Clinical, virological and epidemiological characterization of dengue outbreak in Myanmar, 2015.

Hospital-based surveillance was conducted at two widely separated regions in Myanmar during the 2015 dengue epidemic. Acute phase serum samples were collected from 332 clinically diagnosed dengue patients during the peak season of dengue cases. Viremia levels were measured by quantitative real-time PCR and plaque assays using FcγRIIA-expressing and non-FcγRIIA-expressing BHK cells to specifically determine the infectious virus particles. By serology and molecular techniques, 280/332 (84·3%) were confirmed as dengue patients. All four serotypes of dengue virus (DENV) were isolated from among 104 laboratory-confirmed patients including two cases infected with two DENV serotypes. High percentage of primary infection was noted among the severe dengue patients. Patients with primary infection or DENV IgM negative demonstrated significantly higher viral loads but there was no significant difference among the severity groups. Viremia levels among dengue patients were notably high for a long period which was assumed to support the spread of the virus by the mosquito vector during epidemic. Phylogenetic analyses of the envelope gene of the epidemic strains revealed close similarity with the strains previously isolated in Myanmar and neighboring countries. DENV-1 dominated the epidemic in 2015 and the serotype (except DENV-3) and genotype distributions were similar in both study sites.

Retrospective seroepidemiological study of chikungunya infection in South Asia, Southeast Asia and the Pacific region.

Chikungunya virus (CHIKV) and Ross River virus (RRV) of the genus Alphavirus, family Togaviridae are mainly transmitted by Aedes mosquitoes and the symptoms they cause in patients are similar to dengue. A chikungunya (CHIK) outbreak re-emerged in several Asian countries during 2005-2006. This study aimed to clarify the prevalence of CHIKV infection in suspected dengue patients in six countries in South Asia and Southeast Asia. Seven hundred forty-eight serum samples were from dengue-suspected patients in South Asia and Southeast Asia, and 52 were from patients in Fiji. The samples were analysed by CHIKV IgM capture ELISA, CHIKV IgG indirect ELISA and focus reduction neutralization test against CHIKV or RRV. CHIK-confirmed cases in South Asia, particularly Myanmar and Sri Lanka, were 4·6%, and 6·1%, respectively; and in Southeast Asia, particularly Indonesia, the Philippines and Vietnam, were 27·4%, 26·8% and 25·0%, respectively. It suggests that CHIK was widely spread in these five countries in Asia. In Fiji, no CHIK cases were confirmed; however, RRV-confirmed cases represented 53·6% of suspected dengue cases. It suggests that RRV is being maintained or occasionally entering from neighbouring countries and should be considered when determining a causative agent for dengue-like illness in Fiji.

Cumulative ecological impacts of two successive annual treatments of imidacloprid and fipronil on aquatic communities of paddy mesocosms.

Agricultural landscapes, including paddies, play an important role in maintaining biodiversity, but this biodiversity has been under the threat of toxic agro-chemicals. Our knowledge about how aquatic communities react to, and recover from, pesticides, particularly in relation to their residues, is deficient, despite the importance of such information for realistic environmental impact assessment of pesticides. The cumulative ecological impacts on aquatic paddy communities and their recovery processes after two successive annual applications of two systemic insecticides, imidacloprid and fipronil, were monitored between mid-May and mid-September each year. The abundance of benthic organisms during both years was significantly lower in both insecticide-treated fields than in the controls. Large-impacts of fipronil on aquatic arthropods were found after the two years. Growth of medaka fish, both adults and their juveniles, was affected by the application of the two insecticides. A Principal Response Curve analysis (PRC) showed the escalation and prolongation of changes in aquatic community composition by the successive annual treatments of each insecticide over two years. Residues of fipronil in soil, which are more persistent than those of imidacloprid, had a high level of impact on aquatic communities over time. For some taxonomic groups, particularly for water surface-dwelling and water-borne arthropods, the second annual treatment had far greater impacts than the initial treatment, indicating that impacts of these insecticides under normal use patterns cannot be accurately assessed during short-term monitoring studies, i.e., lasting less than one year. It is concluded that realistic prediction and assessment of pesticide effects at the community level should also include the long-term ecological risks of their residues whenever these persist in paddies over a year.

Epidemiology of tick-borne encephalitis (TBE) and phylogenetic analysis of TBE viruses in Japan and Far Eastern Russia.

In Oshima, the southern part of Hokkaido, a tick-borne encephalitis (TBE) patient was found in 1993; in addition TBE virus was isolated from the blood samples of sentinel dogs, ticks pools, and rodents spleens in 1995 and 1996 by suckling mice. To identify when these TBE viruses emerged in Hokkaido, the times of divergence of TBE virus strains isolated in Oshima and Far Eastern Russia were estimated. TBE virus was isolated in Khabarovsk in 1998, and the nucleotide sequences of viral envelope protein genes of isolates from Oshima and Khabarovsk were compared. Based on the synonymous substitution rates of these virus E-protein genes, the lineage-divergence times of these TBE virus strains were predicted phylogenetically to be approximately 260-430 years ago. Furthermore, the virulence of TBE virus isolates from Oshima and Khabarovsk were compared in a mouse model. The results showed that the isolates possessed very similar virulence in mice. European TBE vaccine was found to be effective in TBE virus, Hokkaido strain. This review provides evidence that the Oshima strains of TBE virus in Hokkaido emerged from the Far Eastern Russia a few hundred years ago, which explains why the virulence of these strains is similar to that of TBE viruses isolated in Russia. Practical application of the vaccine should be considered in Japan.

Enhancement of Borna disease virus transcription in persistently infected cells by serum starvation.

Transcription of Borna disease virus (BDV) in persistently infected MDCK (MDCK/BDV) cells increased in the fetal bovine serum free media as detected by Northern blot analysis. Especially, the amount of 1.9-kb RNA without cap formation at the 5' end and polyadenylation at the 3' end, increased as compared to other mRNA molecules of BDV. Growth arrest of MDCK/BDV cells observed in the condition of serum starvation might be important for increasing viral transcription. Since N-cadherin is the responsible factor for cell-to-cell contact, MDCK/BDV cells were cultured in calcium free medium which inhibits the interaction of N-cadherin. However, inhibition of cell-to-cell contact by N-cadherin is not effective on up regulation of viral transcription. Our finding in this study indicates that enhancement of BDV transcription by serum starvation is a useful technique for further investigation in understanding of mechanisms of BDV transcription.

Single-step reverse transcriptase-polymerase chain reaction for detection of Borna disease virus RNA in vitro and in vivo.

There are few copies of Borna disease virus (BDV) genome in peripheral blood mononuclear cells and no reliable standard reverse transcriptase-polymerase chain reaction (RT-PCR) method for the detection of BDV RNA, which is both highly sensitive and free of contamination. Single-step RT-PCR, in which both reverse transcription and amplification by Taq DNA polymerase work efficiently in a single buffer, was applied to detect the p24 region of BDV RNA in vitro and in vivo. Using in vitro synthesized RNA, it was demonstrated that at least 100 copies of BDV RNA could be detected and the sensitivity and specificity were nearly equal to those obtained by RT-nested PCR. We could detect BDV RNA from more than 1 pg of cellular RNA obtained from BDV-persistently infected MDCK cells. Furthermore, this method was successfully performed on brain specimens obtained from a BDV-infected rat at 11 weeks post-inoculation. This single-step RT-PCR method will be convenient for detecting limited amounts of BDV RNA in various cells and tissue samples.

Characterization of monoclonal antibodies against Hokkaido strain tick-borne encephalitis virus.

A tick-borne encephalitis (TBE) patient was found in Hokkaido in 1993, and TBE viruses were isolated from animals and ticks in our previous studies. To develop a diagnostic reagent to identify TBE viruses, monoclonal antibodies (Mabs) were produced against the TBE virus strain Hokkaido (Oshima 5-10). Seven Mabs were obtained which reacted with the envelope protein of the Oshima 5-10 strain. These Mabs were flavivirus genus-specific, TBE virus complex-specific or TBE virus type-specific. The Mabs are applicable for identification of TBE virus strains.

Evaluation of European tick-borne encephalitis virus vaccine against recent Siberian and far-eastern subtype strains.

To evaluate the efficacy of the European TBE vaccine in east-Siberian and far-eastern regions of Russia, we examined the immune responses of the vaccine against recent TBE virus Siberian (Irkutsk) and far-eastern (Khabarovsk and Vladivostok) isolates. The sera of vaccinated humans showed efficient neutralizing antibody titers (> or =20) against Siberian and far-eastern strains. To evaluate the efficacy of the vaccine in vivo, mice were vaccinated and challenged with lethal doses of the viruses. All vaccinated mice survived each virus challenge. These results suggest that the European vaccine can prevent the TBE virus infection in east-Siberian and far-eastern regions of Russia.

Sub-genomic replicons of Tick-borne encephalitis virus.

We constructed three sub-genomic replicons of Tick-borne encephalitis virus (TBEV) (Oshima REP, Oshima REP-GFP and Oshima REP-Neo) by deleting genes coding for structural proteins without or with insertion of green fluorescent protein (GFP) or Neo genes, respectively. BHK cells transfected with Oshima REP expressed the viral non-structural antigens in immunofluorescent and western blot analyses. GFP and viral antigens were co-expressed in the transfected cells with Oshima REP-GFP. G418-resistant cells harboring Oshima REP-Neo consistently expressed the antigens without showing any apparent CPE. These replicons constructed in this study will be useful in studies on the replication, assembly and packaging of TBEV, and to develop vaccines and gene-delivering systems.

The mechanism of actinomycin D-mediated increase of Borna disease virus (BDV) RNA in cells persistently infected by BDV.

The transcriptional mechanism of Borna disease virus (BDV) has been poorly understood. We have analyzed transcription of the virus upon various stimuli in Madin-Darby canine kidney cells which were persistently infected by BDV (MDCK/BDV). Treatment with actinomycin D (ActD) increased the level of BDV RNA, shifting the size of RNA from 1.9 kb to 2.3 kb beginning 5 hr after the treatment. To understand the mechanism of this unique modulation of BDV RNA, we conducted several experiments. The RNA increase occurred at the stage in which synthesis of cellular intrinsic mRNA was intact, suggesting BDV does not compete with cellular transcriptional machinery for intrinsic RNA polymerase II. The BDV transcription was also enhanced by cycloheximide treatment, indicating that newly synthesized viral or cellular proteins are not necessary for viral transcription. However, a shift in the RNA size was not observed for cycloheximide-induced BDV RNA. The increase in viral transcription persisted during the cellular apoptotic process consequent to p53 gene accumulation beginning 1 hr after ActD treatment. Caspase inhibitors Z-VAD and DEVD-CHO repressed the apoptotic process but failed to block the increase in BDV transcription. In addition, adenovirus-mediated transduction of wild-type p53 did not alter the BDV transcription, indicating that the increase in BDV transcription was independent of the p53-mediated apoptotic process. Other various stimuli that evoke cellular signal transductions failed to alter BDV transcription. Agents inhibitory to topoisomerase except adriamycin failed to enhance BDV transcription, indicating that the increase in BDV transcription is not mediated by an inhibitory action to the topoisomerase II of ActD. Adriamycin showed an increase and size-shift of BDV RNA similar to ActD. These results suggest that intercalation of the viral genome itself with ActD is related to the stabilization of viral RNA and alteration of RNA size rather than secondary host cell changes.

Transcriptional control of Borna disease virus (BDV) in persistently BDV-infected cells.

Regulation of viral RNA levels in infected cells is considered important in the investigation of viral transcription and replication. Amounts of Borna disease virus (BDV) RNAs were increased in confluent persistently BDV-infected MDCK cells (MDCK/BDV) cells, while maintained at low levels in growing cells. The amount of 1.9-kb RNA without cap formation and polyadenylation at the 5' and 3' ends respectively were remarkably increased (200% per day) in confluent MDCK/BDV cells. Both the full-length genomic and anti-genomic RNAs were increased accompained by 1.9-kb RNA, suggesting the transcription of the 1.9-kb RNA was important for replication of BDV. Ribavirin has an inhibitory effect on replication and transcription of BDV at concentrations from 1 to 10 microgram/ml [Mizutani T et al., Arch Virol (1998)143: 2039-2 044]. BDV transcripts were decreased with ribavirin treatment and increased after its removal which indicated that ribavirin has a reversible inhibitory effect on BDV transcription. Furthermore, BDV transcription was also decreased by two agents, RMNPA and EICAR, which selectively inhibit enzyme activity related to cap formation at the 5' end of mRNA. On the contrary, when the growing MDCK/BDV cells were treated with actinomycin D, transcripts of BDV RNA were increased for 24 h. These agents and culture conditions in this study were found to be useful tools for up-and down-regulation of BDV transcription in persistently BDV-infected cells.

A case of tick-borne encephalitis in Japan and isolation of the the virus.

A case of tick-borne encephalitis (TBE) has not been reported for many years in Japan, although a serological survey of sera from domestic animals suggested the presence of TBE foci in Hokkaido, the northern island of Japan. Studies were conducted to prove the presence of an endemic focus of TBE virus in Japan by means of serology and virus isolation. In October 1993 in Hokkaido, a severe case of encephalitis in a dairy farmer's wife was diagnosed as TBE. Serological examination of paired serum specimens showed a rise in the neutralization antibody titer to Russian spring summer encephalitis virus. A seroepizootiological survey of dogs showed that the TBE-related virus was prevalent in the area. Three virus isolates were obtained from the blood of sentinel dogs, and antigenic analysis grouped the isolates into TBE-related viruses. Sequence analysis of the envelope protein gene identified one of the isolates as being of the same subtype as the Russian spring summer encephalitis (Far Eastern TBE) virus. The results provide evidence that TBE is endemic in a certain area of Japan.

Characterization of Oita virus 296/1972 of Rhabdoviridae isolated from a horseshoe bat bearing characteristics of both lyssavirus and vesiculovirus.

Oita virus 296/1972 was isolated from the blood of a wild horseshoe bat, Rhinolophus cornutus (Temminck) in 1972. We investigated the pathogenicity of this virus in mice in relation to its histological, immunohistochemical and ultrastructural characteristics and the entire sequence of nucleoprotein gene. This virus caused lethal encephalitis in mice through intracerebral route. This susceptibility of mice was until 3 weeks of age. Immunohistochemical analysis using the convalescent sera obtained from survived adult mice after intracerebral inoculation revealed that many neurons were positive in the cytoplasm, besides no cross reactivity with normal and rabies virus-infected mouse brain tissues to this anti-sera. Ultrastructural analysis disclosed many bullet-shaped and enveloped virions in neurons. These morphological characteristics of the virions are consistent of that of viruses in the family Rhabdoviridae. Budding from endoplasmic membrane suggests that this virus has a similarity with lyssaviruses. Molecular analysis of cDNA coding a tentative nucleoprotein sequence revealed homology with those of viruses in the family Rhabdoviridae. Distance matrix analysis of this gene sequence with those of other rhabdoviruses isolated from mammals disclosed the discrete position of this virus in the phylogenic tree of rhabdoviridae infecting mammals and we renamed this virus as Oita rhabdovirus.

Serological analysis of hemorrhagic fever with renal syndrome (HFRS) patients in Far Eastern Russia and identification of the causative hantavirus genotype.

Hemorrhagic fever with renal syndrome (HFRS) is endemic in East Asia and Europe. The disease is caused by several viruses belonging to the genus Hantavirus, including the Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava Belgrade virus (DOBV), and Puumala virus (PUUV). Recently, HTNV-related viruses, Amur (AMR) and Far East (FE) genotypes were identified as causative agents of HFRS in Far Eastern Russia. To investigate the epidemiology of HFRS and virus transmission, we collected sera from 17 acute and 32 convalescent patients who were clinically diagnosed with HFRS in the Khabarovsk region of Far Eastern Russia, and detected anti-hantavirus antibodies using an ELISA that can differentiate the infected virus serotype using truncated hantavirus nucleocapsid protein antigen. Sixteen of the 17 acute phase patients had antibodies to hantavirus, and all the positive sera had higher optical densities for HTNV-specific antigen than for SEOV-, DOBV-, or PUUV-specific antigens. The partial M segment of the viral genome was amplified from blood clots from three acute patients by PCR. The nucleotide sequences had closer identities to the FE genotype (>96%) than to the prototype HTNV (88 to 89%) or AMR genotype (81 to 83%). A phylogenetic analysis found that the virus sequences from the patients clustered with the FE type, and were distinct from the AMR type. Thirty-one of 32 convalescent patient sera had antibodies to HTNV-specific antigen. These data suggest that our ELISA system can detect HTNV-specific antibodies to the FE type, which may be responsible for most of the HFRS in Khabarovsk.