Ming-Tsan Liu

Human infection with avian influenza A H6N1 virus: an epidemiological analysis

Summary Background Avian influenza A H6N1 virus is one of the most common viruses isolated from wild and domestic avian species, but human infection with this virus has not been previously reported. We report the clinical presentation, contact, and environmental investigations of a patient infected with this virus, and assess the origin and genetic characteristics of the isolated virus. Methods A 20-year-old woman with an influenza-like illness presented to a hospital with shortness of breath in May, 2013. An unsubtyped influenza A virus was isolated from her throat-swab specimen and was transferred to the Taiwan Centres for Disease Control (CDC) for identification. The medical records were reviewed to assess the clinical presentation. We did a contact and environmental investigation and collected clinical specimens from the case and symptomatic contacts to test for influenza virus. The genomic sequences of the isolated virus were determined and characterised. Findings The unsubtyped influenza A virus was identified as the H6N1 subtype, based on sequences of the genes encoding haemagglutinin and neuraminidase. The source of infection was not established. Sequence analyses showed that this human isolate was highly homologous to chicken H6N1 viruses in Taiwan and had been generated through interclade reassortment. Notably, the virus had a G228S substitution in the haemagglutinin protein that might increase its affinity for the human α2-6 linked sialic acid receptor. Interpretation This is the first report of human infection with a wild avian influenza A H6N1 virus. A unique clade of H6N1 viruses with a G228S substitution of haemagglutinin have circulated persistently in poultry in Taiwan. These viruses continue to evolve and accumulate changes, increasing the potential risk of human-to-human transmission. Our report highlights the continuous need for preparedness for a pandemic of unpredictable and complex avian influenza. Funding Taiwan Centres for Disease Control.

Mammalian Expression of Virus-Like Particles for Advanced Mimicry of Authentic Influenza Virus

Background Influenza A viruses are major human and animal pathogens with huge economic and societal impact from illness, hospitalizations, and deaths. Virus-like particles (VLPs) of influenza virus have been suggested as a vaccine candidate offering improved safety and efficacy. To develop this concept further, we established a flexible platform to efficiently generate different subtypes of mammalian-expressed influenza VLPs. Here we demonstrate that these mammalian VLPs strongly resemble the authentic viruses in structure, particle size and composition of host factors, and even glycosylation of viral antigens. Methodology/Principal Findings In this study, a mammalian VLP system was established by stable co-expression of four influenza structural proteins (HA, NA, M1, and M2) in a Vero cell line. By replacing the surface glycoproteins of HA and NA, we converted the H3N2-VLP subtype to H5N1-VLP. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs were characterized, and the VLP constituents were identified by LC/MS/MS. Protease protection assays demonstrated that specific cellular proteins that co-purified with influenza virions were integrated into mammalian VLPs. The glycosylation profiles of mammalian VLPs as revealed by deglycosylation assays were similar to that of progeny viruses produced from Vero cells. Vaccination of mice with 2.5 µg and above of H5N1-VLP elicited H5-specific IgG1 antibodies and resulted in full protection against lethal infection with homologous virus. These results provide compelling evidence that mammalian VLPs closely emulate the exterior of authentic virus particles not only in antigen presentation but also in biological properties and should provide promising vaccine candidates. Conclusions/Significance This flexible mammalian influenza VLP system offers a superior alternative to the conventional reverse genetic vaccine platform without concerns over inadequate presentation of immune antigens or limitations imposed by the manipulation of real viruses.

Characterization of Drug-Resistant Influenza A(H7N9) Variants Isolated From an Oseltamivir-Treated Patient in Taiwan

BACKGROUND Patients contracting influenza A(H7N9) infection often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but information on drug-resistance markers for influenza A(H7N9) is limited. METHODS Four NA variants of A/Taiwan/1/2013(H7N9) virus containing a single substitution (NA-E119V, NA-I222K, NA-I222R, or NA-R292K) recovered from an oseltamivir-treated patient were tested for NAI susceptibility in vitro; their replicative fitness was evaluated in cell culture, mice, and ferrets. RESULTS NA-R292K led to highly reduced inhibition by oseltamivir and peramivir, while NA-E119V, NA-I222K, and NA-I222R caused reduced inhibition by oseltamivir. Mice infected with any virus showed severe clinical signs with high mortality rates. NA-I222K virus was the most virulent in mice, whereas virus lacking NA change (NA-WT) and NA-R292K virus seemed the least virulent. Sequence analysis suggests that PB2-S714N increased virulence of NA-I222K virus in mice; NS1-K126R, alone or in combination with PB2-V227M, produced contrasting effects in NA-WT and NA-R292K viruses. In ferrets, all viruses replicated to high titers in the upper respiratory tract but produced only mild illness. NA-R292K virus, showed reduced replicative fitness in this animal model. CONCLUSIONS Our data highlight challenges in assessment of the replicative fitness of H7N9 NA variants that emerged in NAI-treated patients.

Rapid SYBR Green I and Modified Probe Real-Time Reverse Transcription-PCR Assays Identify Influenza H1N1 Viruses and Distinguish between Pandemic and Seasonal Strains

ABSTRACT 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.

A VLP Vaccine Induces Broad-Spectrum Cross-Protective Antibody Immunity against H5N1 and H1N1 Subtypes of Influenza A Virus

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.

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.

New Variants and Age Shift to High Fatality Groups Contribute to Severe Successive Waves in the 2009 Influenza Pandemic in Taiwan

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.

Early findings of oseltamivir-resistant pandemic (H1N1) 2009 influenza A viruses in Taiwan

In this study, we investigated the frequency of oseltamivir resistance in pandemic (H1N1) 2009 influenza A viruses in Taiwan and characterized the resistant viruses. From May 2009 to January 2010, 1187 pandemic H1N1 virus-positive cases in Taiwan were tested for the H275Y substitution in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among them, eight hospitalized cases were found to be infected with virus encoding the H275Y substitution in their original specimens collected after oseltamivir treatment. The epidemiologic investigation indicated that each of the cases occurred sporadically and there was no evidence of further transmission. We monitored the variation of amino acid residues at position 275 of the NA gene in a series of specimens taken at various time-points and observed that viruses encoding the H275Y substitution differ in their fitness in vivo and in MDCK cells. Phylogenetic analysis indicated that the hemagglutinin (HA) sequences of oseltamivir-resistant pandemic H1N1 viruses exhibited greater diversity than the NA sequences and progressive changes of the HA genes from clade A1 into A2 and from there into clade A3 were observed. The resistant viruses seemed to occur in combination with diverse HA genes and a dominant NA gene. Enzymatic analysis of the viruses revealed that the ratio of NA/HA activities in oseltamivir-resistant viruses was reduced considerably compared to those in wild-type ones.

Newly Emerging Mutations in the Matrix Genes of the Human Influenza A(H1N1)pdm09 and A(H3N2) Viruses Reduce the Detection Sensitivity of Real-Time Reverse Transcription-PCR

ABSTRACT New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.

Phylogenetic and Evolutionary History of Influenza B Viruses, which Caused a Large Epidemic in 2011–2012, Taiwan

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.