Li-Ching Hsu

Serologic and Molecular Biologic Methods for SARS-associated Coronavirus Infection, Taiwan

Severe acute respiratory syndrome (SARS) has raised a global alert since March 2003. After its causative agent, SARS-associated coronavirus (SARS-CoV), was confirmed, laboratory methods, including virus isolation, reverse transcriptase–polymerase chain reaction (RT-PCR), and serologic methods, have been quickly developed. In this study, we evaluated four serologic tests ( neutralization test, enzyme-linked immunosorbent assay [ELISA], immunofluorescent assay [IFA], and immunochromatographic test [ICT]) for detecting antibodies to SARS-CoV in sera of 537 probable SARS case-patients with correlation to the RT-PCR . With the neutralization test as a reference method, the sensitivity, specificity, positive predictive value, and negative predictive value were 98.2%, 98.7%, 98.7%, and 98.4% for ELISA; 99.1%, 87.8%, 88.1% and 99.1% for IFA; 33.6%, 98.2%, 95.7%, and 56.1% for ICT, respectively. We also compared the recombinant-based western blot with the whole virus–based IFA and ELISA; the data showed a high correlation between these methods, with an overall agreement of >90%. Our results provide a systematic analysis of serologic and molecular methods for evaluating SARS-CoV infection.

Genetic diversity and C2-like subgenogroup strains of enterovirus 71, Taiwan, 2008

BackgroundHuman enterovirus 71 (EV-71) is known of having caused numerous outbreaks of hand-foot-mouth disease, and other clinical manifestations globally. In 2008, 989 EV-71 strains were isolated in Taiwan.ResultsIn this study, the genetic and antigenic properties of these strains were analyzed and the genetic diversity of EV-71 subgenogroups surfacing in Taiwan was depicted, which includes 3 previously reported subgenogroups of C5, B5, and C4, and one C2-like subgenogroup. Based on the phylogenetic analyses using their complete genome nucleotide sequences and neutralization tests, the C2-like subgenogroup forms a genetically distinct cluster from other subgenogroups, and the antisera show a maximum of 128-fold decrease of neutralization titer against this subgenogroup. In addition, the subgenogroup C4 isolates of 2008 were found quite similar genetically to the Chinese strains that caused outbreaks in recent years and thus they should be carefully watched.ConclusionsOther than to be the first report describing the existence of C2-like subgenogroup of EV-71 in Taiwan, this article also foresees a potential of subgenogroup C4 outbreaks in Taiwan in the near future.

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.

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.

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.

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.

Characterization of oseltamivir-resistant influenza A(H1N1)pdm09 viruses in Taiwan in 2009–2011

The early isolated swine‐origin influenza A(H1N1)pdm09 viruses were susceptible to oseltamivir; however, there is a concern about whether oseltamivir‐resistant influenza A(H1N1)pdm09 viruses will spread worldwide as did the oseltamivir‐resistant seasonal influenza A(H1N1) viruses in 2007–2008. In this study, the frequency of oseltamivir resistance in influenza A(H1N1)pdm09 viruses was determined in Taiwan. From May 2009 to April 2011, 1,335 A(H1N1)pdm09‐positive cases in Taiwan were tested for the H275Y mutation in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among these, 15 patients (1.1%) were found to be infected with H275Y virus. All the resistant viruses were detected after the patients have received the oseltamivir. The overall monthly ratio of H275Y‐harboring viruses ranged between 0% and 2.88%, and the peak was correlated with influenza epidemics. The genetic analysis revealed that the oseltamivir‐resistant A(H1N1)pdm09 viruses can emerged from different variants with a great diversity under drug pressure. The ratio of NA/HA activities in different clades of oseltamivir‐resistant viruses was reduced compared to those in the wild‐type viruses, indicating that the balance of NA/HA in the current oseltamivir‐resistant influenza A(H1N1)pdm09 viruses was interfered. It is possible that H275Y‐bearing A(H1N1)pdm09 virus has not yet spread globally because it lacks the essential permissive mutations that can compensate for the negative impact on fitness by the H275Y amino acid substitution in NA. Continuous monitoring the evolution patterns of sensitive and resistant viruses is required to respond to possible emergence of resistant viruses with permissive genetic background which enable the wide spread of resistance. J. Med. Virol. 85:379–387, 2013.

Secular trend of genome types of respiratory adenovirus type 3 during 1983–2005: a study from Taiwan

Genome type analysis of adenovirus type 3 (Ad3) in Taiwan identified four types (Ad3a, Ad3a2, Ad3a1, Ad3–7) during 1983–2005. Ad3a was the major type during 1983–1999, while Ad3a2 was the predominant type from 2001 to 2005. Phylogenetic analysis of the hexon gene of 23 isolates revealed that most Ad3a2 and Ad3–7 isolates belonged to one cluster, and most Ad3a isolates to the other cluster. The clinical manifestations included respiratory tract infections, acute gastroenteritis, hand-foot-and-mouth disease, febrile convulsion and pharyngoconjunctival fever. In conclusion, Ad3a2 has replaced Ad3a as the most common genome type in Taiwan since 2001.