On 25 April 2009, the WHO declared the outbreak of a novel influenza A virus (H1N1) as a ‘Public Health Event of International Concern’ under the International Health Regulations (2005) [202
]. As of 11 June 2009, the WHO raised the level of pandemic alert to phase 6 after global spread of the new virus. With our limited knowledge on the virulence and evolving features of this novel H1N1 virus, early detection and genetic characterization are critical for heightened surveillance, policy decisions, vaccine drug treatment and hospital management.
In the USA, the National Respiratory and Enteric Virus Surveillance System laboratories participate in the influenza surveillance system. At the beginning of the 2009 outbreak, the serological assays most commonly used for influenza A virus detection were unable to detect the newly emerged swine-origin influenza A H1N1 virus. The CDC developed a RRT-PCR subtyping assay that targeted the novel H1N1 virus HA and matrix genes. This RRT-PCR Swine Flu Panel assay was then approved by the FDA for clinical influenza test applications and was rapidly distributed to research and clinical laboratories worldwide to ensure that information on novel influenza A H1N1 virus infection was released in a timely and accurate manner in the international community. The sensitivity of this real-time PCR to detect viral antigens in respiratory clinical specimens was evaluated and compared with RIDTs [66
]. The results showed that the sensitivity of RIDTs compared to the RRT-PCR is 60–80% for seasonal A (H1N1), 80–83% for seasonal A (H3N2) and 40–69% for novel influenza A (H1N1) [65
In addition, other PCR-based molecular protocols were also rapidly developed and used in research and clinical laboratories during the outbreak [67
]. When the large outbreak of novel influenza A H1N1 virus occurred in Milwaukee (WI, USA) in late April 2009, He et al.
developed a rapid and accurate multiplex RT-PCR-enzyme hybridization assay (FluPlex [Medical College of Wisconsin, WI, USA]) to confirm the first infected patients in the state. This assay was designed specifically for differentiating novel influenza A viruses H1N1, and for typing influenza A and B virus, as well as subtyping H1, H2, H3, H5, H7, H9, N1 (human and animal), N2 or N7 of influenza viruses [68
]. As a semiautomated system, FluPlex was demonstrated to be a useful platform for high-throughput detection of influenza virus. For the purpose of clinical surveillance and control, Wang et al.
developed a real-time protocol that can detect and distinguish both seasonal human H1N1 virus and the 2009 pandemic H1N1 virus in a single tube reaction [69
]. In Hong Kong, SYBR Green I real-time PCR was employed to confirm the first human infection with 2009 pandemic H1N1 virus [70
]. The 83-bp fragment of the HA gene of the virus was amplified from patient samples following a positive result with an RT-PCR test of influenza A virus matrix gene, and negative result of RT-PCR detection for influenza A virus subtypes H1 (seasonal) and H3 [69
In response to the 2009 H1N1 pandemic, most countries have enhanced and strengthened their virological surveillance systems. PCR-based molecular methodology is employed by many health officials for tracking, detecting and characterizing H1N1 virus infection [71
]. As part of the enhanced surveillance from the Chicago Department of Public Health, hospital laboratories in Chicago are now initiating RRT-PCR testing to characterize influenza strains [74
]. National Influenza Reference Centers in France developed a H1N1 virus RT-PCR protocol to identify and investigate clusters of influenza-like illness [75
]. Belgium, New Zealand, Greece and many other countries have also adopted RRT-PCR techniques for typing and subtyping influenza A, B, seasonal A (H1N1 and H3N2) and 2009 pandemic influenza A (H1N1) [76
]. Recently, the CDC RRT-PCR assay and another three RRT-PCR assays were compared and evaluated for their specificity and sensitivity for detecting pandemic influenza A (H1N1) virus [79
]. It is critical that the molecular laboratory capacity for distinguishing 2009 pandemic influenza A (H1N1) virus from other circulating influenza A viruses be expanded globally.