Quasispecies of dengue virus

Pathogenic viruses have RNA genomes that cause acute and chronic infections. These viruses replicate with high mutation rates and exhibit significant genetic diversity, so-called viral quasispecies. Viral quasispecies play an important role in chronic infectious diseases, but little is known about their involvement in acute infectious diseases such as dengue virus (DENV) infection. DENV, the most important human arbovirus, is a causative agent of dengue fever (DF) and dengue hemorrhagic fever (DHF). Accumulating observations suggest that DENV exists as an extremely diverse virus population, but its biological significance is unclear. In other virus diseases, quasispecies affect the therapeutic strategies using drugs and vaccines. Here, I describe the quasispecies of DENV and discuss the possible role of quasispecies in the pathogenesis of and therapeutic strategy against DENV infection in comparison with other viruses such as Hepatitis C virus, human immunodeficiency virus type 1, and poliovirus

Resting CD4+ T Cells with CD38+CD62L+ Produce Interleukin-4 Which Contributes to Enhanced Replication of T-Tropic Human Immunodeficiency Virus Type 1

AbstractA significant increase in the CD38+ population among T lymphocytes has been observed in human immunodeficiency virus type 1 (HIV-1)-infected carriers. We previously reported a higher replication rate of T-tropic HIV-1 in the CD4+CD38+CD62L+ than CD38− subset under conditions of mitogen stimulation after infection. Here, we revealed a similarly high susceptibility in the CD38+ subset on culture with conditioned medium containing Th2 cytokine, interleukin (IL)-4 that was produced endogenously from this subset on stimulation with mitogen or anti-CD3 antibody for 3 days. The contribution of IL-4 to the upregulated production of virus in the CD38+ subset was confirmed by culture of this subset with recombinant human IL-4. In contrast, the rate of replication in the CD38− subset was not augmented in the conditioned medium from either subset or with IL-4. However, there were no differences in the surface expression of IL-4 receptor or HIV-1 receptors CD4 and CXCR4 between the two subsets. Thus, the CD4+CD38+CD62L+ subset comprises a specific cell population secreting endogenous Th2 cytokine that contributes to the efficient production of T-tropic HIV-1 through upregulation at a certain stage of the viral life cycle, probably after the adsorption step

RNAウイルスに対する迅速な全ゲノム決定法の構築

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5′ and 3′ terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences’ accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.権利情報：© 2023 Misu, Yoshikawa, Sugimoto, Takamatsu, Kurosu, Ouji, Yoshikawa, Shimojima, Ebihara and Saijo. This is an open-access article distributed under　the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is　permitted which does not comply with these terms

Chikungunya virus was isolated in Thailand, 2010

Chikungunya fever (CHIKF) is an acute febrile illness caused by a mosquito-borne alphavirus, chikungunya virus (CHIKV). This disease re-emerged in Kenya in 2004, and spread to the countries in and around the Indian Ocean. The re-emerging epidemics rapidly spread to regions like India and Southeast Asia, and it was subsequently identified in Europe in 2007, probably as a result of importation of chikungunya cases. On the one hand, chikungunya is one of the neglected diseases and has only attracted strong attention during large outbreaks. In 2008–2009, there was a major outbreak of chikungunya fever in Thailand, resulting in the highest number of infections in any country in the region. However, no update of CHIKV circulating in Thailand has been published since 2009. In this study, we examined the viral growth kinetics and sequences of the structural genes derived from CHIKV clinical isolates obtained from the serum specimens of CHIKF-suspected patients in Central Thailand in 2010. We identified the CHIKV harboring two mutations E1-A226V and E2-I211T, indicating that the East, Central, and South African lineage of CHIKV was continuously circulating as an indigenous population in Thailand. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-014-1105-5) contains supplementary material, which is available to authorized users

A New View of Alcohol Metabolism and Alcoholism—Role of the High-Km Class III Alcohol Dehydrogenase (ADH3)

The conventional view is that alcohol metabolism is carried out by ADH1 (Class I) in the liver. However, it has been suggested that another pathway plays an important role in alcohol metabolism, especially when the level of blood ethanol is high or when drinking is chronic. Over the past three decades, vigorous attempts to identify the enzyme responsible for the non-ADH1 pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. Recently, using ADH3-null mutant mice, we demonstrated that ADH3 (Class III), which has a high Km and is a ubiquitous enzyme of ancient origin, contributes to systemic alcohol metabolism in a dose-dependent manner, thereby diminishing acute alcohol intoxication. Although the activity of ADH3 toward ethanol is usually low in vitro due to its very high Km, the catalytic efficiency (kcat/Km) is markedly enhanced when the solution hydrophobicity of the reaction medium increases. Activation of ADH3 by increasing hydrophobicity should also occur in liver cells; a cytoplasmic solution of mouse liver cells was shown to be much more hydrophobic than a buffer solution when using Nile red as a hydrophobicity probe. When various doses of ethanol are administered to mice, liver ADH3 activity is dynamically regulated through induction or kinetic activation, while ADH1 activity is markedly lower at high doses (3–5 g/kg). These data suggest that ADH3 plays a dynamic role in alcohol metabolism, either collaborating with ADH1 or compensating for the reduced role of ADH1. A complex two-ADH model that ascribes total liver ADH activity to both ADH1 and ADH3 explains the dose-dependent changes in the pharmacokinetic parameters (β, CLT, AUC) of blood ethanol very well, suggesting that alcohol metabolism in mice is primarily governed by these two ADHs. In patients with alcoholic liver disease, liver ADH3 activity increases, while ADH1 activity decreases, as alcohol intake increases. Furthermore, ADH3 is induced in damaged cells that have greater hydrophobicity, whereas ADH1 activity is lower when there is severe liver disease. These data suggest that chronic binge drinking and the resulting liver disease shifts the key enzyme in alcohol metabolism from low-Km ADH1 to high-Km ADH3, thereby reducing the rate of alcohol metabolism. The interdependent increase in the ADH3/ADH1 activity ratio and AUC may be a factor in the development of alcoholic liver disease. However, the adaptive increase in ADH3 sustains alcohol metabolism, even in patients with alcoholic liver cirrhosis, which makes it possible for them to drink themselves to death. Thus, the regulation of ADH3 activity may be important in preventing alcoholism development

Detection of viral RNA in diverse body fluids in an SFTS patient with encephalopathy, gastrointestinal bleeding and pneumonia: a case report and literature review

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease that commonly has a lethal course caused by the tick-borne Huaiyangshan banyang virus [former SFTS virus (SFTSV)]. The viral load in various body fluids in SFTS patients and the best infection control measure for SFTS patients have not been fully established. CASE PRESENTATION: A 79-year-old man was bitten by a tick while working in the bamboo grove in Nagasaki Prefecture in the southwest part of Japan. Due to the occurrence of impaired consciousness, he was referred to Nagasaki University Hospital for treatment. The serum sample tested positive for SFTSV-RNA in the genome amplification assay, and he was diagnosed with SFTS. Furthermore, SFTSV-RNA was detected from the tick that had bitten the patient. He was treated with multimodal therapy, including platelet transfusion, antimicrobials, antifungals, steroids, and continuous hemodiafiltration. His respiration was assisted with mechanical ventilation. On day 5, taking the day on which he was hospitalized as day 0, serum SFTSV-RNA levels reached a peak and then decreased. However, the cerebrospinal fluid collected on day 13 was positive for SFTSV-RNA. In addition, although serum SFTSV-RNA levels decreased below the detectable level on day 16, he was diagnosed with pneumonia with computed tomography. SFTSV-RNA was detected in the bronchoalveolar lavage fluid on day 21. By day 31, he recovered consciousness completely. The pneumonia improved by day 51, but SFTSV-RNA in the sputum remained positive for approximately 4 months after disease onset. Strict countermeasures against droplet/contact infection were continuously conducted. CONCLUSIONS: Even when SFTSV genome levels become undetectable in the serum of SFTS patients in the convalescent phase, the virus genome remains in body fluids and tissues. It may be possible that body fluids such as respiratory excretions become a source of infection to others; thus, careful infection control management is needed

Runx1 Binds Positive Transcription Elongation Factor b and Represses Transcriptional Elongation by RNA Polymerase II: Possible Mechanism of CD4 Silencing

Runx1 binds the silencer and represses CD4 transcription in immature thymocytes. In this study, we found that Runx1 inhibits P-TEFb, which contains CycT1, CycT2, or CycK and Cdk9 and stimulates transcriptional elongation by RNA polymerase II (RNAPII) in eukaryotic cells. Indeed, its inhibitory domain, spanning positions 371 to 411, not only bound CycT1 but was required for silencing CD4 transcription in vivo. Our chromatin immunoprecipitation assays revealed that Runx1 inhibits the elongation but not initiation of transcription and that RNAPII is engaged at the CD4 promoter but is unable to elongate in CD4(−) CD8(+) thymoma cells. These results suggest that active repression by Runx1 occurs by blocking the elongation by RNAPII, which may contribute to CD4 silencing during T-cell development

Acute Systemic Infection with Dengue Virus Leads to Vascular Leakage and Death through Tumor Necrosis Factor-α and Tie2/Angiopoietin Signaling in Mice Lacking Type I and II Interferon Receptors.

Severe dengue is caused by host responses to viral infection, but the pathogenesis remains unknown. This is, in part, due to the lack of suitable animal models. Here, we report a non-mouse-adapted low-passage DENV-3 clinical isolate, DV3P12/08, derived from recently infected patients. DV3P12/08 caused a lethal systemic infection in type I and II IFN receptor KO mice (IFN-α/β/γR KO mice), which have the C57/BL6 background. Infection with DV3P12/08 induced a cytokine storm, resulting in severe vascular leakage (mainly in the liver, kidney and intestine) and organ damage, leading to extensive hemorrhage and rapid death. DV3P12/08 infection triggered the release of large amounts of TNF-α, IL-6, and MCP-1. Treatment with a neutralizing anti-TNF-α antibody (Ab) extended survival and reduced liver damage without affecting virus production. Anti-IL-6 neutralizing Ab partly prolonged mouse survival. The anti-TNF-α Ab suppressed IL-6, MCP-1, and IFN-γ levels, suggesting that the severe response to infection was triggered by TNF-α. High levels of TNF-α mRNA were expressed in the liver and kidneys, but not in the small intestine, of infected mice. Conversely, high levels of IL-6 mRNA were expressed in the intestine. Importantly, treatment with Angiopoietin-1, which is known to stabilize blood vessels, prolonged the survival of DV3P12/08-infected mice. Taken together, the results suggest that an increased level of TNF-α together with concomitant upregulation of Tie2/Angiopoietin signaling have critical roles in severe dengue infection

Efficient functional screening of a cellular cDNA library to identify severe fever with thrombocytopenia syndrome virus entry factors

The identification of host cell factors for virus entry is useful for the molecular explanation of viral tropisms and often leads to a more profound understanding of virus-induced diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus. No countermeasures against the disease exist. In this report, we show an efficient method using virus-like particles for the functional screening of a cellular cDNA library to identify SFTS virus entry factors. Two variants encoding dendritic cell-specific ICAM-3 grabbing non-integrin related (DC-SIGNR), a calcium-dependent lectin known to enhance SFTS virus infection, were successfully identified from a human liver cDNA library. We will discuss applications for yet unidentified factor(s) for SFTS virus entry and for entry factor(s) for other viruses related to SFTS virus