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Taiwan experienced two waves of imported infections with Coronavirus Disease 2019 (COVID-19). This study aimed at investigating the genomic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Taiwan and compared their evolutionary trajectories with the global strains. We performed culture and full-genome sequencing of SARS-CoV-2 strains followed by phylogenetic analysis. A 382-nucleotides deletion in open reading frame 8 (ORF8) was found in a Taiwanese strain isolated from a patient on February 4, 2020 who had a travel history to Wuhan. Patients in the first wave also included several sporadic, local transmission cases. Genomes of 5 strains sequenced from clustered infections were classified into a new clade with ORF1ab-V378I mutation, in addition to 3 dominant clades ORF8-L84S, ORF3a-G251V and S-D614G. This highlighted clade also included some strains isolated from patients who had a travel history to Turkey and Iran. The second wave mostly resulted from patients who had a travel history to Europe and Americas. All Taiwanese viruses were classified into various clades. Genomic surveillance of SARS-CoV-2 in Taiwan revealed a new ORF8-deletion mutant and a virus clade that may be associated with infections in the Middle East, which contributed to a better understanding of the global SARS-CoV-2 transmission dynamics.

H10 subtype avian influenza viruses primarily circulate among wild waterfowl but can occasionally infect mammals, including humans, and recent sporadic human cases have raised significant public health concerns. In this study, we sequenced and analysed 59 H10 subtype viruses isolated from wild birds in Taiwan. Results showed that all isolates were genetically distinct from human and other mammalian H10 subtype isolates. Taiwanese isolates exhibited high genetic diversity and could be categorized into 34 distinct genotypes, with each genotype circulating only in a single migratory season and not recurring during subsequent seasons. Additional analyses revealed that certain gene pools frequently circulate in the Pacific Rim, with evidence of North American lineage genes establishing long-term populations in Eurasia and vice versa. Although no characteristics indicative of mammalian adaptation was found in the Taiwanese isolates, temporal changes in the haemagglutinin cleavage site sequences were observed. This study provides a comprehensive overview of the evolutionary dynamics of H10 avian influenza viruses isolated from wild birds in Taiwan, emphasizing the complexity of intercontinental gene flow and viral reassortment. Currently, no consistent molecular markers indicating the impact of H10 avian influenza viruses on mammals or poultry have been identified. Notably, the observed changes in the haemagglutinin cleavage site sequences among Eurasian viruses suggest potential antigenic variations, indicating a need for further investigation into these changes. This potential highlights the necessity of ongoing surveillance to track the evolution and transmission dynamics of these viruses.

An avian influenza A H7N9 virus emerged in March 2013 and caused a remarkable number of human fatalities. Genome variability in these viruses may provide insights into host adaptability. We scanned over 140 genomes of the H7N9 viruses isolated from humans and identified 104 positions that exhibited seven or more amino acid substitutions. Approximately half of these substitutions were identified in the influenza ribonucleoprotein (RNP) complex. Although PB2 627K of the avian virus promotes replication in humans, 45 of the 147 investigated PB2 sequences retained the E signature at this position, which is an avian characteristic. We discovered 10 PB2 substitutions that covaried with K627E. An RNP activity assay showed that Q591K, D701N, and M535L restored the polymerase activity in human cells when 627K transformed to an avian-like E. Genomic analysis of the human-isolated avian influenza virus is crucial in assessing genome variability, because relationships between position-specific variations can be observed and explored. In this study, we observed alternative positions that can potentially compensate for PB2 627K, a well-known marker for cross-species infection. An RNP assay suggested Q591K, D701N, and M535L as potential markers for an H7N9 virus capable of infecting humans.

This review presents a comprehensive perspective on the genomic surveillance of SARS-CoV-2 in Taiwan, with a focus on next-generation sequencing and phylogenetic interpretation. This article aimed to explore how Taiwan has utilized genomic sequencing technologies and surveillance to monitor and mitigate the spread of COVID-19. We examined databases and sources of genomic sequences and highlighted the role of data science methodologies in the explanation and analyses of evolutionary data. This review addressed the challenges and limitations inherent in genomic surveillance, such as concerns regarding data quality and the necessity for interdisciplinary expertise for accurate data interpretation. Special attention was given to the unique challenges faced by Taiwan, including its high population density and major transit destination for international travelers. We underscored the far-reaching implications of genomic surveillance data for public health policy, particularly in influencing decisions regarding travel restrictions, vaccine administration, and public health decision-making. Studies were examined to demonstrate the effectiveness of using genomic data to implement public health measures. Future research should prioritize the integration of methodologies and technologies in evolutionary data science, particularly focusing on phylodynamic analytics. This integration is crucial to enhance the precision and applicability of genomic data. Overall, we have provided an overview of the significance of genomic surveillance in tracking SARS-CoV-2 variants globally and the pivotal role of data science methodologies in interpreting these data for effective public health interventions.

Forty-two cytopathic effect (CPE)-positive isolates were collected from 2008 to 2012. All isolates could not be identified for known viral pathogens by routine diagnostic assays. They were pooled into 8 groups of 5-6 isolates to reduce the sequencing cost. Next-generation sequencing (NGS) was conducted for each group of mixed samples, and the proposed data analysis pipeline was used to identify viral pathogens in these mixed samples. Polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA) was individually conducted for each of these 42 isolates depending on the predicted viral types in each group. Two isolates remained unknown after these tests. Moreover, iteration mapping was implemented for each of these 2 isolates, and predicted human parechovirus (HPeV) in both. In summary, our NGS pipeline detected the following viruses among the 42 isolates: 29 human rhinoviruses (HRVs), 10 HPeVs, 1 human adenovirus (HAdV), 1 echovirus and 1 rotavirus. We then focused on the 10 identified Taiwanese HPeVs because of their reported clinical significance over HRVs. Their genomes were assembled and their genetic diversity was explored. One novel 6-bp deletion was found in one HPeV-1 virus. In terms of nucleotide heterogeneity, 64 genetic variants were detected from these HPeVs using the mapped NGS reads. Most importantly, a recombination event was found between our HPeV-3 and a known HPeV-4 strain in the database. Similar event was detected in the other HPeV-3 strains in the same clade of the phylogenetic tree. These findings demonstrated that the proposed NGS data analysis pipeline identified unknown viruses from the mixed clinical samples, revealed their genetic identity and variants, and characterized their genetic features in terms of viral evolution.

Background Mutations in the PB1 subunit of RNA-dependent RNA polymerase (RdRp) of influenza A virus can affect replication fidelity. Before the influenza A/H1N1 pandemic in 2009, most human influenza A/H1N1 viruses contained the avian-associated residue, serine, at position 216 in PB1. However, near the onset of the 2009 pandemic, human viruses began to acquire the mammalian-associated residue, glycine, at PB1–216, and PB1–216G became predominant in human viruses thereafter. Methods Using entropy-based analysis algorithm, we have previously identified several host-specific amino-acid signatures that separated avian and swine viruses from human influenza viruses. The presence of these host-specific signatures in human influenza A/H1N1 viruses suggested that these mutations were the result of adaptive genetic evolution that enabled these influenza viruses to circumvent host barriers, which resulted in cross-species transmission. We investigated the biological impact of this natural avian-to-mammalian signature substitution at PB1–216 in human influenza A/H1N1 viruses. Results We found that PB1–216G viruses had greater mutation potential, and were more sensitive to ribavirin than PB1–216S viruses. In oseltamivir-treated HEK293 cells, PB1–216G viruses generated mutations in viral neuraminidase at a higher rate than PB1–216S viruses. By contrast, PB1–216S viruses were more virulent in mice than PB1–216G viruses. These results suggest that the PB1-S216G substitution enhances viral epidemiological fitness by increasing the frequency of adaptive mutations in human influenza A/H1N1 viruses. Conclusions Our results thus suggest that the increased adaptability and epidemiological fitness of naturally arising human PB1–216G viruses, which have a canonical low-fidelity replicase, were the biological mechanisms underlying the replacement of PB1–216S viruses with a high-fidelity replicase following the emergence of pdmH1N1. We think that continued surveillance of such naturally occurring PB1–216 variants among others is warranted to assess the potential impact of changes in RdRp fidelity on the adaptability and epidemiological fitness of human A/H1N1 influenza viruses.

The influenza A virus contains 8 segmented genomic RNAs and was considered to encode 10 viral proteins until investigators identified the 11th viral protein, PB1-F2, which uses an alternative reading frame of the PB1 gene. The recently identified PB1-N40, PA-N155 and PA-N182 influenza A proteins have shown the potential for using a leaking ribosomal scanning mechanism to generate novel open reading frames (ORFs). These novel ORFs provide examples of the manner in which the influenza A virus expands its coding capacity by using overlapping reading frames. In this study, we performed a computational search, based on a ribosome scanning mechanism, on all influenza A coding sequences to identify possible forward-reading ORFs that could be translated into novel viral proteins. We specified that the translated products had a prevalence ≥5% to eliminate sporadic ORFs. A total of 1,982 ORFs were thus identified and presented in terms of their locations, lengths and Kozak sequence strengths. We further provided an abridged list of ORFs by requiring every candidate an upstream start codon (within the upstream third of the primary transcript), a strong Kozak consensus sequence and high prevalence (≥95% and ≥50% for in-frame and alternative-frame ORFs, respectively). The PB1-F2, PB1-N40, PA-N155 and PA-N182 proteins all fulfilled our filtering criteria. Subject to these three stringent settings, we additionally named 16 novel ORFs for all influenza A genomes except for HA and NA, for which 43 HA and 11 NA ORFs from their respective subtypes were also recognized.

Metagenomic approaches to detect viral genomes and variants in clinical samples have various challenges, including low viral titers and bacterial and human genome contamination. To address these limitations, we examined a next-generation sequencing (NGS) and iterative mapping approach for virus detection in clinical samples. We analyzed 40 clinical specimens from hospitalized children diagnosed with acute bronchiolitis, croup, or respiratory tract infections in which virus identification by viral culture or polymerase chain reaction (PCR) was unsuccessful. For our NGS data analysis pipeline, clinical samples were pooled into two NGS groups to reduce sequencing costs, and the depth and coverage of assembled contigs were effectively increased using an iterative mapping approach. PCR was individually performed for each specimen according to the NGS-predicted viral type. We successfully detected previously unidentified respiratory viruses in 26 of 40 specimens using our proposed NGS pipeline. Two dominant populations within the detected viruses were human rhinoviruses (HRVs; n = 14) and human coronavirus NL63 ( n = 8), followed by human parainfluenza virus (HPIV), human parechovirus, influenza A virus, respiratory syncytial virus (RSV), and human metapneumovirus. This is the first study reporting the complete genome sequences of HRV-A101, HRV-C3, HPIV-4a, and RSV, as well as an analysis of their genetic variants, in Taiwan. These results demonstrate that this NGS pipeline allows to detect viruses which were not identified by routine diagnostic assays, directly from clinical samples.

The history of influenza in Taiwan can be traced up to the 1918 H1N1 Spanish flu pandemic, followed by several others including the 1957 H2N2, 1968 H3N2, and the 2009 new H1N1. A couple of avian influenza viruses of H5N1 and H7N9 also posed threats to the general public in Taiwan in the two recent decades. Nevertheless, two seasonal influenza A viruses and two lineages of influenza B viruses continue causing annual endemics one after the other, or appearing simultaneously. Their interplay provided interesting evolutionary trajectories for these viruses, allowing us to computationally model their global migrations together with the data collected elsewhere from different geographical locations. An island-wide laboratory-based surveillance network was also established since 2000 for systematically collecting and managing the disease and molecular epidemiology. Experiences learned from this network helped in encountering and managing newly emerging infectious diseases, including the 2003 SARS and 2009 H1N1 outbreaks.

Herein, we present a new approach for serological testing for SARS-CoV-2 antibodies using innovative laboratory methods that demonstrate a combination of biology and mathematics. The traditional virus neutralization test is the gold standard method; however, it is time-consuming and poses a risk to medical personnel. Considering the urgent demand for faster methods to quantify neutralizing antibody titers in patients with coronavirus (CoV) disease 2019 (COVID-19), developing an analytical model or method to replace the conventional virus neutralization test (NT) is essential. Moreover, a “COVID-19 immunity passport” is currently being proposed as a certification for people who travel internationally. Therefore, an enzyme-linked immunosorbent assay (ELISA) was designed to detect severe acute respiratory syndrome CoV 2 (SARS-CoV-2)-neutralizing antibodies in serum, which is based on the binding affinity of SARS-CoV-2 viral spike protein 1 (S1) and the viral spike protein receptor-binding domain (RBD) to antibodies. The RBD is considered the major binding region of neutralizing antibodies. Furthermore, S1 covers the RBD and several other regions, which are also important for neutralizing antibody binding. In this study, we assessed 144 clinical specimens, including those from patients with PCR-confirmed SARS-CoV-2 infections and healthy donors, using both the NT and ELISA. The ELISA results analyzed by spline regression and the two-variable generalized additive model precisely reflected the NT value, and the correlation between predicted and actual NT values was as high as 0.917. Therefore, our method serves as a surrogate to quantify neutralizing antibody titer. The analytic method and platform used in this study present a new perspective for serological testing of SARS-CoV-2 infection and have clinical potential to assess vaccine efficacy. IMPORTANCE Herein, we present a new approach for serological testing for SARS-CoV-2 antibodies using innovative laboratory methods that demonstrate a combination of biology and mathematics. The traditional virus neutralization test is the gold standard method; however, it is time-consuming and poses a risk to medical personnel. Thus, there is a demand for methods that rapidly quantify neutralizing antibody titers in patients with COVID-19 or examine vaccine efficacy at a biosafety level 2 containment facility. Therefore, we used a two-variable generalized additive model to analyze the results of the enzyme-linked immunosorbent assay and found the method to serve as a surrogate to quantify neutralizing antibody titers. This methodology has potential for clinical use in assessing vaccine efficacy.