Ji-Rong Yang

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.

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.

Hospital-based surveillance and molecular epidemiology of rotavirus infection in Taiwan, 2005-2007.

To determine the distribution of rotavirus strains and facilitate vaccine policy decisions in Taiwan, active hospital-based gastroenteritis surveillance was conducted in three sentinel hospitals. From 1 January 2005 to 31 December 2007, a total of 3435 children less than 5 years old with gastroenteritis were enrolled. The presence of rotavirus was documented by enzyme immunoassay (EIA), and the G and P genotypes were determined by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing methods. Results confirmed that 856 (25%) of these gastroenteritis admissions were EIA-positive for rotavirus and 448 (52%) of the rotavirus positive admissions were less than 2 years old. The most prevalent rotavirus genotypes were G1P[8] (40%), followed by strains G3P[8] (27%), and G9P[8] (17%). These data will help inform decisions as to whether rotavirus vaccine should be considered for inclusion into Taiwans National Immunisation Programme.

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.

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.