An in vitro poliovirus RNA-synthesizing system derived from a crude membrane fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VYlg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing nucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase Tj-resistant oligonufleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUU4AAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a Ifjlqple fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VP&-pUpU in the presence of UTP. Our data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.
Abstract. We present our surveillance results on imported dengue cases in Taiwan during [2008][2009][2010]. A total of 734 imported dengue patients were identified. The travelers were arriving from 18 countries, including Southeast Asia, the Indian subcontinent, South Pacific islands, and Latin America. Gene sequences from 358 dengue virus (DENV) isolates were used to perform phylogenetic analyses, thus, providing an updated view of the geographic distribution and dynamic transmission of DENV strains circulating in these countries. Our results suggest that the DENV-1 genotype I and DENV-2 Cosmopolitan genotype comprise the predominant DENV strains circulating in Southeast Asian countries. The DENV-3 Genotype III strain was found to be newly emerging in several Southeast Asian countries, however, the Asian genotype 2 and the Asian/American genotype of DENV-2 strains appeared to be less prevalent in Southeast Asia. Furthermore, imported dengue viruses are representative of the overall patterns of serotype/genotype frequencies of dengue outbreaks that occurred in Taiwan.
Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Pigs and water birds are the main amplifying and maintenance hosts of the virus. In this study, we conducted a JEV survey in mosquitoes captured in pig farms and water bird wetland habitats in Taiwan during 2005 to 2012. A total of 102,633 mosquitoes were collected. Culex tritaeniorhynchus was the most common mosquito species found in the pig farms and wetlands. Among the 26 mosquito species collected, 11 tested positive for JEV by RT-PCR, including Cx. tritaeniorhynchus, Cx. annulus, Anopheles sinensis, Armigeres subalbatus, and Cx. fuscocephala. Among those testing positive, Cx. tritaeniorhynchus was the predominant vector species for the transmission of JEV genotypes I and III in Taiwan. The JEV infection rate was significantly higher in the mosquitoes from the pig farms than those from the wetlands. A phylogenetic analysis of the JEV envelope gene sequences isolated from the captured mosquitoes demonstrated that the predominant JEV genotype has shifted from genotype III to genotype I (GI), providing evidence for transmission cycle maintenance and multiple introductions of the GI strains in Taiwan during 2008 to 2012. This study demonstrates the intense JEV transmission activity in Taiwan, highlights the importance of JE vaccination for controlling the epidemic, and provides valuable information for the assessment of the vaccine's efficacy.
We used the dengue virus NS1 antigen (Ag) rapid test for on-site detection of imported dengue cases at airports. Among 22 positive cases of dengue identified from 850 patients with a fever suspected to have dengue, 17 were NS1 Ag test positive. These findings demonstrate the usefulness of the NS1 Ag rapid test in screening imported dengue cases at airports.Rapid and accurate detection of dengue virus (DENV) infection from acute-phase viremic blood samples from patients with a fever contributes greatly to patient management in hospitals and control measures in public health. In addition, rapid detection of imported dengue cases at airports can help to reduce the annual local outbreaks in a country where dengue is not endemic, such as Taiwan. We have previously reported on screening for fever at airports in Taiwan as part of active surveillance for a panel of notifiable infectious diseases, such as dengue, gastroenteritis caused by enteric bacteria, malaria, and chikungunya (10, 12). A total of 298 imported dengue cases were identified at airports in Taiwan from July 2003 to August 2008 using real-time one-step reverse transcription-PCR (RT-PCR) and envelope/membrane-specific capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assays (ELISAs) (8, 9, 11).For molecular diagnosis, a real-time one-step RT-PCR was performed using two sets of consensus primers, one primer set targeting a region of the nonstructural protein 5 (NS5) genes to detect all flaviviruses and the other primer set targeting a region of the capsid gene to detect all DENV serotypes. Positive samples were then confirmed by DENV serotyping using four sets of serotype-specific primers targeting the capsid gene to differentiate the DENV serotypes (8, 11). For serological diagnosis, envelope/membrane-specific capture IgM and IgG ELISAs were used to detect and differentiate primary and secondary DENV infections in acute-phase and convalescentphase serum samples (9, 11). Differentiation of primary and secondary DENV infections was defined by the ratio of the IgM-to-IgG readings, Ն1.2 or Ͻ1.2, respectively. In addition, a simple and sensitive nonstructural protein 1 (NS1) serotypespecific IgG ELISA was used to differentiate the immunological status of individuals into naïve, primary, or secondary DENV infections using acute-phase, convalescent-phase, or postinfection serum samples (3, 9, 11). A unique feature of this assay is that differentiation of primary and secondary DENV infections can be made when DENV-specific IgG antibody has not been produced in the early acute-phase serum samples.Most of these imported dengue cases were viremic when arriving at airports. It would be desirable to reduce the time gap between clinical and laboratory diagnoses to prevent the local transmission of the imported DENVs. Recent advances on the development of DENV NS1 antigen (Ag) assay offer promising new perspectives on the rapid diagnosis of dengue, although limited sensitivity was reported in patients with acute febrile illness in an area where dengue is...
We report two cases of imported infection in patients who had returned to Taiwan from Singapore: one was coinfected with chikungunya virus and dengue virus type 2, and the other was infected with the same dengue virus. Both viruses were successfully isolated from the coinfected case by using antibody neutralization and a plaque purification technique.Dengue fever, caused by a flavivirus in the family Flaviviridae, is the most prevalent arboviral disease in tropical and subtropical regions of Asia, the Pacific and Caribbean islands, and Central and South America (9). Chikungunya, caused by an alphavirus in the family Togaviridae, is endemic to Africa and Asia (12). Both diseases are transmitted to humans by day-biting Aedes aegypti and Aedes albopictus mosquitoes, and both diseases have similar clinical symptoms, including fever, rash, and joint pains as well as headache, fatigue, nausea, vomiting, and muscle pain; a laboratory test is required to distinguish between the two diseases. Thus, many risk factors for chikungunya virus (CHIKV) and dengue virus (DENV) infections are the same or similar. The urban mosquito Aedes aegypti is the primary vector of both viruses throughout most of their geographic range, although Aedes albopictus was recently identified as the main vector of the recently emerged CHIKV E1-226V variant of the African genotype (17).The explosive epidemics of chikungunya in Indian Ocean islands and India since 2004 and the worldwide increase in travel have facilitated the expansion of different strains of CHIKV of the African genotype into overlap areas where DENV is endemic (13). As a result, cocirculation of CHIKV and DENV has been reported in various geographic areas, including India, Sri Lanka, Gabon, Cameroon, Madagascar, Malaysia, Indonesia, Singapore, and Thailand. Consequently, a few studies showing patients coinfected with CHIKV and DENV have been reported in India, Sri Lanka, Malaysia, and Gabon (1,5,8,11,14). Although molecular and serologic evidence demonstrated or suggested coinfections in the abovementioned reports, neither CHIKV nor DENV was isolated from these patients. Successful isolation of both viruses is needed to conduct basic and applied research on CHIKV and DENV biology, immunology, and pathogenesis, as well as the development of laboratory diagnosis, antiviral drugs, and vaccines.The first and only concurrent isolation of CHIKV and DENV-2, from a single blood specimen taken from a patient in the acute phase of a dengue-like illness in southern India in 1964, was reported by Myers and Carey (10). In their study, the dominance of CHIKV in the coinfected patient's serum, along with growth competition, prevented the initial isolation of DENV-2; isolation was finally accomplished through pretreatment of the acute-phase serum sample with a CHIKV-specific mouse antibody, followed by inoculation into infant mice for in vivo growth. Here we report only the second case confirmed by actual isolation of CHIKV and DENV-2, from a patient returned from Singapore, using an in vitro cell...
60:43-53, 1986). Viruses used to produce the CRC were poliovirus type 1 (Mahoney), [PV-1(M)], poliovirus type 1 (Sabin) [PV-1(S)], and four in vitro recombinants that were constructed from infectious cDNA clones. RNA synthesis in CRC was studied. No end-linked, full-length double-stranded poliovirus RNA was detected in CRC regardless of whether nonionic detergent (Nonidet P-40) was added prior to incubation. Synthesis of VPg-pU and VPg-pUpU, two nucleotidyl proteins presumed to be involved in the initiation of RNA synthesis, was slower at 30°C in CRC induced by PV-1(S) than by PV-1(M). This observation was used to design a pulse-chase experiment whose result suggested that synthesis of VPg-pUpU occurred by uridylylation of VPg-pU. Synthesis of VPg-pU(pU) was thermosensitive in CRC induced by PV-1(S). With CRC of recombinant viruses, the thermosensitive block covaried to nucleotide substitutions in PV-1(S) that mapped to the virus-induced RNA polymerase 3DPJ0. We conclude that plus-stranded RNA synthesis in CRC does not proceed via hairpin structures. The results of VPg-pU -> VPg-pUpU synthesis are consistent with a model in which VPg-pU is the primer of RNA synthesis mediated by 3DP01. The data suggest that uridylylation of VPg or a precursor thereof may be catalyzed by 31Y°' itself, a mechanism resembling events occurring in adenovirus DNA replication.The mechanism of genome replication of eucaryotic RNA viruses has not been solved, and that of poliovirus is no exception. As reviewed recently (14a, 39;
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