Jih-Hui Lin

Rapid Differentiation of Influenza A Virus Subtypes and Genetic Screening for Virus Variants by High-Resolution Melting Analysis

ABSTRACT We assessed the use of high-resolution melting (HRM) analysis for the rapid identification of influenza A virus subtypes and the detection of newly emerging virus variants. The viral matrix gene was amplified by LightCycler real-time reverse transcription-PCR (RT-PCR) in the presence of the LCGreen I fluorescent dye. Upon optimization of the assay conditions, all the major influenza A virus subtypes, including H1N1, H3N2, H5N1, H7N3, and H9N2, were amplifiable by this method and had a PCR product length of 179 bp. Real-time RT-PCR of in vitro-transcribed H3N2 RNA revealed a standard curve for quantification with a linear range (correlation coefficient = 0.9935) across at least 8 log units of RNA concentrations and a detection limit of 103 copies of viral RNA. We performed HRM analysis of the PCR products with the HR-1 instrument and used the melting profiles as molecular fingerprints for virus subtyping. The virus subtypes were identified from the high-resolution derivative plot obtained by heteroduplex formation between the PCR products of the viral isolates tested and those of the reference viral isolates. The melting profiles were consistent with minimal interassay variability. Hence, an HRM database and a working protocol were established for the identification of these five influenza A virus subtypes. When this protocol was used to test 21 clinical influenza A virus isolates, the results were comparable to those obtained by RT-PCR with hemagglutinin-specific primer sets. Sequence variants of the clinical isolates (n = 4) were also revealed by our HRM analytical scheme. This assay requires no multiplexing or hybridization probes and provides a new approach for influenza A virus subtyping and genetic screening of virus variants in a clinical virology laboratory.

Clinical and genetic analysis of Human Bocavirus in children with lower respiratory tract infection in Taiwan

Abstract Background Human Bocavirus (HBoV) is a likely etiologic agent of acute respiratory disease in children. The prevalence of this virus has been studied in several sites worldwide. We conducted the first clinical and molecular study of HBoV in Taiwan at the Centers for Diseases Control, Taiwan. Objectives To investigate the genomic and epidemiologic profiles of HBoV infection in Taiwan. Study design Throat swabs or nasopharyngeal aspirates were obtained from hospitalized pediatric patients with acute lower respiratory tract infections. Specimens negative for other respiratory viruses by molecular screening were examined for HBoV. Results HBoV was the only virus detected in 30 (5.6%) of 531 samples. Of these positive cases, 56.7% were from children less than 2 years old. Two groups of HBoV co-circulated in Taiwan during the study. Results of evolutionary networks evaluation suggest that HBoV might have had an opportunity for interbreeding of viruses and genetic recombinations among the different genes. Conclusion HBoV may have circulated in Taiwan for some time and it appears to be one of the etiological agents responsible for lower respiratory tract infection in children.

Exploring the Molecular Epidemiology and Evolutionary Dynamics of Influenza A Virus in Taiwan

The evolution and population dynamics of human influenza in Taiwan is a microcosm of the viruses circulating worldwide, which has not yet been studied in detail. We collected 343 representative full genome sequences of human influenza A viruses isolated in Taiwan between 1979 and 2009. Phylogenetic and antigenic data analysis revealed that H1N1 and H3N2 viruses consistently co-circulated in Taiwan, although they were characterized by different temporal dynamics and degrees of genetic diversity. Moreover, influenza A viruses of both subtypes underwent internal gene reassortment involving all eight segments of the viral genome, some of which also occurred during non-epidemic periods. The patterns of gene reassortment were different in the two subtypes. The internal genes of H1N1 viruses moved as a unit, separately from the co-evolving HA and NA genes. On the other hand, the HA and NA genes of H3N2 viruses tended to segregate consistently with different sets of internal gene segments. In particular, as reassortment occurred, H3HA always segregated as a group with the PB1, PA and M genes, while N2NA consistently segregated with PB2 and NP. Finally, the analysis showed that new phylogenetic lineages and antigenic variants emerging in summer were likely to be the progenitors of the epidemic strains in the following season. The synchronized seasonal patterns and high genetic diversity of influenza A viruses observed in Taiwan make possible to capture the evolutionary dynamic and epidemiological rules governing antigenic drift and reassortment and may serve as a “warning” system that recapitulates the global epidemic.

Phylodynamics and Molecular Evolution of Influenza A Virus Nucleoprotein Genes in Taiwan between 1979 and 2009

Background Many studies concentrate on variation in the hemagglutinin glycoprotein (HA) because of its significance in host immune response, the evolution of this virus is even more complex when other genome segments are considered. Recently, it was found that cytotoxic T lymphocytes (CTL) play an important role in immunity against influenza and most CTL epitopes of human influenza viruses were remarkably conserved. The NP gene has evolved independently in human and avian hosts after 1918 flu pandemic and it has been assigned a putative role as a determinant of host range. Methods and Findings Phylodynamic patterns of the genes encoding nucleoprotein (NP) of influenza A viruses isolated from 1979–2009 were analyzed by applying the Bayesian Markov Chain Monte Carlo framework to better understand the evolutionary mechanisms of these Taiwanese isolates. Phylogenetic analysis of the NP gene showed that all available H3 worldwide isolates collected so far were genetically similar and divided into two major clades after the year 2004. We compared the deduced amino acid sequences of the NP sequences from human, avian and swine hosts to investigate the emergence of potential adaptive mutations. Overall, selective pressure on the NP gene of human influenza A viruses appeared to be dominated by purifying selection with a mean dN/dS ratio of 0.105. Site-selection analysis of 488 codons, however, also revealed 3 positively selected sites in addition to 139 negatively selected ones. Conclusions The demographic history inferred by Bayesian skyline plot showed that the effective number of infections underwent a period of smooth and steady growth from 1998 to 2001, followed by a more recent rise in the rate of spread. Further understanding the correlates of interspecies transmission of influenza A virus genes from other host reservoirs to the human population may help to elucidate the mechanisms of variability among influenza A virus.

Molecular epidemiologic and clinical characteristics of influenza B-associated complications among hospitalized patients during an outbreak in Taiwan

OBJECTIVES To study the resurgence of influenza B/Yam in Taiwan and summarize clinical findings of influenza B-associated complications among hospitalized patients, in particular the link between clinical and molecular epidemiologic characteristics. METHODS Clinical information and isolates were collected through the national surveillance system of the Taiwan Centers for Disease Control. Potential risk factors associated with severe illness were analyzed. Antigenic and genetic analysis of representative hemagglutinin (HA) nucleotide sequences was performed. RESULTS Of 326 patients admitted to the intensive care unit (ICU), 63.2% were aged ≤18 years or ≥65 years and 12.9% were adults aged 19-49 years. Most of the cases had underlying medical conditions before admission, and more fatal cases had chronic medical conditions than those who convalesced in the ICU. Results of the phylogenetic analysis showed that the majority of isolates from fatal cases in Taiwan were in group 2 (represented by B/Massachusetts/2/2012-like) rather than group 3, which was the predominant group of strains circulating in other Asian countries. CONCLUSIONS Our findings suggest a regional trend of influenza B viruses and showed that new phylogenetic lineages and antigenic variants emerging in neighboring countries were likely to be the progenitors of the epidemic strains in the following seasons.

Molecular Epidemiology and Phylogenetic Analysis of Human Adenovirus Caused an Outbreak in Taiwan during 2011.

An outbreak of adenovirus has been surveyed in Taiwan in 2011. To better understand the evolution and epidemiology of adenovirus in Taiwan, full-length sequence of hexon and fiber coapsid protein was analyzed using series of phylogenetic and dynamic evolution tools. Six different serotypes were identified in this outbreak and the species B was predominant (HAdV-3, 71.50%; HAdV-7, 15.46%). The most frequent diagnosis was acute tonsillitis (54.59%) and bronchitis (47.83%). Phylogenetic analysis revealed that hexon protein gene sequences were highly conserved for HAdV-3 and HAdV-7 circulation in Taiwan. However, comparison of restriction fragment length polymorphism (RFLP) analysis and phylogenetic trees of fiber gene in HAdV-7 clearly indicated that the predominant genotype in Taiwan has shifted from 7b to 7d. Several positive selection sites were observed in hexon protein. The estimated nucleotide substitution rates of hexon protein of HAdV-3 and HAdV-7 were 0.234×10-3 substitutions/site/year (95% HPD: 0.387~0.095×10-3) and 1.107×10-3 (95% HPD: 0. 541~1.604) respectively; those of the fiber protein of HAdV-3 and HAdV-7 were 1.085×10-3 (95% HPD: 1.767~0.486) and 0.132×10-3 (95% HPD: 0.283~0.014) respectively. Phylodynamic analysis by Bayesian skyline plot (BSP) suggested that using individual gene to evaluate the effective population size might possibly cause miscalculation. In summary, the virus evolution is ongoing, and continuous surveillance of this virus evolution will contribute to the control of the epidemic.

Spatial Temporal Dynamics and Molecular Evolution of Re-Emerging Rabies Virus in Taiwan

Taiwan has been recognized by the World Organization for Animal Health as rabies-free since 1961. Surprisingly, rabies virus (RABV) was identified in a dead Formosan ferret badger in July 2013. Later, more infected ferret badgers were reported from different geographic regions of Taiwan. In order to know its evolutionary history and spatial temporal dynamics of this virus, phylogeny was reconstructed by maximum likelihood and Bayesian methods based on the full-length of glycoprotein (G), matrix protein (M), and nucleoprotein (N) genes. The evolutionary rates and phylogeographic were determined using Beast and SPREAD software. Phylogenetic trees showed a monophyletic group containing all of RABV isolates from Taiwan and it further separated into three sub-groups. The estimated nucleotide substitution rates of G, M, and N genes were between 2.49 × 10−4–4.75 × 10−4 substitutions/site/year, and the mean ratio of dN/dS was significantly low. The time of the most recent common ancestor was estimated around 75, 89, and 170 years, respectively. Phylogeographic analysis suggested the origin of the epidemic could be in Eastern Taiwan, then the Formosan ferret badger moved across the Central Range of Taiwan to western regions and separated into two branches. In this study, we illustrated the evolution history and phylogeographic of RABV in Formosan ferret badgers.

Molecular epidemiology and antigenic analyses of influenza A viruses H3N2 in Taiwan

The severity of an influenza epidemic season may be influenced not only by variability in the surface glycoproteins, but also by differences in the internal proteins of circulating influenza viruses. To better understand viral antigenic evolution, all eight gene segments from 44 human H3N2 epidemic strains isolated during 2004-2008 in Taiwan were analyzed to provide a profile of protein variability. Comparison of the evolutionary profiles of the HA, NA and PB2 genes of influenza A (H3N2) viruses indicated that they were derived from a group of H3N2 isolates first seen in 2004. However, the PA, M and PB1 genes were derived from a different group of H3N2 isolates from 2004. Tree topology revealed the NP and NS genes could each be segregated into two groups similar to those for the polymerase genes. In addition, new genetic variants occurred during the non-epidemic period and become the dominant strain after one or two seasons. Comparison of evolutionary patterns in consecutive years is necessary to correlate viral genetic changes with antigenic changes as multiple lineages co-circulate.

Surveillance of upper respiratory infections using a new multiplex PCR assay compared to conventional methods during the influenza season in Taiwan

Abstract Objectives To improve diagnosis as part of laboratory surveillance in Taiwan, influenza-like illness (ILI) surveillance was conducted using a new multiplex PCR assay (FilmArray) and the results compared to those of conventional methods The study was performed during the winter months. Methods Throat swabs from patients with an ILI presenting to physicians in sentinel practices were collected during the 2016–2017 influenza season. Results A total of 52 samples tested positive by FilmArray Respiratory Panel. Forty percent were influenza A virus, and subtype H3N2 virus was the major epidemic strain. However, nearly 60% of ILI cases seen at sentinel sites were caused by non-influenza pathogens. The results of the FilmArray assay and cell culture were identical, and this assay was more sensitive than a rapid influenza diagnostic test. Genetic analyses revealed new influenza A H3N2 variants belonging to a novel subclade 3C.2a2. Conclusions The FilmArray assay facilitates urgent testing and laboratory surveillance for common viral and bacterial respiratory pathogens. This study demonstrated the use of a highly sensitive assay using clinical samples that is feasible for application worldwide. This may lead to an increased rate of diagnosis of viral infections and to improved patient outcomes, and in particular to a reduction in the overuse of antibiotics and antivirals.

Molecular epidemiological analysis of Saffold cardiovirus genotype 3 from upper respiratory infection patients in Taiwan

BACKGROUND Saffold cardiovirus (SAFV) belongs to the Cardiovirus genus of Picornaviridae family, and may be a relevant new human pathogen; Thus far, eleven genotypes have been identified. The SAFV type 3 (SAFV-3) is thought to be the major genotype and is detected relatively frequently in children with acute gastroenteritis and respiratory illness. The epidemiology and pathogenicity of SAFV-3 remain unclear. OBJECTIVES To investigate the genomic and epidemiologic profiles of SAFV-3 infection in Taiwan. STUDY DESIGN Virus was detected in respiratory samples from children suffering for URI. SAFV-3 isolates were detected by isolation on cell culture and IF assay. The molecular typing was performed by RT-PCR and was sequenced to compare with reference strains available in the NCBI GeneBank. Serum samples were collected from 2005 to 2013 in Taiwan for seroprevalence investigation. RESULTS A total of 226 specimens collected from children with URIs, 22 (9.73%) were positive for SAFV-3. The majority of SAFV-3 infections were found in children less than 6 years of age (14 of 22, 63.6%). Genetic analysis of VP1 coding region of Taiwanese isolates shown an 83.2-97.7% difference from other available SAFV-3 sequences in NCBI GenBank. Phylogenetic analysis revealed there is three genetic groups of SAFV-3 co-circulated in Taiwan during the study period. In addition, seroprevalence investigation results indicated that SAFV-3 infection occurs early in life and 43.7-77.8% of children aged between 6 months to 9 years old, had neutralizing antibodies against SAFV-3. CONCLUSION SAFV-3 may have circulated in Taiwan for some time and it appears to be one of the etiological agents responsible for URIs in children.