Dengue viruses (DEN), mosquito-borne members of the family Flaviviridae, are human pathogens of global significance. The effects of mycophenolic acid (MPA) and ribavirin (RBV) on DEN replication in monkey kidney (LLC-MK2) cells were examined. MPA (IC 50 =0?4±0?3 mM) and RBV (IC 50 =50?9±18 mM) inhibited DEN2 replication. Quantitative real-time RT-PCR of viral RNA and plaque assays of virions from DEN2-infected and MPA (10 mM)-and RBV (¢200 mM)-treated cells showed a fivefold increase in defective viral RNA production by cells treated with each drug. Moreover, a dramatic reduction of intracellular viral replicase activity was seen by in vitro replicase assays. Guanosine reversed the inhibition of these compounds, suggesting that one mode of antiviral action of MPA and RBV is by inhibition of inosine monophosphate dehydrogenase and thereby depletion of the intracellular GTP pool. In addition, RBV may act by competing with guanine-nucleotide precursors in viral RNA translation, replication and 59 capping.Dengue viruses (DEN1-4), mosquito-borne members of the family Flaviviridae, are human pathogens of global significance. Of the 1 million annual cases of dengue haemorrhagic fever/dengue shock syndrome, about 2-5 % are fatal. Currently, there is no vaccine or antiviral drug to treat DEN infections (Barrett, 2001;Gubler, 1998;Halstead & Deen, 2002). DEN2 New Guinea C strain, used in this study, has a single-stranded RNA genome (10 723 nt) of positive polarity (Irie et al., 1989). The aim of this study was to examine the antiviral action of mycophenolic acid (MPA) and ribavirin (RBV) on DEN2-infected LLC-MK2 cells by determining the number of infectious particles, levels of virion-associated RNAs and intracellular viral replicase activity by plaque assays, quantitative real-time RT-PCR (qRT-PCR) and in vitro replicase assays, respectively. MPA, a non-nucleoside analogue, is a potent, non-competitive inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key enzyme required for biosynthesis of guanine nucleotides (reviewed by Allison & Eugui, 2000). GTP is required for translation, transcription and replication processes. Therefore, inhibition of IMPDH is expected to inhibit not only proliferation of eukaryotic cells, but also replication of DNA and RNA viruses (Markland et al., 2000). However, RBV, a nucleoside analogue, is a competitive inhibitor of IMPDH.It is approved as an inhaled drug for treatment of respiratory syncytial virus infection, as well as orally, together with alpha interferon, for treatment of hepatitis C virus (HCV) infections.DEN2 was propagated in mosquito (C6/36) cells as described previously (Charnsilpa et al., 2005). LLC-MK2 cells were infected with DEN2 under single-step growth conditions (Dulbecco & Vogt, 1954) at an m.o.i. of 10 and incubated for 72 h with 1 % fetal bovine serum. The plaque assay was performed essentially as described previously (Charnsilpa et al., 2005).To quantify the virus-associated RNA, qRT-PCR was used as described previously (Houng et al., 2000). The detection of...

BackgroundJapanese 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.FindingsTwo regions of the JEV genome were sequenced from 19 pools; the envelope gene and the nonstructural protein 5 (NS5)/3'-untranslated region (UTR). Eighteen pools of Culex tritaeniorhynchus and one pool of Cx. bitaeniorhynchus were positive for genotype I and genotype V, respectively. Sequence alignment of the complete E gene from Cx. bitaeniorhynchus showed high amino acid similarity (98.8%) to the Muar strain, characterized as the first report of genotype V, isolated from an encephalitis patient in Malaysia in 1952.ConclusionThis study represents the first report of JEV genotype V in the ROK. The reemergence of genotype V in Asia (China and ROK) after more than a half-century and its discovery in Cx. bitaeniorhynchus, a mosquito species previously unknown to carry JEV in the ROK, emphasizes the need for enhanced JE surveillance to monitor the dynamics of JEV strains within the region. Future findings may have implications with regard to JEV vaccination/prevention strategies.

Bioisosteric deaza analogues of 6-methyl-9-β-D-ribofuranosylpurine, a hydrophobic analogue of adenosine, were synthesized and evaluated for antiviral activity. Whereas the 1-deaza and 3-deaza analogues were essentially inactive in plaque assays of infectivity, a novel 7-deaza-6-methyl-9-β-D-ribofuranosylpurine analogue, structurally related to the natural product tubercidin, potently inhibited replication of poliovirus (PV) in HeLa cells (IC 50 = 11 nM) and dengue virus (DENV) in Vero cells (IC 50 = 62 nM). Selectivity against PV over cytotoxic effects to HeLa cells was >100-fold after incubation for 7 h. Mechanistic studies of the 5'-triphosphate of 7-deaza-6-methyl-9-β-D-ribofuranosylpurine revealed that this compound is an efficient substrate of PV RNA-dependent RNA polymerase (RdRP) and is incorporated into RNA mimicking both ATP and GTP.

High mobility group box 1 (HMGB1) protein functions in regulation of transcription, cellular activation and pro-inflammatory responses. However, the potential role of HMGB1 during viral infection has not been investigated. This study attempted to elucidate whether the HMGB1-mediated inflammatory response contributes to the pathogenesis of dengue virus (DENV) infection. Our data showed that HMGB1 was released at low DENV infection levels (m.o.i. of 1) under non-necrotic conditions by human dendritic cells (DCs). When DENV-infected DCs were co-cultured with autologous T cells, there was increased production of HMGB1 by both cell types. HMGB1 regulated tumour necrosis factor alpha, interleukin (IL)-6, IL-8 and alpha interferon secretion in DENV-infected DCs. Additionally, increased HMGB1 production was associated with reduced DENV replication titres in DCs. These results suggest that HMGB1 production influences DENV infection in susceptible hosts.

In this study, we used a metagenomic approach to analyze bacterial communities from diverse populations (humans, animals, and vectors) to investigate the role of these microorganisms as causative agents of disease in human and animal populations. Wild rodents and ectoparasites were collected from 2014 to 2018 in Nan province, Thailand where scrub typhus is highly endemic. Samples from undifferentiated febrile illness (UFI) patients were obtained from a local hospital. A total of 200 UFI patient samples were obtained and 309 rodents and 420 pools of ectoparasites were collected from rodents ( n = 285) and domestic animals ( n = 135). The bacterial 16S rRNA gene was amplified and sequenced with the Illumina. Real-time PCR and Sanger sequencing were used to confirm the next-generation sequencing (NGS) results and to characterize pathogen species. Several pathogens were detected by NGS in all populations studied and the most common pathogens identified included Bartonella spp., Rickettsia spp., Leptospira spp., and Orientia tsutsugamushi . Interestingly, Anaplasma spp. was detected in patient, rodent and tick populations, although they were not previously known to cause human disease from this region. Candidatus Neoehrlichia, Neorickettsia spp., Borrelia spp., and Ehrlichia spp. were detected in rodents and their associated ectoparasites. The same O. tsutsugamushi genotypes were shared among UFI patients, rodents, and chiggers in a single district indicating that the chiggers found on rodents were also likely responsible for transmitting to people. Serological testing using immunofluorescence assays in UFI samples showed high prevalence (IgM/IgG) of Rickettsia and Orientia pathogens, most notably among samples collected during September–November. Additionally, a higher number of seropositive samples belonged to patients in the working age population (20–60 years old). The results presented in this study demonstrate that the increased risk of human infection or exposure to chiggers and their associated pathogen ( O. tsutsugamushi ) resulted in part from two important factors; working age group and seasons for rice cultivation and harvesting. Evidence of pathogen exposure was shown to occur as there was seropositivity (IgG) in UFI patients for bartonellosis as well as for anaplasmosis. Using a metagenomic approach, this study demonstrated the circulation and transmission of several pathogens in the environment, some of which are known causative agents of illness in human populations.

Recently, nitric oxide (NO) has been shown to suppress dengue virus (DENV) RNA and protein accumulation in infected cells. In this report, the potential target of the inhibitory effect of NO was studied at the molecular level. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), showed an inhibitory effect on RNA accumulation at around 8-14 h post-infection, which corresponded to the step of viral RNA synthesis in the DENV life cycle. The activity of the viral replicase isolated from SNAP-treated DENV-2-infected cells was suppressed significantly compared with that of the negative-control N-acetyl-DL-penicillamine (NAP)-treated cells. Further investigations on the molecular target of NO action showed that the activity of recombinant DENV-2 NS5 in negative-strand RNA synthesis was affected in the presence of 5 mM SNAP in in vitro RNA-dependent RNA polymerase (RdRp) assays, whereas the RNA helicase activity of DENV-2 NS3 was not inhibited up to a concentration of 15 mM SNAP. These results suggest that the inhibitory effect of NO on DENV infection is partly via inhibition of the RdRp activity, which then downregulates viral RNA synthesis. INTRODUCTIONDengue viruses (DENVs) (serotypes 1-4) are members of the genus Flavivirus in the family Flaviviridae. The flavivirus genome is a single-stranded RNA (~11 kb) of positive polarity, which has a type I cap structure at the 59 end, but no poly(A) tail at the 39 end. The genome encodes a single polyprotein, which is processed co-and posttranslationally into at least ten mature proteins consisting of three structural proteins (C, prM and E) that form the virion and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) that are important for virus replication (Lindenbach & Rice, 2003). NS5 is an RNA-dependent RNA polymerase (RdRp) required for viral RNA replication (O'Reilly & Kao, 1998;Poch et al., 1989). NS5 also has a 29-O-methyltransferase activity thought to be important for 59 capping (Egloff et al., 2002;Rozanov et al., 1992). NS3 is a multifunctional protein; it has the active site of a trypsin-like serine protease and, together with the NS2B co-factor, forms an active protease that is required for polyprotein processing. NS3 also has an RNA-stimulated NTPase and RNA helicase, thought to be important for viral RNA replication, as well as a 59 RNA triphosphatase activity, which is required in the first step of 59 capping. In flavivirus-infected cells, NS3 and NS5 exist in a complex and are thought to be components of the viral RNA replicase complex (Kapoor et al., 1995;Westaway et al., 2002Westaway et al., , 2003.The endoplasmic reticulum-associated replicase complex is composed of the consensus components NS1, NS2A, NS3, NS4A and NS5 (Westaway et al., 1997), as well as putative host proteins such as La protein (García-Montalvo et al., 2004;Yocupicio-Monroy et al., 2003). Although the binding of several host proteins to the 39 and 59 untranslated regions (UTRs) has been shown, their definitive role in virus replication is still obscure. The enzymic...

Our study highlights the surveillance of Bartonella species among rodents and their associated ectoparasites (ticks, fleas, lice, and mites) in several regions across Thailand. A total of 619 rodents and 554 pooled ectoparasites (287 mite pools, 62 flea pools, 35 louse pools, and 170 tick pools) were collected from 8 provinces within 4 regions of Thailand. Bandicota indica (279), Rattus rattus (163), and R. exulans (96) were the most prevalent species of rats collected in this study. Real-time PCR assay targeting Bartonella-specific ssrA gene was used for screening and each positive sample was confirmed by PCR using nuoG gene. The prevalence of Bartonella DNA in rodent (around 17%) was recorded in all regions. The highest prevalence of Bartonella species was found in B. savilei and R. rattus with the rate of 35.7% (5/14) and 32.5% (53/163), respectively. High prevalence of Bartonella-positive rodent was also found in B. indica (15.1%, 42/279), and R. norvegicus (12.5%, 5/40). In contrast, the prevalence of Bartonella species in ectoparasites collected from the rats varied significantly according to types of ectoparasites. A high prevalence of Bartonella DNA was found in louse pools (Polyplax spp. and Hoplopleura spp., 57.1%) and flea pools (Xenopsylla cheopis, 25.8%), while a low prevalence was found in pools of mites (Leptotrombidium spp. and Ascoschoengastia spp., 1.7%) and ticks (Haemaphysalis spp., 3.5%). Prevalence of Bartonella DNA in ectoparasites collected from Bartonella-positive rodents (19.4%) was significantly higher comparing to ectoparasites from Bartonella-negative rodents (8.7%). The phylogenetic analysis of 41 gltA sequences of 16 Bartonella isolates from rodent blood and 25 Bartonella-positive ectoparasites revealed a wide range of diversity among Bartonella species with a majority of sequences (61.0%) belonging to Bartonella elizabethae complex (11 rodents, 1 mite pool, and 5 louse pools), while the remaining sequences were identical to B. phoceensis (17.1%, 1 mite pool, 5 louse pools, and 1 tick pool), B. coopersplainensis (19.5%, 5 rodents, 1 louse pool, and 2 tick pools), and one previously unidentified Bartonella species (2.4%, 1 louse pool).

Nitric oxide is well accepted as one of the defenses for inhibiting viral dissemination. Macrophages and cells in the macrophage lineage are professional nitric oxide producers which sub-serve as target for dengue virus. The interaction between nitric oxide and dengue virus in such target cell is unknown. In this report, the impact of nitric oxide on infectious dengue virus serotype 2 production and RNA replication was investigated in vitro. Primary isolates of dengue virus serotype 2 from dengue patients were replicated in mouse neuroblastoma cells in the presence of an exogenous nitric oxide donor, s-nitroso-N-acethylpennicillamine, SNAP, at the concentration of 50 or 75 or 100 microM. Nitric oxide inhibited viral replication in a dose and a multiplicity of infection dependent manner. Nitric oxide from 50 and 75 microM SNAP delayed and suppressed replication of dengue virus isolates while higher concentration of nitric oxide, 100 microM SNAP, completely inhibited production of infectious particles up to 36 hr study. Twenty-four out of forty tested isolates, 60%, were susceptible to 50 microM SNAP inhibitory effect. The mechanism of inhibition was investigated at the level of RNA synthesis and was found that RNA production was suppressed which correlated to production of the infectious particles. Down-regulation of the RNA synthesis resulted in reduction of protein synthesis which was detected by lower level of NS1 protein synthesis using immunoblotting. In conclusion, nitric oxide from exogenous nitric oxide donor down regulated replication of dengue virus serotype 2 isolates from dengue patients. The suppression was clearly shown at the level of viral RNA and protein synthesis resulting in reduction of viral progenies production. This phenomenon implies that nitric oxide may serve as a defense which diminishes viral load in patients.