeviruses' genes, and not in phylogenetically related plant viruses' genes. In this report, we first describe the three-dimensional electron cryo-microscopy (cryo-EM) structure of Tanay virus (TANAV), one of the nege-like viruses. TANAV particle demonstrates a periodical envelope structure consisting of three layers surrounding the centred viral RNA. The elliptical core dynamically changes its shape under acidic and even low detergent conditions to form bullet-like or tubular shapes. The further cryo-EM studies on these transformed TANAV particles reveal their overall structural rearrangement. These findings suggest putative geometries of TANAV and its transformation in the life cycle, and the potential importance of the short projection for enabling cell entry to the insect hosts.

An outbreak of a novel lineage of dengue virus 2 in Vietnam in 2022.

In 2022, a large dengue outbreak was reported in Vietnam, where dengue was endemic. A total of 1889 acute-phase serum samples were collected from patients with suspected dengue at Vung Tau General Hospital, the core hospital in Vung Tau Province, southern Vietnam. Among the 1889 samples analyzed for laboratory confirmation of dengue virus (DENV) infection, 339 positive cases were identified, of which 130 were primary infections and 209 were secondary infections. DENV-2 was the dominant serotype in both primary and secondary infection groups. Phylogenetic analysis based on sequences of the envelope protein-coding region revealed the emergence of a new DENV-2 lineage during this outbreak.

Differential Infectivity of Human Neural Cell Lines by a Dengue Virus Serotype-3 Genotype-III with a Distinct Nonstructural Protein 2A (NS2A) Amino Acid Substitution Isolated from the Cerebrospinal Fluid of a Dengue Encephalitis Patient.

Dengue encephalitis is considered as a severe but unusual clinical presentation of dengue infection. Limited molecular information is available on the neurotropism of dengue virus (DENV), highlighting the need for further research. During a dengue outbreak in Vietnam in 2013, two DENV-3 strains were isolated, in which one was isolated from cerebrospinal fluid (CSF) samples from a dengue encephalitis patient and another strain was isolated from a patient with classical dengue fever in Hai Phong, Vietnam. DENV serotype-3 (DENV-3) isolated from these samples belonged to genotype III, marking the first report of this genotype in the country at that time. Genetic variation between both strains was elucidated by using a full genome sequencing by next-generation sequencing (NGS). The infectivity of the isolated DENV-3 strains was further characterized using human and mouse neuronal cell lines. Phylogenetic analysis of the isolates demonstrated high homogeneity between the CSF-derived and serum-derived DENV-3, in which the full genome sequences of the CSF-derived DENV-3 presented a Thr-1339-Ile mutation in the nonstructural 2A (NS2A) protein. The CSF-derived DENV-3 isolate grew preferentially in human neuronal cells, with a significant proportion of cells that were positive for nonstructural 1 (NS1), nonstructural 4B (NS4B), and nonstructural 5 (NS5) antigens. These results suggest that NS2A may be a crucial region in the neuropathogenesis of DENV-3 and its growth in human neuronal cells. Taken together, our results demonstrate that a CSF-derived DENV-3 has unique infectivity characteristics for human neuronal cells, which might play a crucial role in the neuropathogenesis of DENV infection.

Discrepancy of SARS-CoV-2 PCR results due to the sample collection sites and possible improper sampling.

Polymerase chain reaction (PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is necessary for confirming a diagnosis of Coronavirus disease 2019 (COVID-19). Here we present a COVID-19 case of an elderly woman whose SARS-CoV-2 PCR tests showed false negative repeatedly by evaluating with different sampling sites and procedures. Nasopharyngeal swabs, suctioned sputum, and tongue swabs were collected for SARS-CoV-2-PCR. As for tongue swabs, we compared between two different sample conditions; one obtained with dry condition and the other obtained with moistened condition inside the oral cavity. SARS-CoV-2-PCR showed positive for an extended period with suctioned sputum samples compared with nasopharyngeal swabs and tongue swabs. No SARS-CoV-2 from a nasopharyngeal swab sample obtained on day 46 after symptoms onset was isolated despite high viral load (183740.5 copies/5µL). An adequate production of neutralizing antibody in a serum sample on day 46 was also confirmed. The number of RNA copies of the tongue swab samples was higher with moistened condition than with dry condition. The present case suggests that the difference of sampling site or sample condition can affect PCR results. High loads viral RNA detection does not always correlate with infectivity.

Chikungunya Virus Infection in Blood Donors and Patients During Outbreak, Mandalay, Myanmar, 2019.

In 2019, an outbreak of chikungunya virus infection occurred in Mandalay, Myanmar, and 3.2% of blood donors and 20.5% of patients who were children were confirmed as being infected. The prevalence rate was up to 6.3% among blood donors. The East Central/South African genotype was predominantly circulating during this outbreak.

Severe Acute Respiratory Syndrome Coronavirus 2 Shedding by Travelers, Vietnam, 2020.

We analyzed 2 clusters of 12 patients in Vietnam with severe acute respiratory syndrome coronavirus 2 infection during January-February 2020. Analysis indicated virus transmission from a traveler from China. One asymptomatic patient demonstrated virus shedding, indicating potential virus transmission in the absence of clinical signs and symptoms.

A single amino acid substitution in the NS4B protein of Dengue virus confers enhanced virus growth and fitness in human cells in vitro through IFN-dependent host response.

Dengue virus (DENV) replication between mosquito and human hosts is hypothesized to be associated with viral determinants that interact in a differential manner between hosts. However, the understanding of inter-host viral determinants that drive DENV replication and growth between hosts is limited. Through the use of clinical isolates, we identified an amino acid variation of Ala, Met and Val at position 116 of DENV-1 NS4B. While the proportion of virus with the NS4B-116V variant remained constantly high in serial passages in a mosquito cell line, populations of the NS4B-116M and NS4B-116A variants became dominant after serial passages in mammalian cell lines. Using recombinant DENV-1 viruses, the Val to Ala or Met alteration at position NS4B-116 (rDENV-1-NS4B-116A and rDENV-1-NS4B-116M) resulted in enhanced virus growth in human cells in comparison to the clone with Val at NS4B-116 (rDENV-1-NS4B-116V). However, the reverse phenomenon was observed in a mosquito cell line. Additionally, in a human cell line, differential levels of IFN-α/ß and IFN-stimulated gene expressions (IFIT3, IFI44L, OAS1) suggested that the enhanced viral growth was dependent on the ability of the NS4B protein to hamper host IFN response during the early phase of infection. Overall, we identified a novel and critical viral determinant at the pTMD3 of NS4B region that displayed differential effects on DENV replication and fitness in human and mosquito cell lines. Taken together, the results suggest the importance of the NS4B protein in virus replication and adaptation between hosts.

Detection of east/central/south African genotype of chikungunya virus in Myanmar, 2010.

In 2010, chikungunya virus of the East Central South African genotype was isolated from 4 children in Myanmyar who had dengue-like symptoms. Phylogenetic analysis of the E1 gene revealed that the isolates were closely related to isolates from China, Thailand, and Malaysia that harbor the A226V mutation in this gene.

NS1' protein expression facilitates production of Japanese encephalitis virus in avian cells and embryonated chicken eggs.

Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus of the family Flaviviridae, is a leading cause of meningo-encephalitis in Asian countries. The flavivirus non-structural protein 1 (NS1) plays a role in virus replication and in the elicitation of an immune response. The NS1' protein found among the members of the JEV subgroup is an extended form of NS1 and is generated by a -1 ribosomal frameshift. This protein is known to be involved in viral pathogenicity; however, its specific function is still unknown. Here, we describe an investigation of the molecular function of NS1' protein through the production of JEV NS1'-expressing and -non-expressing clones and their infection of avian and mammalian cells. Efficient NS1' protein expression was observed in avian cells and was found to facilitate JEV production in both avian cultured cells and embryonated chicken eggs. NS1' protein was observed to co-localize with NS5 protein and resulted in increased viral RNA levels in avian cells. These findings clearly indicate that NS1' enhances the production of JEV in avian cells and may facilitate the amplification/maintenance role of birds in the virus transmission cycle in nature.

Tanay virus, a new species of virus isolated from mosquitoes in the Philippines.

In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.

The Development of New Primer Sets for the Amplification and Sequencing of the Envelope Gene of All Dengue Virus Serotypes.

Dengue virus (DENV) poses a significant threat to global health, infecting approximately 390 million people annually. This virus comprises four serotypes capable of causing severe disease. Genetic analyses are crucial for understanding the epidemiology, evolution, and spread of DENV. Although previous studies have focused on the envelope protein-coding (E) gene, only a few primers can efficiently detect and amplify the viral genes from multiple endemic countries simultaneously. In this study, we designed degenerate primer pairs for each DENV serotype to amplify and sequence the entire E gene, using globally representative sequences for each serotype. These primers were validated using DENV isolates from various Asian countries and demonstrated broad-spectrum detection capabilities and high-quality sequences. The primers provide effective tools for genetic analysis in the regions affected by dengue, aiding strain identification and epidemiological studies during outbreaks.

Clinical Factors Associated with SFTS Diagnosis and Severity in Cats.

Severe fever with thrombocytopenia syndrome (SFTS) is a potentially fatal tick-borne zoonosis caused by SFTS virus (SFTSV). In addition to tick bites, animal-to-human transmission of SFTSV has been reported, but little is known about feline SFTSV infection. In this study, we analyzed data on 187 cats with suspected SFTS to identify biomarkers for SFTS diagnosis and clinical outcome. Body weight, red and white blood cell and platelet counts, and serum aspartate aminotransferase and total bilirubin levels were useful for SFTS diagnosis, whereas alanine aminotransferase, aspartate aminotransferase and serum SFTSV RNA levels were associated with clinical outcome. We developed a scoring model to predict SFTSV infection. In addition, we performed a phylogenetic analysis to reveal the relationship between disease severity and viral strain. This study provides comprehensive information on feline SFTS and could contribute to the protection of cat owners, community members, and veterinarians from the risk of cat-transmitted SFTSV infection.

Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes.

Nidoviruses with large genomes (26.3-31.7 kb; 'large nidoviruses'), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7-15.7 kb; 'small nidoviruses'). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3'-5'exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60-80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3'-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3'-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2'-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that - in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

Survey of causative agents for acute respiratory infections among patients in Khartoum-State, Sudan, 2010-2011.

BACKGROUND: This study was carried out to determine causative agents of acute respiratory illness of patients in Khartoum State, Sudan. METHODS: Four hundred patients experiencing respiratory infections within January-March 2010 and January-March 2011 were admitted at Khartoum Hospital and had their throat swab samples subjected to multiplex real-time RT-PCR to detect influenza viruses (including subtypes) and other viral agents. Isolation, nucleotide sequence and phylogenetic analysis on some influenza viruses based on the HA gene were done. RESULTS: Out of 400 patients, 66 were found to have influenza viruses (35, 27, 2, and 2 with types A, B, C, and A and B co-infections, respectively). Influenza viruses were detected in 28, 33 and 5 patients in the age groups <1, 1-10, and 11-30 years old, respectively but none in the 31-50 years old group. Out of 334 patients negative for influenza viruses, 27, 14, and 2 were positive for human respiratory syncytial virus, rhinovirus and adenovirus, respectively. Phylogenetic tree on influenza A (H1N1) pdm09 subtype shows that Sudan strains belong to the same clade and are related to those strains from several countries such as USA, Japan, Italy, United Kingdom, Germany, Russia, Greece, Denmark, Taiwan, Turkey and Kenya. Seasonal A H3 subtypes have close similarity to strains from Singapore, Brazil, Canada, Denmark, USA and Nicaragua. For influenza B, Sudan strains belong to two different clades, and just like influenza A (H1N1) pdm09 and A H3 subtypes, seem to be part of worldwide endemic population (Kenya, USA, Brazil, Russia, Taiwan and Singapore). CONCLUSIONS: In Sudan, the existence of respiratory viruses in patients with acute respiratory infection was confirmed and characterized for the first time by using molecular techniques.

An approach for differentiating echovirus 30 and Japanese encephalitis virus infections in acute meningitis/encephalitis: a retrospective study of 103 cases in Vietnam.

BACKGROUND: In recent decades, Echovirus 30 (E30) and Japanese encephalitis virus (JEV) have been reported to be the common causative agents of acute meningitis among patients in South East Asia. An E30 outbreak in Vietnam in 2001-2002 gained our interest because the initial clinical diagnosis of infected patients was due to JEV infection. There are few clinical insights regarding E30 cases, and there are no reports comparing E30 and JEV acute meningitis/encephalitis cases based on clinical symptoms and case histories. We therefore aimed to identify reliable clinical methods to differentiate E30 and JEV acute meningitis/encephalitis. METHODS: A retrospective, cross-sectional study was conducted to compare E30 and JEV acute meningitis/encephalitis cases. We collected and analyzed the clinical records of 43 E30 confirmed cases (E30 group) and 60 JEV confirmed cases (JEV group). Clinical data were compared between the E30 and the JEV groups. Differences of clinical parameters were analyzed by certain statistical tests. RESULTS: Fever, headache, and vomiting were the most common symptoms in both the E30 and the JEV groups. Combined symptoms of headache and vomiting and the triad of symptoms of fever, headache, and vomiting were observed in more patients in the E30 group (E30 vs. JEV: 19% vs. 0%, p < 0.001; 74% vs. 27%, p < 0.001, respectively). On the other hand, strong neurological symptoms such as seizure (5% vs. 73%, p < 0.001) and altered consciousness (12% vs. 97%, p < 0.001) were manifested primarily in the JEV group. CSF leukocytosis was observed predominantly in the E30 group (80 vs. 18 cells/µL, p = 0.003), whereas decreasing CSF sugar level was observed predominantly in the JEV group (58.7 vs. 46.9 mg/dL, p < 0.001). CONCLUSION: Fever, headache, vomiting, absence of neurological symptoms (seizure, altered consciousness), and presence of CSF leukocytosis are important parameters to consider in differentiating E30 from JEV cases during early infection. Then, proper measures can be adopted immediately to prevent the spread of the disease in the affected areas.

Clinical, Virological, and Immunological Features in Cosmopolitan Genotype DENV-2-Infected Patients during a Large Dengue Outbreak in Sri Lanka in 2017.

In 2017, Sri Lanka experienced its largest dengue epidemic and reported severe and unusual presentations of dengue with high morbidity. This outbreak was associated with the reemergence of dengue virus-2 (DENV-2), with the responsible strain identified as a variant of the previously circulating DENV-2 cosmopolitan genotype. In this study, we characterized the DENV-2 cosmopolitan genotype from patients during this epidemic. Also, we identified host factors that contributed to the severity of dengue infection in patients infected with this particular virus. Ninety-one acute serum samples from patients at the National Hospital in Kandy were randomly selected. Of these, 40.2% and 48.9% were positive for dengue IgM and IgG, respectively. NS1 antigen levels were significantly higher in primary infections. The severe dengue (SD) and dengue with warning signs (DWWS) groups exhibited significantly higher viral genome and infectivity titers than the dengue without warning signs (DWoWS) group. The highest viremia level was observed in SD patients. As for host cytokine response, interferon α (IFN-α) levels were significantly higher in the DWoWS group than in the DWWS and SD groups, whereas interleukin (IL)-12p40 and tumor necrosis factor α (TNF-α) levels in SD patients were significantly higher than in the other two groups. The TNF-α, IL-4, and monocyte chemoattractant protein-1 concentrations were positively correlated with NS1 antigen levels. From whole-genome analysis, NS4 had the highest frequency of amino acid variants, followed by the E gene. Our study suggests that viremia levels and immune responses contributed to SD outcomes, and these findings may help in identifying an effective therapeutic strategy against SD infection.

Coinfection and circulation of chikungunya virus and dengue virus in pediatric patients in Myanmar, 2019.

Myanmar is an endemic country for arboviruses, and outbreaks occur frequently. A cross-sectional analytical study was conducted during the peak season of the chikungunya virus (CHIKV) outbreak in 2019. A total of 201 patients with acute febrile illness who were admitted to the 550-bedded Mandalay Children Hospital in Myanmar were enrolled in the study, and virus isolation, serological tests, and molecular tests for the dengue virus (DENV) and CHIKV were performed for all samples. Out of 201 patients, 71 (35.3%) were only DENV-infected, 30 (14.9%) were only CHIKV-infected and 59 (29.4%) were coinfected with DENV and CHIKV. The viremia levels of the DENV- and CHIKV- mono-infected groups were significantly higher than those of the group coinfected with DENV and CHIKV. Genotype I of DENV-1, genotypes I and III of DENV-3, genotype I of DENV-4 and the East/Central/South African genotype of CHIKV were co-circulating during the study period. Two novel epistatic mutations of CHIKV (E1:K211E and E2:V264A) were noted. This study highlighted that there were many coinfection cases during the outbreak and that the co-circulation of both viruses in DENV-endemic regions warrants effective monitoring of these emerging pathogens via comprehensive surveillance to facilitate the implementation of effective control measures.

The 2017 Dengue virus 1 outbreak in northern Vietnam was caused by a locally circulating virus group.

BACKGROUND: Dengue virus (DENV) is a member of insect vector-borne viruses, and it causes dengue fever. Southeast Asia is the epi-center of dengue fever in the world. The characterization of the virus is essential to identify the transmission and evolution of DENV. OBJECTIVES: In 2017, there was an outbreak of Dengue virus type 1 (DENV1) in northern Vietnam and the neighboring countries. To identify the genetic character of the outbreak virus in the area, we conducted whole-genome sequencing analysis on the samples positive for the DENV1 along with real-time PCR. STUDY DESIGN: In total, 1026 blood samples were collected from patients with suspected dengue fever in Ha Nam and Hai Duong province, nearby areas of the capital of Vietnam. After screening by real-time PCR, 40 of DENV1 positive samples were subjected to whole-genome sequencing, and 28 complete coding sequences were obtained. RESULTS: All 28 sequences were genotype I of DENV1, which is dominant in the southeast and East Asian countries. The phylogenetic analysis of the E region showed that they fell into a single cluster with the reported sequences from Vietnam between 2009 and 2016, in which the isolates from other countries are very rare. Our results suggested that the 2017 outbreak in the area was caused by locally circulating viruses.

Two hidden taxa in the Japanese encephalitis vector mosquito, Culex tritaeniorhynchus, and the potential for long-distance migration from overseas to Japan.

The Culex vishnui subgroups, particularly Culex tritaeniorhynchus, are considered the primary vectors of the Japanese encephalitis virus (JEV) in Asia. Recent molecular phylogenetic analyses of JEV isolates from Asian countries have shown that JEVs with diverse genetic variants are present in Asia. Furthermore, some JEV strains have been found to have crossed the East China Sea and been introduced into Japan. In this study, the possibility of overseas migration of the JE vector mosquito, Cx. tritaeniorhynchus was examined from the genetic, physical, and meteorological perspectives. Molecular phylogenetic analysis was performed based on both whole coding sequences and on the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) gene of Cx. vishnui subgroups collected from Asian countries. Culex tritaeniorhymchus was classified into two genetically independent taxa by COI sequences: the Japanese type (Ct-J), which inhabits Japan except for the Amami Islands of southern Japan, and the continental type (Ct-C), which inhabits the Asian region except for Japan. It was confirmed that approximately 10% of Cx. tritaeniorhynchus trapped during the summer in western Kyushu were Ct-C, and that they could fly for up to 38 h continuously. The meteorological analysis also confirmed that the atmospheric flow occurring over the continent coincided with the date of Ct-C capture. This is the first report showing the existence of two taxa in Cx. tritaeniorhynchus. Their physical and physiological characteristics suggest the possibility of long-distance migration from overseas regions to Japan across the East China Sea. Future efforts are expected to provide evidence to support the occurrence of long-distance migration of Cx. tritaeniorhynchus with JEV.

Evaluation of a Triage Checklist for Mild COVID-19 Outpatients in Predicting Subsequent Emergency Department Visits and Hospitalization during the Isolation Period: A Single-Center Retrospective Study.

Managing mild illness in COVID-19 and predicting progression to severe disease are concerning issues. Here, we investigated the outcomes of Japanese patients with mild COVID-19, and identified triage risk factors for further hospitalization and emergency department (ED) visits at a single tertiary hospital. A triage checklist with 30 factors was used. Patients recommended for isolation were followed up for 10 days for subsequent ED visits or hospital admission. Overall, 338 patients (median age, 44.0; 45% women) visited the clinic 5.0 days (median) after symptom onset. Thirty-six patients were immediately hospitalized following triage; others were isolated. In total, 72 non-hospitalized patients visited the ED during their isolation, and 30 were hospitalized after evaluation for oxygen desaturation. The median ED visit and hospitalization durations after symptom onset were 5.0 and 8.0 days, respectively. The checklist factors associated with hospitalization during isolation were age > 50 years, body mass index > 25 kg/m2, hypertension, tachycardia with pulse rate > 100/min or blood pressure > 135 mmHg at triage, and >3-day delay in hospital visit after symptom onset. No patients died. Altogether, 80% of patients with mild COVID-19 could be safely isolated at home. Age, BMI, underlying hypertension, date after symptom onset, tachycardia, and systolic blood pressure at triage might be related to later hospitalization.