Development of a reverse transcription-loop-mediated isothermal amplification assay for detection of pandemic (H1N1) 2009 virus as a novel molecular method for diagnosis of pandemic influenza in resource-limited settings.

This paper reports on the development of a one-step, real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the hemagglutinin (HA) gene for the rapid molecular-based detection of pandemic (H1N1) 2009 virus. The detection limit of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay was same as that of the currently used real-time reverse transcription-PCR method. The assay detected the pandemic (H1N1) 2009 virus HA gene in 136 RNA samples extracted from nasopharyngeal swab specimens from Japanese and Vietnamese patients. No cross-reactive amplification with the RNA of other seasonal influenza viruses was observed, and the detection of specific viral genome targets in clinical specimens was achieved in less than 40 min. The sensitivity and specificity of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay obtained in this study were 97.8% and 100%, respectively. Use of the (H1N1) 2009 virus HA-specific RT-LAMP assay will enable the faster and easier diagnosis of pandemic (H1N1) 2009 virus infection, especially in resource-limited situations in developing countries.

Newly emerged enterovirus-A71 C4 sublineage may be more virulent than B5 in the 2015-2016 hand-foot-and-mouth disease outbreak in northern Vietnam.

Enterovirus-A71 (EV-A71) is a common cause of hand-foot-and-mouth disease (HFMD) and, rarely, causes severe neurological disease. This study aimed to elucidate the epidemiological and genetic characteristics and virulence of EV-A71 strains isolated from children diagnosed with HFMD. Rectal and throat swabs were collected from 488 children with HFMD in Hanoi, Vietnam, in 2015-2016. From 391 EV-positive patients, 15 EVs, including coxsackievirus A6 (CV-A6; 47.1%) and EV-A71 (32.5%, n = 127), were identified. Of the 127 EV-A71 strains, 117 (92.1%) were the B5 subgenotype and 10 (7.9%) were the C4 subgenotype. A whole-genome analysis of EV-A71 strains showed that seven of the eight C4a strains isolated in 2016 formed a new lineage, including two possible recombinants between EV-A71 C4 and CV-A8. The proportion of inpatients among C4-infected children was higher than among B5-infected children (80.0% vs. 27.4%; P = 0.002). The virulence of EV-A71 strains was examined in human scavenger receptor class B2 (hSCARB2)-transgenic mice, and EV-A71 C4 strains exhibited higher mortality than B5 strains (80.0% vs. 30.0%, P = 0.0001). Thus, a new EV-A71 C4a-lineage, including two possible recombinants between EV-A71 C4 and CV-A8, appeared in 2016 in Vietnam. The EV-A71 C4 subgenotype may be more virulent than the B5 subgenotype.

Identification of novel antiviral of fungus-derived brefeldin A against dengue viruses.

Microbial natural products possess a wide range of biological and biochemical potential. Among them, fungal secondary metabolites are one of the most important sources for discovering new drugs or lead compounds. In the present study, we explored substances produced by the strain Penicillium sp. FKI-7127 for its antiviral activity. We identified brefeldin A as a novel antiviral agent against dengue viruses. The inhibitory effect of brefeldin A was confirmed by virus titer and immunofluorescence assay. Brefeldin A inhibited dengue viruses regardless of serotypes and other related viruses including Zika virus and Japanese encephalitis virus. Time-of-addition study showed that brefeldin A exerts its antiviral effect at an early stage of the dengue virus (DENV) life cycle. These studies demonstrate that (i) brefeldin A could be used as a lead compound for drug development of anti-DENV and other related viruses and (ii) fungal metabolites are a potential and valuable source for dengue virus drug discovery.

Molecular and serological epidemiology of Japanese encephalitis virus (JEV) in a remote island of western Japan: an implication of JEV migration over the East China Sea.

BACKGROUND: Japanese encephalitis (JE) is a mosquito-borne infectious disease caused by Japanese encephalitis virus (JEV). About 1-10 cases with severe central nervous system symptoms have been constantly reported every year in Japan. To clarify the mechanism of maintenance of JEV, the present study surveyed pigs for serological evidence of JEV infection and isolated JEV strains from pigs and mosquitoes in Isahaya City (Isahaya) and Goto City (Goto) in the islets of Goto in Nagasaki Prefecture from 2008 to 2014. RESULTS: The serological survey of pigs showed the increase of IgM sero-positivity against JEV in July or August, and it was maintained until October or November in both Isahaya and Goto every year. There were 47 JEV strains isolated in Nagasaki from 2001 to 2014 including the isolates in this study, and they belonged to genotype 1. Thirty four of the isolated strains were from pigs in Isahaya and were classified under six subclusters (1-A-1, 1-A-2, 1-A-3, 1-A-4, 1-A-5, and 1-A-9). Thirteen strains were isolated from pigs and mosquitoes in Goto and were classified into three subclusters (1-A-5 (2008); 1-A-1 (2009); and 1-A-2). In the subcluster 1-A-2, three different monophyletic subgroups, 1-A-2-2 (2010), 1-A-2-3 (2011), and 1-A-2-1 (2013, 2014), appeared in Goto. CONCLUSIONS: These data strongly suggested that JEV appearance in Goto seems to depend on the frequent introduction of JEV from outside of the island and this pattern is different from what has been observed in subtropical islands in the East China Sea such as Okinawa and Taiwan, where the same populations of JEV (1-A-7 (1998-2008) in Okinawa; genotype 3 (until 2012) in Taiwan) have been maintained for a long period.

Seroepidemiological evidence of severe fever with thrombocytopenia syndrome virus infections in wild boars in Nagasaki, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging disease in East Asia. It is thought that the SFTS virus (SFTSV) circulates between ticks and animals in nature and that the virus is transmitted to humans by tick bites. SFTS is endemic to Nagasaki in western Japan; however, epidemiological information regarding SFTSV in Nagasaki is not known. In this study, we performed SFTSV IgG ELISAs and neutralization antibody assays for a seroepidemiological survey using samples from wild boars captured in six areas of Nagasaki. SFTSV seropositive animals were found in three areas. Our findings provide epidemiological information on the distribution of SFTSV in Nagasaki.

Insect diapause-specific peptide from the leaf beetle has consensus with a putative iridovirus peptide.

Diapause and hibernation during periods of environmental adversity are essential features of the life cycle in many organisms, yet the molecular basis for these events differs among animals. We have identified an endogenous diapause/hibernation-specific peptide, from the leaf beetle Gastrophysa atrocyanea. This peptide provides antifungal activity, acts as a N-type voltage-gated Ca2+ channel blocker, and has a new consensus sequence with an unknown polypeptide encoded in the insect iridescent virus. These results indicate that the diapause-specific peptide may be utilized as a probe to analyze and compare functional and evolutional aspects of the life cycles of insects and iridoviruses.

Crystal structure of the catalytic domain of Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolution of 1.8 A.

The NS3 protein of Japanese encephalitis virus (JEV) is a large multifunctional protein possessing protease, helicase, and nucleoside 5'-triphosphatase (NTPase) activities, and plays important roles in the processing of a viral polyprotein and replication. To clarify the enzymatic properties of NS3 protein from a structural point of view, an enzymatically active fragment of the JEV NTPase/helicase catalytic domain was expressed in bacteria and the crystal structure was determined at 1.8 A resolution. JEV helicase is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Each of the motifs I (Walker A motif), II (Walker B motif) and VI was composed of an NTP-binding pocket. Mutation analyses revealed that all of the residues in the Walker A motif (Gly(199), Lys(200) and Thr(201)), in addition to the polar residues within the NTP-binding pocket (Gln(457), Arg(461) and Arg(464)), and also Arg(458) in the outside of the pocket in the motif IV were crucial for ATPase and helicase activities and virus replication. Lys(200) was particularly indispensable, and could not be exchanged for other amino acid residues without sacrificing these activities. The structure of the NTP-binding pocket of JEV is well conserved in dengue virus and yellow fever virus, while different from that of hepatitis C virus. The detailed structural comparison among the viruses of the family Flaviviridae should help in clarifying the molecular mechanism of viral replication and in providing rationale for the development of appropriate therapeutics.