Rapid SYBR green I and modified probe real-time reverse transcription-PCR assays identify influenza H1N1 viruses and distinguish between pandemic and seasonal strains.

A rapid SYBR green I real-time reverse transcription-PCR (RT-PCR) assay was developed to identify pandemic influenza H1N1 virus from clinical specimens in less than 1 h. Probe real-time RT-PCR influenza A/B, H1/H3, and swNP/swHA assays were modified into the same PCR program, which allows for rapid and simultaneous typing and subtyping of influenza viruses.

Pandemic H1N1 and seasonal H3N2 influenza infection in the human population show different distributions of viral loads, which substantially affect the performance of rapid influenza tests.

A wide range of sensitivity has been reported for rapid influenza antigen tests (RIAT). In this study, we analyzed the viral loads of 778 pandemic H1N1- and 227 seasonal H3N2-virus positive clinical specimens collected during the same period and found that viral loads in pandemic H1N1 viruses was characterized by lower copy numbers than seasonal H3N2 viruses. Among various factors including the timing of specimen collection, patient age, patient gender and subtype of influenza, we found that the subtype of influenza was the most important determinant of viral load. To investigate whether these different patterns of viral load distribution affect the clinical performance of RIAT, the RIAT reagent itself and the various virus subtypes were considered and analyzed further. Based on three strategies, including cut-off values, performance on a subset of clinical specimens and evaluated performance curve of the Espline influenza A&B-N RIAT, the clinical sensitivities were 48.7-55.9% for pandemic H1N1 and 64.0-70.5% for seasonal H3N2 viruses in this study. These results indicate that the distributions of viral loads of different influenza A subtypes substantially influence the sensitivity of RIAT for clinical specimens. The lower sensitivity of RIAT for pandemic H1N1 than seasonal H3N2 virus is mainly due to differences in viral load in clinical samples rather than a diminished capacity of RIAT itself to detect these two subtypes of influenza A viruses.

Reassortment and mutations associated with emergence and spread of oseltamivir-resistant seasonal influenza A/H1N1 viruses in 2005-2009.

A dramatic increase in the frequency of the H275Y mutation in the neuraminidase (NA), conferring resistance to oseltamivir, has been detected in human seasonal influenza A/H1N1 viruses since the influenza season of 2007-2008. The resistant viruses emerged in the ratio of 14.3% and quickly reached 100% in Taiwan from September to December 2008. To explore the mechanisms responsible for emergence and spread of the resistant viruses, we analyzed the complete genome sequences of 25 viruses collected during 2005-2009 in Taiwan, which were chosen from various clade viruses, 1, 2A, 2B-1, 2B-2, 2C-1 and 2C-2 by the classification of hemagglutinin (HA) sequences. Our data revealed that the dominant variant, clade 2B-1, in the 2007-2008 influenza emerged through an intra-subtype 4+4 reassortment between clade 1 and 2 viruses. The dominant variant acquired additional substitutions, including A206T in HA, H275Y and D354G in NA, L30R and H41P in PB1-F2, and V411I and P453S in basic polymerase 2 (PB2) proteins and subsequently caused the 2008-2009 influenza epidemic in Taiwan, accompanying the widespread oseltamivir-resistant viruses. We also characterized another 3+5 reassortant virus which became double resistant to oseltamivir and amantadine. Comparison of oseltamivir-resistant influenza A/H1N1 viruses belonging to various clades in our study highlighted that both reassortment and mutations were associated with emergence and spread of these viruses and the specific mutation, H275Y, conferring to antiviral resistance, was acquired in a hitch-hiking mechanism during the viral evolutionary processes.

A new antigenic variant of human influenza A (H3N2) virus isolated from airport and community surveillance in Taiwan in early 2009.

A new variant of influenza A H3N2 virus emerged in January 2009 and became the dominant strain in Taiwan in April 2009. The variant was also detected in imported cases from various regions, including East and Southeast Asia and North America, indicating that it has circulated globally. Compared to the 2009-2010 vaccine strain, A/Brisbane/10/2007, the hemagglutinin gene of this variant exhibited five substitutions, E62K, N144K, K158N, K173Q and N189K, which are located in the antigenic sites E, A, B, D and B respectively, and it was antigenically distinct from A/Brisbane/10/2007 with more than eight-fold titer reduction in the hemagglutination inhibition reaction. The A/Perth/16/2009 (H3N2)-like virus recommended by World Health Organization for use in the 2010 southern hemisphere and 2010-2011 northern influenza seasons exhibited the same substitutions like this new variant. In addition to regional or community influenza surveillance, the imported cases or airport fever screening surveillance may be a good resource to monitor the evolution of the virus and benefit the real-time information of global influenza circulation.