Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay

BACKGROUND: Continuous outbreaks of the highly pathogenic H5N1 avian influenza A in Asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. We report here the development of a PCR-based assay that is highly specific for the H5N1 avian influenza A virus. METHODS: A one-step reverse-transcription PCR assay was developed to detect the H5N1 avian influenza A virus. The specificity of the assay was shown by testing sub-types of influenza A virus and other viral and bacterial pathogens; and on field samples. RESULTS: Validation on 145 field specimens from Vietnam and Malaysia showed that the assay was specific without cross reactivity to a number of other infuenza strains as well as human respiratory related pathogens. Detection was 100% from allantoic fluid in H5N1 positive samples, suggesting it to be a reliable sampling source for accurate detection. CONCLUSION: The assay developed from this study indicates that the primers are specific for the H5N1 influenza virus. As shown by the field tested results, this assay would be highly useful as a diagnostic tool to help identify and control influenza epidemics

Altered Immune Response of Interferon Regulatory Factor 1-Deficient Mice against Plasmodium berghei Blood-Stage Malaria Infection

Nitric oxide (NO) is a short-lived biological mediator which can be induced in various cell types and is able to cause many metabolic changes in target cells. Inhibition of tumor cell growth and antimicrobial activity has been attributed to the stimulation of NO production by transcriptional upregulation of inducible nitric oxide synthase. In the present study, we used mice devoid of functional interferon regulatory factor 1 by targeted gene disruption (IRF-1(−/−)) to investigate the role of NO in the host immune response against blood-stage Plasmodium berghei ANKA infection. IRF-1(−/−) mice survived longer with a later onset of and a lower peak parasitemia despite the inability to produce appreciable levels of NO. The administration of exogenous interleukin-12 (IL-12) was able to prolong survival in the wild-type mice with an upregulation in the expression of both gamma interferon (IFN-γ) and NO. However, the administration of IL-12 did not improve the survival of IRF-1(−/−) mice. These studies indicate that while IL-12 is able to mediate protection via an IFN-γ- and NO-dependent pathway in the wild-type mice, such a protective mechanism may not be functional in the IRF-1(−/−) mice. Our results suggest that NO may not be essential for host immunity to the parasite and that IRF-1(−/−) mice are able to induce an IFN-γ- and NO-independent mechanism against P. berghei infection