Among the different hemagglutinin (HA) subtypes of avian influenza (AI) viruses, H5, H7, and H9 are of major interest because of the serious consequences for the poultry industry and the increasing frequency of direct transmission of these viruses to humans. The availability of new tools to rapidly detect and subtype the influenza viruses can enable the immediate application of measures to prevent the widespread transmission of the infection. In this study, a novel one-step real-time reverse transcription-PCR (RRT-PCR) was developed to detect simultaneously the H5, H7, and H9 subtypes of AI viruses from clinical samples of avian origin. The sensitivity of the RRT-PCR assay was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection ranging from 101 to 103 RNA copies and from 101 50% egg infectious dose (EID50)/100 μl to 102.74 EID50/100 μl with titrated viruses. Excellent results were achieved in the intra- and interassay variability tests. The comparison of the results with those obtained from the analysis of 725 avian samples by means of the reference method (virus isolation [VI]) showed a high level of agreement. To date, this is the first real-time PCR protocol available for the simultaneous detection of AI viruses belonging to subtypes H5, H7, and H9, and the results obtained indicate that this method is suitable as a routine laboratory test for the rapid detection and differentiation of the three most-important AI virus subtypes in samples of avian origin.

Development and Validation of a One-Step Real-Time PCR Assay for Simultaneous Detection of Subtype H5, H7, and H9 Avian Influenza Viruses

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.

Development of Real Time RT-PCR Assays for Detection of Type A Influenza Virus and for Subtyping of Avian H5 and H7 Hemagglutinin Subtypes

Manual of Security Sensitive Microbes and Toxins

Characterizing airborne influenza virus exposure is important for infection prevention and exposure control in health care and public settings. Detecting airborne influenza virus is important in assessing infection risk. The virus must also be protected from deterioration during aerosol sampling and long term storage. RNA preservation buffers (RNAPBs) may stabilize influenza virus after sampling and during storage. Bioaerosol samplers are used to collect airborne influenza virus, and many different types of samplers are available. The objectives of this experiment were to: 1) compare influenza virus concentrations across bioaerosol samplers; 2) compare the efficacy of RNAPB over Hanks Balanced Salt Solution (HBSS) as a sample collection media; and 3) determine whether RNAPB stabilizes viral particles stored over time. In this experiment we aerosolized active influenza virus (H1N1) in a bioaerosol chamber and compared sampling efficiencies using two different samplers: the SKC Biosampler and NIOSH Biosampler, and two different medias: Hanks Balanced Salt Solution (HBSS) and an RNAPB. Ten 15-minute experimental trials were completed. We also compared HBSS and RNAPB in terms of the maintenance of virus RNA integrity during storage at room temperatures. Samples were stored at room temperature for 1, 4, 7, and 14 days. Virus concentrations were measured and compared at each time point. Significant differences were found between sampler and media type – the SKC Biosampler collected a higher concentration of virus than the NIOSH Biosampler, and HBSS collected a higher concentration of virus than RNAPB. In storage at room temperature conditions, RNAPB maintained virus in concentrations significantly greater

Sampling for airborne influenza virus using RNA preservation buffer : a new approach

Avian Influenza Virus (Highly Pathogenic)

Avian influenza is a serious threat to both animal and human health. To enable cutting-edge research in this field, we developed a molecular test on the basis of real-time polymerase chain reaction (real-time PCR), which detects influenza virus RNA. The test enables highly sensitive detection of influenza virus A and B strains, including H5N1, and specific identification of influenza A virus H5 subtypes. The kit was tested against a broad panel of influenza A and B subtypes and other respiratory viruses in collaboration with worldwide authoritative laboratories and shows a very high specificity and sensitivity. An internal control verifies RNA extraction as well as real-time PCR success. With this kit, rapid and reliable detection of influenza A and B viruses and identification of H5 subtypes can be achieved.

J. Rossi

Papers

Sensitive and specific detection of influenza virus A subtype H5 with real-time PCR.