A Comprehensive Phylogenetic Analysis of SARS-CoV-2: Utilizing a Novel and Convenient In-House RT-PCR Method for Characterization without Virus Culture and BSL-3 Facilities.

We developed a convenient method for amplifying the complete SARS-CoV-2 sequence using in-house RT-PCR without virus culture. Forty-one stored throat swabs and blood specimens were collected from eight SARS-CoV-2 infections at multiple time points. Total RNA was extracted using the QIAamp viral RNA mini kit and pooled for higher RNA levels. Only those positive specimens by commercial real-time RT-PCR (RT-qPCR) were selected and amplified by in-house RT-PCR for complete sequences, followed by sequencing. Phylogenetic trees and exploratory analyses were performed using MEGA 11 and Simplot 3.5.1 software. Swab samples had significantly higher total RNA concentrations than plasma (p < 0.01). Positive results were found mainly in swabs, but one was found in plasma. Successful gene amplification depended on Ct values (Ct < 38). A non-synonymous substitution was found in ORF1ab/Nsp3 (at NC045512.2 position 6312, C to A) and most spike protein mutations occurred in the S1 subunit (residues 14-685). The proposed method is time-saving and reliable for rapid genomic analysis. Increasing sample volume and pooling them for RNA extraction increases RNA concentration without culture. Combining nucleotide sequences from specific variable regions of the genome is more efficient than conventional methods.

Geographic Transmission and Epidemic History of HIV-1 CRF01_AE, CRF07_BC, and HCV Subtype-6w among Taiwanese Persons Who Inject Drugs.

Persons who inject drugs (PWID) and their risk-related behaviors (e.g., unprotected sex and sharing needles/syringes/other injection equipment) have caused severe public health problems, especially in the rapid spread of HIV-1 and HCV. Here, we reconstructed the epidemic history of HIV-1 circulating recombinant form (CRF) 01_AE, CRF07_BC, and HCV subtype-6w among Taiwanese PWID. The timescales were estimated using phylogenetic and Bayesian coalescent analyses. The results revealed that CRF01_AE started to circulate in the Taiwanese PWID population in central Taiwan at 1992.5 (95% credible region: 1988.8-1995.9) and spread to other regions of Taiwan, while CRF07_BC was first identified in southern Taiwan at 2000.0 (95% CR: 1997.8-2002.2) and then spread northward to central-northern Taiwan. All HCV-6 strains were from Asia (that is, China, Myanmar, Taiwan, and Vietnam) and originated in 1928.1 (95% CR: 1890.2-1966.0). Furthermore, subtype-6w isolates from different regions of Taiwan appeared to share a common source that existed in the mid-1990s (95% CR: 1985.0-2001.8) or thereabouts. The routes of drug trafficking and the resulting high prevalence of HIV-1/HCV co-infections among PWID might have contributed to the virus transmission and promoted its spread worldwide. Long-term monitoring and policy implementation in at-risk populations would be useful for disease control.

Deciphering the evolution of composite-type GSKIP in mitochondria and Wnt signaling pathways.

We previously revealed the origin of mammalian simple-type glycogen synthase kinase interaction protein (GSKIP), which served as a scavenger and a competitor in the Wnt signaling pathway during evolution. In this study, we investigated the conserved and nonconserved regions of the composite-type GSKIP by utilizing bioinformatics tools, site-directed mutagenesis, and yeast two-hybrid methods. The regions were denoted as the pre-GSK3ß binding site, which is located at the front of GSK3ß-binding sites. Our data demonstrated that clustered mitochondria protein 1 (CLU1), a type of composite-type GSKIP that exists in the mitochondria of all eukaryotic organisms, possesses the protein known as domain of unknown function 727 (DUF727), with a pre-GSK3ß-binding site and a mutant GSK3ß-binding flanking region. Another type of composite-type GSKIP, armadillo repeat containing 4 (ARMC4), which is known for cilium movement in vertebrates, contains an unintegrated DUF727 flanking region with a pre-GSK3ß-binding site (115SPxF118) only. In addition, the sequence of the GSK3ß-binding site in CLU1 revealed that Q126L and V130L were not conserved, differing from the ideal GSK3ß-binding sequence of simple-type GSKIP. We further illustrated two exceptions, namely 70 kilodalton heat shock proteins (Hsp70/DnaK) and Mitofilin in nematodes, that presented an unexpected ideal GSK3ß-binding region with a pre-GSK3ß sequence; this composite-type GSKIP could only occur in vertebrate species. Furthermore, we revealed the importance of the pre-GSK3ß-binding site (118F or 118Y) and various mutant GSK3ß-binding sites of composite-type GSKIP. Collectively, our data suggest that the new composite-type GSKIP starts with a DUF727 domain followed by a pre-GSK3ß-binding site, with the subsequent addition of the GSK3ß-binding site, which plays vital roles for CLU1, Mitofilin, and ARMC4 in mitochondria and Wnt signaling pathways during evolution.

Phylodynamic analysis and spike protein mutations in porcine deltacoronavirus with a new variant introduction in Taiwan.

Porcine deltacoronavirus (PDCoV) is a highly transmissible intestinal pathogen that causes mild to severe clinical symptoms, such as anorexia, vomiting, and watery diarrhea in pigs. By comparing the genetic sequences of the spike glycoprotein between historical and current Taiwanese PDCoV strains, we identified a novel PDCoV variant that displaced the PDCoV responsible for the 2015 epidemic. This PDCoV variant belongs to a young population within the US lineage, and infected pigs carry high concentrations of the virus. It also has several critical point mutations and an amino acid insertion at position 52 that may enhance the affinity between the B-cell epitopes located in the N-terminal domain with its complementarity regions, consequently facilitating binding or penetration between the fusion peptide and cellular membrane. Furthermore, viral protein structure prediction demonstrated that these amino acid changes may change the ability of the virus to bind to the receptor, which may consequently alter virus infectivity. Our results hence suggest the emergence of new PDCoV strains in Taiwan with the potential for greater transmission and pathogenesis.

Detection and genetic characterization of the novel torque teno virus group 6 in Taiwanese general population.

Torque teno virus (TTV) is one of the most common human viruses and can infect an individual with multiple genotypes chronically and persistently. TTV group 6 is a recently discovered phylogenetic group first isolated from eastern Taiwan indigenes, but whether the TTV group 6 was also prevalent in the general population still unknown. One hundred and three randomly collected blood samples from general population and 66 TTV positive DNA samples extracted from Taiwan indigenes were included. A group-6-specific PCR was developed for re-screen over TTV positive samples. Two TTV group 6 positive samples from general population were cloned and sequenced for identifying mix-infected TTVs and confirming their classification by maximum-likelihood and Bayesian inference phylogeny. TTV group 6 can be detected in 4.5% (4/89) and 7.6% (5/66) of TTV positive samples from Taiwanese general population and eastern Taiwan indigenes, respectively. Sample VC09 was mix-infected with TTV groups 3 and 6. Sample VC99 was mix-infected with TTV groups 3, 4 and 6. A highly diverse triple overlapping region was observed, which may represent a unique phenomenon of TTV. The group-6-specific PCR can successfully detect TTV group 6. TTV group 6 may be prevalent worldwide regardless of the geographic region and/or ethnic groups.

Source identification of HIV-1 transmission in three lawsuits Using Ultra-Deep pyrosequencing and phylogenetic analysis.

BACKGROUND/PURPOSE: Intentional transmission of HIV-1 is a crime. Identifying the source of transmission between HIV-1 infected cases using phylogenetic analysis has limitations, including delayed examinations after the initiation of infection and ambiguity of phyletic relationships. This study was the first to introduce phylogenetic tree Results as forensic evidence in a trial in Taiwan. METHODS: Three lawsuit cases from different district courts in Taiwan were chosen for this study. We identified the source of transmission between individuals in each lawsuit based on the maximum likelihood and Bayesian phylogenetic tree analyses using the HIV-1 sequences from molecular cloning and ultra-deep pyrosequencing (UDPS). Two gene regions of the HIV genome, env and gag, were involved. RESULTS: The results of phylogenetic analysis using sequences from molecular cloning were clear and evidential enough in lawsuits 1 and 3. Due to the delayed sampling time, the result of sequences from molecular cloning in lawsuit 2 was ambiguous. Combined with the analyzed result of sequences from UDPS and epidemiological information, the source of transmission in lawsuit 2 was further identified. CONCLUSION: Hence phylogenetic analyses cannot exclude the possibility of unsampled intermediaries, the data interpretation should be more careful and conservative, and it should not be considered as the only evidence for the source identification in a trial without epidemiological or serological information. The evaluation of the introduced UDPS method in the identification of transmission source has shown that the validity and evidential effects were still limited and need further optimization.

Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan, 2008-2017.

In this study, we investigated the molecular evolution and phylodynamics of respiratory syncytial virus (RSV) over 10 consecutive seasons (2008-2017) and the genetic variability of the RSV genotypes ON1 and BA in central Taiwan. The ectodomain region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the second hypervariable region of the G protein in RSV ON1 and BA were analyzed. A total of 132 RSV-A and 81 RSV-B isolates were obtained. Phylogenetic analysis revealed that the NA1, ON1, and BA9 genotypes were responsible for the RSV epidemics in central Taiwan in the study period. For RSV-A, the NA1 genotype predominated during the 2008-2011 seasons. The ON1 genotype was first detected in 2011 and replaced NA1 after 2012. For RSV-B, the BA9 and BA10 genotypes cocirculated from 2008 to 2010, but the BA9 genotype has predominated since 2012. Amino acid sequence alignments revealed the continuous evolution of the G gene in the ectodomain region. The predicted N-glycosylation sites were relatively conserved in the ON1 (site 237 and 318) and BA9 (site 296 and 310) genotype strains. Our results contribute to the understanding and prediction of the temporal evolution of RSV at the local level.

Genetic Diversity and Phylogenetic Analysis of Human Bocavirus 2 in Pediatric Patients with Acute Gastroenteritis in Taiwan.

Human bocavirus (HBoV) is a causative agent of respiratory and gastrointestinal diseases worldwide. Four HBoV species (HBoV1-4) have been identified so far. Although a previous report has documented the HBoV association with acute gastroenteritis (AGE) in Taiwan, their epidemiology, genetic diversity, and phylogenetic relationships remain unclear. In this study, we focused on an investigation of these unsolved issues, which will help to reveal molecular epidemiology and phylogeny of the circulating HBoV2 in Taiwan. A total of 176 stool samples were collected from children with AGE for this study. PCR amplification and sequencing on the VP1 gene region were used to identify species. Phylogenetic analysis was conducted by maximum-likelihood and neighbor-joining methods. Selection pressure was also estimated to obtain HBoV evolutionary information. Our results showed the prevalence of HBoV in AGE children was 8.5%, of which HBoV1 was the predominant species (6.3%), followed by HBoV2 (2.3%). Phylogenetic analysis showed those Taiwanese HBoV2 strains have significant genetic variability and can be divided into two clusters. One belongs to HBoV2 genotype A and the other forms an independent unclassified cluster. The nucleotide distance between that independent cluster and the known HBoV2 genotypes was more than 5%, suggesting a new HBoV2 genotype. No positive selection site was found and the virus was under purifying selection. This is the first report to reveal HBoV2 genetic diversity and phylogenetic relationships among AGE children in Taiwan. We find that HBoV2 may have been introduced into the country by multiple origins, and a potential new HBoV2 genotype is proposed.

Increased Detection of Viruses in Children with Respiratory Tract Infection Using PCR.

Respiratory viruses are a common cause of respiratory tract infection (RTI), particularly in neonates and children. Rapid and accurate diagnosis of viral infections could improve clinical outcomes and reduce the use of antibiotics and treatment sessions. Advances in diagnostic technology contribute to the accurate detection of viruses. We performed a multiplex real-time polymerase chain reaction (PCR) to investigate the viral etiology in pediatric patients and compared the detection rates with those determined using traditional antigen tests and virus cultures. Fifteen respiratory viruses were included in our investigation: respiratory syncytial virus A/B (RSV), influenza virus A (FluA) and influenza virus B (FluB), human metapneumovirus (MPV), enterovirus (EV), human parainfluenza virus (PIV) types 1-4, human rhinovirus (RV), human coronavirus OC43, NL63, and 229E, human adenovirus (ADV), and human bocavirus (Boca). In total, 474 specimens were collected and tested. Respiratory viruses were detected more frequently by PCR (357, 75.3%) than they were by traditional tests (229, 49.3%). The leading pathogens were RSV (113, 23.8%), RV (72, 15.2%), PIV3 (53, 11.2%), FluA (51, 10.8%), and ADV (48, 10.1%). For children younger than 5 years, RSV and RV were most prevalent; for children older than 5 years, FluA and ADV were the most frequently detected. Of the specimens, 25.8% (92/357) were coinfected with two or more viruses. RV, Boca, PIV2, FluB, and PIV4 had higher rates of coinfection; MPV and PIV1 had the lowest rates of coinfection (9.1% and 5.3%). To conclude, the detection power of PCR was better than that of traditional antigen tests and virus cultures when considering the detection of respiratory viruses. RSV and RV were the leading viral pathogens identified in the respiratory specimens. One-quarter of the positive specimens were coinfected with two or more viruses. In the future, further application of PCR may contribute to the rapid and accurate diagnosis of respiratory viruses and could improve patient outcomes.

Phylogeographic and genetic characterization of porcine circovirus type 2 in Taiwan from 2001-2017.

Porcine circovirus type 2 (PCV2) is an important pathogen that causes significant economic losses in the swine industry worldwide. Five major PCV2 genotypes have been identified, including PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e. To investigate the prevalence and phylodynamics of the different PCV2 genotypes in Taiwan, 214 PCV2 ORF2 sequences from Taiwan and other countries were analyzed. Genotypic differences were observed among PCV2a, 2b, and 2d at amino acid position 89 in ORF2, with isoleucine (I), arginine (R), and leucine (L), respectively. Similar to other countries, a genotypic shift was also observed in Taiwan, where the predominant genotype shifted from PCV2b to 2d after 2010. The estimated nucleotide substitution rate of Taiwanese strains in the ORF2 region was 8.467 × 10-4 substitutions per site per year. This rapid evolution rate of PCV2 may lead to the genotypic shift observed in Taiwan. The times to the most recent common ancestor (TMRCA) for PCV2a, -2b, and -2d-2 was dated to 1970, 1992 and 2004, respectively. Thus, the PCV2a, -2b, and -2d genotypes were already present in Taiwan before the introduction of the PCV2 vaccine.

Persistence and continuous evolution of the human respiratory syncytial virus in northern Taiwan for two decades.

The study aimed to characterize the molecular epidemiology, phylogenetic relationship, and population dynamics of the G protein gene in clinical respiratory syncytial virus (RSV) strains isolated from northern Taiwan. We analyzed a total of 160 and 116 G protein gene sequences of RSV-A and RSV-B representative strains, respectively, from 804 clinical viral stocks collected between July 2000 and June 2016. Population dynamic patterns of the RSV G protein gene were analyzed using Bayesian inference through the Markov chain Monte Carlo framework. A phylogenetic analysis revealed that RSV-A from Taiwan could be categorized into GA2, GA5, and GA7 lineages. GA2 of RSV-A could be further divided into NA1, NA2, NA4, and ON1 clades. These RSV-A lineages has been replaced over time, whereas RSV-B strains from Taiwan continually evolved from a single lineage with significant time-dependent waves. Four putative positive selection sites were observed in both RSV-A and RSV-B. The Bayesian skyline plot revealed that the local population dynamics of RSV were associated with lineage displacement events. Both circulating subtypes and population dynamics represented a unique local pattern. Our results affirm the necessity of continuing molecular surveillance of RSV to attain a more comprehensive understanding of epidemics.

Origin and dissemination of hepatitis B virus genotype C in East Asia revealed by phylodynamic analysis and historical correlates.

Hepatitis B virus disease progression in East Asia is most frequently associated with genotype C (HBV/C). The increasing availability of HBV/C genetic sequences and detailed annotations provides an opportunity to investigate the epidemiological factors underlying its evolutionary history. In this study, the Bayesian phylogeography framework was used to investigate the origins and patterns in spatial dissemination of HBV/C by analyzing East Asian sequences obtained from 1992 to 2010. The most recent common ancestor of HBV/C was traced back to the early 1900s in China, where it eventually diverged into two major lineages during the 1930s-1960s that gave rise to distinct epidemic waves spreading exponentially to other East Asian countries and the USA. Demographic inference of viral effective population size over time indicated similar dynamics for both lineages, characterized by exponential growth since the early 1980s, followed by a significant bottleneck in 2003 and another increase after 2004. Although additional factors cannot be ruled out, we provide evidence to suggest this bottleneck was the result of limited human movement from/to China during the SARS outbreak in 2003. This is the first extensive evolutionary study of HBV/C in East Asia as well as the first to assess more realistic spatial ecological influences between co-circulating infectious diseases.

The origin of GSKIP, a multifaceted regulatory factor in the mammalian Wnt pathway.

GSK3ß interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3ß and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3ß interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3ß binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3ß in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3ß binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3ß compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3ß activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3ß interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3ß away from the ß-catenin destruction complex and as a competitor to compete for GSK3ß binding, resulting in accumulation of S675 phosphorylated ß-catenin.

Phylodynamic and Genetic Diversity of Canine Parvovirus Type 2c in Taiwan.

Canine parvovirus type 2c (CPV-2c) emerged in 2000 and is known for causing a more severe disease than other CPV-2 variants in puppies. In 2015, the emerging CPV-2c variant was isolated in Taiwan and it subsequently became the predominant variant. To trace the evolution of Taiwanese CPV-2c, we compared complete VP2 genes of CPV-2c from Taiwan and sequences obtained from GenBank. The evolutionary rate of CPV-2c was estimated to be 4.586 × 10-4 substitutions per site per year (95% highest posterior density (HPD) was 3.284-6.076 × 10-4). The time to the most recent common ancestor (TMRCA) dated to 1990 (95% HPD: 1984-1996) and 2011 (95% HPD: 2010-2013) for the CPV-2c variant and Taiwanese isolates, respectively. The CPV-2c variant isolated from Taiwan was clustered with CPV-2c from China. This phylogenetic clade began to branch off in approximately 2010 (95% HPD was 3.823-6.497). Notably, two unique mutations of Taiwanese CPV-2c were found, Q383R and P410L. In summary, this is the first report on the genome evolution of CPV-2c in Taiwan, revealing that this CPV-2c variant shares a common evolutionary origin with strains from China. The demographic history inferred by the Bayesian skyline plot showed that the effective population of CPV-2c increased until 2006 and then slowly declined until 2011.

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

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.

Indigenous Wildlife Rabies in Taiwan: Ferret Badgers, a Long Term Terrestrial Reservoir.

The emerging disease of rabies was confirmed in Taiwan ferret badgers (FBs) and reported to the World Organization for Animal Health (OIE) on July 17, 2013. The spread of wildlife rabies can be related to neighborhood countries in Asia. The phylogenetic analysis was conducted by maximum likelihood (ML) methods and the Bayesian coalescent approach based on the glycoprotein (G) and nucleoprotein (N) genes. The phylogeographic and spatial temporal dynamics of viral transmission were determined by using SPREAD, QGIS. Therefore, the origin and the change with time of the viruses can be identified. Results showed the rabies virus of FB strains in Taiwan is a unique clade among other strains in Asia. According to the phylogeographic coalescent tree, three major genotypes of the FB rabies virus have circulated in three different geographical areas in Taiwan. Two genotypes have distributed into central and southern Taiwan between two ecological river barriers. The third genotype has been limited in southeastern Taiwan by the natural mountain barrier. The diversity of FB rabies viruses indicates that the biological profile of FBs could vary in different geographical areas in Taiwan. An enhanced surveillance system needs to be established near the currently identified natural barriers for early warnings of the rabies virus outbreak in Taiwan.

Molecular Evolution and Phylodynamics of Acute Hepatitis B Virus in Japan.

Hepatitis B virus (HBV) is prevalent worldwide and causes liver diseases, including acute and chronic hepatitis. Ten HBV genotypes (A-J) with distinct geographic distributions have been reported. Cases of acute HBV infection with genotype A have increased in Japan nationwide since the 1990s, mainly through sexual transmission. To investigate the molecular evolution and phylodynamics of HBV genotypes, we collected acute HBV isolates acquired in Japan from 1992-2002. Full genomes were obtained for comprehensive phylogenetic and phylodynamic analysis, with other Japanese HBV sequences from GenBank that were isolated during 1991-2010. HBV genotypes were classified using the maximum-likelihood and Bayesian methods. The GMRF Bayesian Skyride was used to estimate the evolution and population dynamics of HBV. Four HBV genotypes (A, B, C, and H) were identified, of which C was the major genotype. The phylodynamic results indicated an exponential growth between the 1960s and early 1990s; this was followed by a population bottleneck after 1995, possibly linked with successful implementation of a nationwide vaccination program. However, HBV/A increased from 1990 to 2003-2004, and then started to decrease. The prevalence of genotype A has increased over the past 10 years. Phylodynamic inference clearly demonstrates a steady population growth compatible with an ongoing subepidemic; this might be due to the loss of immunity to HBV in adolescents and people being born before the vaccination program. This is the first phylodynamic study of HBV infection in Japan and will facilitate understanding the molecular epidemiology and long-term evolutionary dynamics of this virus in Japan.

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.

New Phylogenetic Groups of Torque Teno Virus Identified in Eastern Taiwan Indigenes.

Torque teno virus (TTV) is a single-stranded DNA virus highly prevalent in the world. It has been detected in eastern Taiwan indigenes with a low prevalence of 11% by using N22 region of which known to underestimate TTV prevalence excessively. In order to clarify their realistic epidemiology, we re-analyzed TTV prevalence with UTR region. One hundred and forty serum samples from eastern Taiwanese indigenous population were collected and TTV DNA was detected in 133 (95%) samples. Direct sequencing revealed an extensive mix-infection of different TTV strains within the infected individual. Entire TTV open reading frame 1 was amplified and cloned from a TTV positive individual to distinguish mix-infected strains. Phylogenetic analysis showed eleven isolates were clustered into a monophyletic group that is distinct from all known groups. In addition, another our isolate was clustered with recently described Hebei-1 strain and formed an independent clade. Based on the distribution pattern of pairwise distances, both new clusters were placed at phylogenetic group level, designed as the 6th and 7th phylogenetic group. In present study, we showed a very high prevalence of TTV infection in eastern Taiwan indigenes and indentified new phylogenetic groups from the infected individual. Both intra- and inter-phylogenetic group mix-infections can be found from one healthy person. Our study has further broadened the field of human TTVs and proposed a robust criterion for classification of the major TTV phylogenetic groups.

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.