Kridsada Chaichoun

Differential Modulation of prM Cleavage, Extracellular Particle Distribution, and Virus Infectivity by Conserved Residues at Nonfurin Consensus Positions of the Dengue Virus pr-M Junction

ABSTRACT In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.

Ecologic Risk Factor Investigation of Clusters of Avian Influenza A (H5N1) Virus Infection in Thailand

This study was conducted to investigate space and time clusters of highly pathogenic avian influenza A (H5N1) virus infection and to determine risk factors at the subdistrict level in Thailand. Highly pathogenic avian influenza A (H5N1) was diagnosed in 1890 poultry flocks located in 953 subdistricts during 2004-2007. The ecologic risk for H5N1 virus infection was assessed on the basis of a spatial-based case-control study involving 824 case subdistricts and 3296 control subdistricts from 6 study periods. Risk factors investigated in clustered areas of H5N1 included human and animal demographic characteristics, poultry production systems, and wild birds and their habitats. Six variables remained statistically significant in the final model: flock density of backyard chickens (odds ratio [OR], 0.98), flock density of fighting cocks (OR, 1.02), low and high human density (OR, 0.60), presence of quail flocks (OR, 1.21), free-grazing duck flocks (OR, 2.17), and a poultry slaughterhouse (OR, 1.33). We observed a strong association between subdistricts with H5N1 virus-infected poultry flocks and evidence of prior and concomitant H5N1 infection in wild birds in the same subdistrict.

Multispecies detection of antibodies to influenza A viruses by a double-antigen sandwich ELISA.

A double-antigen sandwich ELISA was developed for the detection of antibodies to influenza A viruses. A recombinant nucleoprotein (rNP) of influenza A virus was used as a capture antigen and an HRP-conjugate for detecting the antibodies. A total of 125 serum samples from birds of different species including chickens, geese, open-billed storks, Khaki Campbell ducks, lesser whistling ducks, and pigeons with known antibodies were tested by ELISA. The sensitivity and the specificity of ELISA were found to be 98% and 97.3%, respectively. The assay was able to detect the presence of influenza A antibodies as early as the fourth day post-inoculation in ducks infected experimentally with influenza A (H5N1) virus. Excellent agreement (97.6%) was obtained between this sandwich ELISA and the hemagglutination inhibition (HI) tests (kappa=0.95). The double-antigen sandwich ELISA correlated well with a commercial avian influenza (AI) multispecies ELISA and was slightly more sensitive than the AI multispecies ELISA. These findings indicate that the double-antigen sandwich ELISA based on rNP may offer an effective screening method for serodiagnosis of influenza A virus. The double-antigen sandwich ELISA also enables the detection of antibodies to influenza A viruses in different species without the need for species-specific secondary antibodies.

Targeted Small Interfering RNA-Immunoliposomes as a Promising Therapeutic Agent against Highly Pathogenic Avian Influenza A (H5N1) Virus Infection

ABSTRACT This study describes a proof-of-concept study on the use of small interfering RNA (siRNA)-immunoliposomes as a therapeutic agent against H5N1 influenza virus infection. siRNA specific for influenza virus nucleoprotein (NP) mRNA was employed as the key antiviral agent to inhibit viral replication in this study. A humanized single-chain Fv antibody (huscFv) against the hemagglutinin (HA) of H5N1 highly pathogenic avian influenza virus (HPAI) was used as the targeting molecule to HA of H5N1 virus, which is abundantly expressed on the surface of infected cells (the HA target cells). The huscFv was applied to cationic polyethylene glycol-conjugated 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol–dioleoylphosphatidyl ethanolamine (PEGylated DC-Chol–DOPE) liposomes to generate immunoliposomes for siRNA delivery. The immunoliposomes were shown to specifically bind HA-expressing Sf9 cells and demonstrated enhanced siRNA transfection efficiency. The siRNA transfection efficiency was significantly reduced after preincubation of the HA target cells with an excess amount of free huscFv. These results therefore demonstrated that the enhanced siRNA delivery by use of immunoliposomes was mediated via targeting by huscFv. Furthermore, the siRNA silencing effect was more pronounced when the immunoliposomes were administered 6 to 12 h post-H5N1 infection in MDCK cells compared with the nontargeted liposomes. This proof-of-concept study may contribute to the future design and development of an siRNA delivery system for combating viral infectious diseases in humans.

Development of a SYBR Green I-based real-time PCR for detection of elephant endotheliotropic herpesvirus 1 infection in Asian elephants (Elephas maximus).

Elephant endotheliotropic herpesvirus 1 (EEHV1) can cause fatal hemorrhagic disease in Asian elephants (Elephas maximus). Several studies have described this virus as a major threat to young Asian elephants. A SYBR Green I-based real-time polymerase chain reaction (PCR) was developed to identify EEHV1 on trunk swabs and necropsied tissues. Two of 29 (6.9%) trunk swab samples from healthy Asian elephants were positive for EEHV1. The viruses were analyzed and classified as EEHV1A based on 231 nucleotides of the terminase gene. Necropsied spleen and heart tissue showed the highest level and second highest levels of DNA virus copy accumulation, respectively. The detection limit of the test was 276 copies/μl of DNA. There was no cross-reaction with other mammalian herpesviruses, such as herpes simplex virus 1 and equine herpesvirus 2. Inter- and intra-assay showed low coefficients of variation values indicating the reproducibility of the test. The results indicated that the test can be practically used for epidemiological study, clinical diagnosis, and management and control of EEHV1.

Serosurveillance for pandemic influenza A (H1N1) 2009 virus infection in domestic elephants, Thailand

The present study conducted serosurveillance for the presence of antibody to pandemic influenza A (H1N1) 2009 virus (H1N1pdm virus) in archival serum samples collected between 2009 and 2013 from 317 domestic elephants living in 19 provinces situated in various parts of Thailand. To obtain the most accurate data, hemagglutination-inhibition (HI) assay was employed as the screening test; and sera with HI antibody titers ≥20 were further confirmed by other methods, including cytopathic effect/hemagglutination based-microneutralization (microNT) and Western blot (WB) assays using H1N1pdm matrix 1 (M1) or hemagglutinin (HA) recombinant protein as the test antigen. Conclusively, the appropriate assays using HI in conjunction with WB assays for HA antibody revealed an overall seropositive rate of 8.5% (27 of 317). The prevalence of antibody to H1N1pdm virus was 2% (4/172) in 2009, 32% (17/53) in 2010, 9% (2/22) in 2011, 12% (1/8) in 2012, and 5% (3/62) in 2013. Notably, these positive serum samples were collected from elephants living in 7 tourist provinces of Thailand. The highest seropositive rate was obtained from elephants in Phuket, a popular tourist beach city. Young elephants had higher seropositive rate than older elephants. The source of H1N1pdm viral infection in these elephants was not explored, but most likely came from close contact with the infected mahouts or from the infected tourists who engaged in activities such as elephant riding and feeding. Nevertheless, it could not be excluded that elephant-to-elephant transmission did occur.