Taiwan Centres for Disease Control.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens, including SARS-CoV-2, in nasopharyngeal specimens from individuals with symptoms of upper respiratory tract infection. We developed and validated a rapid total nucleic acid extraction method based on real-time reverse transcription-polymerase chain reaction (RT-PCR) for the reliable, high-throughput simultaneous detection of common cold viral pathogens using the BD MAX Platform. The system was evaluated using 205 nasopharyngeal swab clinical samples. For assessment of the limit of detection (LoD), we used SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) RNA standards. The BD MAX dual multiplex real-time RT-PCR panel demonstrated a sensitivity comparable to that of the World Health Organization-recommended SARS-CoV-2 assay with an LoD of 50 copies/PCR. The LoD of influenza A/B and RSV was 100-200 copies/PCR. The overall percent agreement between the BD MAX panel and laboratory-developed RT-PCR test on 55 SARS-CoV-2-positive clinical samples was 100%; no cross-reactions were encountered. Among the 55 positive cases of COVID-19 analysed, no coinfection was detected. The BD MAX rapid multiplex PCR provides a highly sensitive, robust, and accurate assay for the rapid detection of SARS-CoV-2, influenza A/B, and RSV. Our assay could accurately identify SARS-CoV-2 and other common respiratory viral infections, shortening the turnaround time, and could thus increase the effectiveness of control and prevention measures for this emerging infectious disease.

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.

The annual recurrence of the influenza epidemic is considered to be primarily associated with immune escape due to changes to the virus. In 2011–2012, the influenza B epidemic in Taiwan was unusually large, and influenza B was predominant for a long time. To investigate the genetic dynamics of influenza B viruses during the 2011–2012 epidemic, we analyzed the sequences of 4,386 influenza B viruses collected in Taiwan from 2004 to 2012. The data provided detailed insight into the flux patterns of multiple genotypes. We found that a re-emergent TW08-I virus, which was the major genotype and had co-circulated with the two others, TW08-II and TW08-III, from 2007 to 2009 in Taiwan, successively overtook TW08-II in March and then underwent a lineage switch in July 2011. This lineage switch was followed by the large epidemic in Taiwan. The whole-genome compositions and phylogenetic relationships of the representative viruses of various genotypes were compared to determine the viral evolutionary histories. We demonstrated that the large influenza B epidemic of 2011–2012 was caused by Yamagata lineage TW08-I viruses that were derived from TW04-II viruses in 2004–2005 through genetic drifts without detectable reassortments. The TW08-I viruses isolated in both 2011–2012 and 2007–2009 were antigenically similar, indicating that an influenza B virus have persisted for 5 years in antigenic stasis before causing a large epidemic. The results suggest that in addition to the emergence of new variants with mutations or reassortments, other factors, including the interference of multi-types or lineages of influenza viruses and the accumulation of susceptible hosts, can also affect the scale and time of an influenza B epidemic.

Past influenza pandemics have been characterized by the signature feature of multiple waves. However, the reasons for multiple waves in a pandemic are not understood. Successive waves in the 2009 influenza pandemic, with a sharp increase in hospitalized and fatal cases, occurred in Taiwan during the winter of 2010. In this study, we sought to discover possible contributors to the multiple waves in this influenza pandemic. We conducted a large-scale analysis of 4703 isolates in an unbiased manner to monitor the emergence, dominance and replacement of various variants. Based on the data from influenza surveillance and epidemic curves of each variant clade, we defined virologically and temporally distinct waves of the 2009 pandemic in Taiwan from May 2009 to April 2011 as waves 1 and 2, an interwave period and wave 3. Except for wave 3, each wave was dominated by one distinct variant. In wave 3, three variants emerged and co-circulated, and formed distinct phylogenetic clades, based on the hemagglutinin (HA) genes and other segments. The severity of influenza was represented as the case fatality ratio (CFR) in the hospitalized cases. The CFRs in waves 1 and 2, the interwave period and wave 3 were 6.4%, 5.1%, 15.2% and 9.8%, respectively. The results highlight the association of virus evolution and variable influenza severity. Further analysis revealed that the major affected groups were shifted in the waves to older individuals, who had higher age-specific CFRs. The successive pandemic waves create challenges for the strategic preparedness of health authorities and make the pandemic uncertain and variable. Our findings indicate that the emergence of new variants and age shift to high fatality groups might contribute potentially to the occurrence of successive severe pandemic waves and offer insights into the adjustment of national responses to mitigate influenza pandemics.

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The recent threats of influenza epidemics and pandemics have prioritized the development of a universal vaccine that offers protection against a wider variety of influenza infections. Here, we demonstrate a genetically modified virus-like particle (VLP) vaccine, referred to as H5M2eN1-VLP, that increased the antigenic content of NA and induced rapid recall of antibody against HA2 after viral infection. As a result, H5M2eN1-VLP vaccination elicited a broad humoral immune response against multiple viral proteins and caused significant protection against homologous RG-14 (H5N1) and heterologous A/California/07/2009 H1N1 (CA/07) and A/PR/8/34 H1N1 (PR8) viral lethal challenges. Moreover, the N1-VLP (lacking HA) induced production of a strong NA antibody that also conferred significant cross protection against H5N1 and heterologous CA/07 but not PR8, suggesting the protection against N1-serotyped viruses can be extended from avian-origin to CA/07 strain isolated in humans, but not to evolutionally distant strains of human-derived. By comparative vaccine study of an HA-based VLP (H5N1-VLP) and NA-based VLPs, we found that H5N1-VLP vaccination induced specific and strong protective antibodies against the HA1 subunit of H5, thus restricting the breadth of cross-protection. In summary, we present a feasible example of direction of VLP vaccine immunity toward NA and HA2, which resulted in cross protection against both seasonal and pandemic influenza strains, that could form the basis for future design of a better universal vaccine.

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