Prasert Auewarakul

Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2–4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003–04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.

Studies of H5N1 Influenza Virus Infection of Pigs by Using Viruses Isolated in Vietnam and Thailand in 2004

ABSTRACT To determine whether avian H5N1 influenza viruses associated with human infections in Vietnam had transmitted to pigs, we investigated serologic evidence of exposure to H5N1 influenza virus in Vietnamese pigs in 2004. Of the 3,175 pig sera tested, 8 (0.25%) were positive for avian H5N1 influenza viruses isolated in 2004 by virus neutralization assay and Western blot analysis. Experimental studies of replication and transmissibility of the 2004 Asian H5N1 viruses in pigs revealed that all viruses tested replicated in the swine respiratory tract but none were transmitted to contact pigs. Virus titers from nasal swabs peaked on day 2, and low titers were detected in the liver of two of the four pigs tested. Our findings indicate that pigs can be infected with highly lethal Asian H5N1 viruses but that these viruses are not readily transmitted between pigs under experimental conditions.

An Avian Influenza H5N1 Virus That Binds to a Human-Type Receptor

ABSTRACT Avian influenza viruses preferentially recognize sialosugar chains terminating in sialic acid-α2,3-galactose (SAα2,3Gal), whereas human influenza viruses preferentially recognize SAα2,6Gal. A conversion to SAα2,6Gal specificity is believed to be one of the changes required for the introduction of new hemagglutinin (HA) subtypes to the human population, which can lead to pandemics. Avian influenza H5N1 virus is a major threat for the emergence of a pandemic virus. As of 12 June 2007, the virus has been reported in 45 countries, and 312 human cases with 190 deaths have been confirmed. We describe here substitutions at position 129 and 134 identified in a virus isolated from a fatal human case that could change the receptor-binding preference of HA of H5N1 virus from SAα2,3Gal to both SAα2,3Gal and SAα2,6Gal. Molecular modeling demonstrated that the mutation may stabilize SAα2,6Gal in its optimal cis conformation in the binding pocket. The mutation was found in approximately half of the viral sequences directly amplified from a respiratory specimen of the patient. Our data confirm the presence of H5N1 virus with the ability to bind to a human-type receptor in this patient and suggest the selection and expansion of the mutant with human-type receptor specificity in the human host environment.

Apoptosis and Pathogenesis of Avian Influenza A (H5N1) Virus in Humans

Apoptosis may play a crucial role in the pathogenesis of pneumonia and lymphopenia caused by this virus in humans.

Identification of prohibitin as a Chikungunya virus receptor protein

Chikungunya virus (CHIKV) has recently re‐emerged causing millions of infections in countries around the Indian Ocean. While CHIKV has a broad host cell range and productively infects a number of different cell types, macrophages have been identified as a potential viral reservoir serving to increase the duration of symptoms. To date no CHIKV interacting protein has been characterized and this study sought to identify CHIKV binding proteins expressed on target cell membranes. Two‐dimensional virus overlay identified prohibitin (PHB) as a microglial cell expressed CHIKV binding protein. Co‐localization, co‐immunoprecipitation as well as antibody and siRNA mediated infection inhibition studies all confirmed a role for PHB in mediating internalization of CHIKV into microglial cells. PHB is the first identified CHIKV receptor protein, and this study is evidence that PHB may play a role in the internalization of multiple viruses. J. Med. Virol. 84:1757–1770, 2012.

Double-Stranded RNA Adenosine Deaminases Enhance Expression of Human Immunodeficiency Virus Type 1 Proteins

ABSTRACT ADARs (adenosine deaminases that act on double-stranded RNA) are RNA editing enzymes that catalyze a change from adenosine to inosine, which is then recognized as guanosine by translational machinery. We demonstrate here that overexpression of ADARs but not of an ADAR mutant lacking editing activity could upregulate human immunodeficiency virus type 1 (HIV-1) structural protein expression and viral production. Knockdown of ADAR1 by RNA silencing inhibited HIV-1 production. Viral RNA harvested from transfected ADAR1-knocked-down cells showed a decrease in the level of unspliced RNA transcripts. Overexpression of ADAR1 induced editing at a specific site in the env gene, and a mutant with the edited sequence was expressed more efficiently than the wild-type viral genome. These data suggested the role of ADAR in modulation of HIV-1 replication. Our data demonstrate a novel mechanism in which HIV-1 employs host RNA modification machinery for posttranscriptional regulation of viral protein expression.

Chikungunya in Southeast Asia: understanding the emergence and finding solutions

In the last few years, chikungunya has become a major problem in Southeast Asia, with large numbers of cases being reported in Singapore, Malaysia, and Thailand. Much of the current epidemic of chikungunya in Southeast Asia is being driven by the emergence of a strain of chikungunya virus that originated in Africa and spread to islands in the Indian Ocean, as well as to India and Sri Lanka, and then onwards to Southeast Asia. There is currently no specific treatment for chikungunya and no vaccine is available for this disease. This review seeks to provide a short update on the reemergence of chikungunya in Southeast Asia and the prospects for control of this disease.

Targeted expression of the E6 and E7 oncogenes of human papillomavirus type 16 in the epidermis of transgenic mice elicits generalized epidermal hyperplasia involving autocrine factors.

The E6 and E7 early genes of human papillomavirus type 16 have been shown in vitro to play a central role in the transforming capability of this virus. To explore their effects on differentiating epithelial cells in vivo, we used a bovine cytokeratin 10 (K10) promoter to target the expression of E6 and E7 to the suprabasal layers of the epidermis of transgenic mice. In two different lines of mice efficiently expressing the transgene, animals displayed generalized epidermal hyperplasia, hyperkeratosis and parakeratosis in the skin and the forestomach, both known to be sites of K10 expression. Northern (RNA) blot analysis revealed high levels of E6 and E7 transcripts, and in situ hybridizations localized these transcripts to the suprabasal strata of epidermis. In vivo labeling of proliferating cells showed two distinct effects of E6 and E7 expression in the epidermis: (i) an increase in the number of growing cells in the undifferentiated basal layer and (ii) abnormal proliferation of differentiated cells in the suprabasal strata. The expression of c-myc in the skin of transgenics was higher than that in control animals. The induction of c-myc transcription by topical application of tetradecanoyl phorbol acetate was prevented by simultaneous treatment with transforming growth factor beta 1 in nontransgenic skin but not in transgenic skin. In addition, transforming growth factor alpha was found to be overexpressed in the suprabasal layers of the transgenic epidermis. These findings suggest that autocrine mechanisms are involved in the development and maintenance of epidermal hyperplasia. Animals of both lines developed papillomas in skin sites exposed to mechanical irritation and wounding, suggesting that secondary events are necessary for progression to neoplasia. Collectively, these results provide new insights into the tumor promoter activities of human papillomavirus type 16 in epithelial cells in vivo.

A simplified and economical intradermal regimen of purified chick embryo cell rabies vaccine for postexposure prophylaxis

Healthy volunteers were randomized to receive either intradermal purified chick embryo cell rabies vaccine (PCEC) alone (0.1 ml at each of two sites on days 0, 3 and 7, and at one site on days 28 and 90) (n = 81), or intradermal PCEC with one dose of human rabies immunoglobulin (HRIG) intramuscularly at 20 IU kg-1 on day 0 (n = 52). Neutralizing antibody (NAB) was detectable in every volunteer, in both groups, from day 14 up to day 365. The peak NAB occurred on day 28 in both groups. No significant suppressive effects of HRIG on NAB response were observed. Side-effects were mild and self-limiting. These preliminary results suggest that this simplified low-dose intradermal regimen could be an alternative schedule in rabies postexposure prophylaxis, resulting in lower overall costs.

Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms.