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Autoantibodies against interferon-gamma (AutoAbs-IFN-γ) can cause the immunodeficiency condition following various opportunistic infections. Gut microbiota can affect the human immune system in many ways. Many studies have shown that gut dysbiosis was associated with some immune diseases, such as autoimmune diseases and human immunodeficiency virus (HIV) infection, while its relationship at anti-IFN-γ AAbs remains unknown. We aimed to identify the anti-IFN-γ AAbs specific microbiome and the possible association with immunodeficiency. We profiled fecal microbiome for two cohorts of forty subjects, including seven patients with anti-IFN-γ AAbs and 33 individuals with competent immune. The study shows that patients with anti-IFN-γ AAbs have characterized the gut microbiome and have lower alpha diversity indexes than healthy controls (HC). There are significant differences in the microbiome structure at both the family and genera level between the two cohorts. The anti-IFN-γ AAbs cohort featured some microbiome such as Clostridium, including the possible opportunistic pathogen and fewer genera including Bacteroides, Ruminococcus, and Faecalibacterium, some of them with possible immune-related genera. The PICRUSt2 pathway demonstrated the decreased abundance of some immune-related pathways and one potential pathway related to the immune alternations in the anti- IFN-γ AAbs cohort. This was the first study to examine the gut microbiome characteristics in patients with anti-IFN-γ AAbs. It could be involved in the pathogenesis of anti-IFN-γ AAbs and contribute to the derived immune condition in this disease. This could lead to new strategies for treating and preventing patients suffering from this disease.

A novel coronavirus (CoV) that emerged in Wuhan, Hubei province in China, in December 2019, has rapidly spread worldwide. Named as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this virus has been responsible for infecting about 153 million people and causing 3 million deaths by May 2021. There is obvious interest in gaining novel insights into the epidemiologic evolution of this virus; however, inappropriate application and interpretation of genomic and phylogenetic analyses has led to dangerous outcomes and misunderstandings. This chapter focuses on not only introducing this virus, its genomic characteristics and molecular mechanisms but also describing the application and interpretation of phylogenetic tree analyses, in order to provide useful information to better understand the evolution and epidemiology of this virus. In addition, recombinant region and genetic ancestry of SARS-CoV-2 remain unknown. It is urgently required to collect samples and obtain related viral genetic data from animal sources for identifying the intermediate host of SARS-CoV-2 that is responsible for its cross-species transmission.

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.

Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae, poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

Incidence of invasive pneumococcal disease caused by antimicrobial-resistant Streptococcus pneumoniae types not included in pneumococcal conjugate vaccines has increased, including a penicillin- and meropenem-resistant serotype 15A-ST63 clone in Japan. During 2013-2017, we collected 206 invasive pneumococcal isolates in Taiwan for penicillin and meropenem susceptibility testing. We found serotypes 15B/C-ST83 and 15A-ST63 were the most prevalent penicillin- and meropenem-resistant clones. A transformation study confirmed that penicillin-binding protein (PBP) 2b was the primary meropenem resistance determinant, and PBP1a was essential for high-level resistance. The rate of serotype 15B/C-ST83 increased during the study. All 15B/C-ST83 isolates showed an ermB macrolide resistance genotype. Prediction analysis of recombination sites revealed 12 recombination regions in 15B/C-ST83 compared with the S. pneumoniae Spain23F-ST81 genome. Pneumococcal clones rapidly recombine to acquire survival advantages and undergo local expansion under the selective pressure exerted by vaccines and antimicrobial drugs. The spread of 15B/C-ST83 is alarming for countries with high antimicrobial pressure.

Influenza A virus mutates rapidly, allowing it to escape natural and vaccine-induced immunity. Neuraminidase (NA) is a surface protein capable of cleaving the glycosidic linkages of neuraminic acids to release newly formed virions from infected cells. Genetic variants within a viral population can influence the emergence of pandemic viruses as well as drug susceptibility and vaccine effectiveness. In the present study, 55 clinical specimens from patients infected with the 2009 pandemic influenza A/H1N1 virus, abbreviated as A(H1N1)pdm09, during the 2015-2016 outbreak season in Taiwan were collected. Whole genomes were obtained through next-generation sequencing. Based on the published sequences from A(H1N1)pdm09 strains worldwide, a mixed population of two distinct variants at NA position 151 was revealed. We initially reasoned that such a mixed population may have emerged during cell culture. However, additional investigations confirmed that these mixed variants were detectable in the specimens of patients. To further investigate the role of the two NA-151 variants in a dynamic population, a reverse genetics system was employed to generate recombinant A(H1N1)pdm09 viruses. It was observed that the mixture of the two distinct variants was characterized by a higher replication rate compared to the recombinant viruses harbouring a single variant. Moreover, an NA inhibition assay revealed that a high frequency of the minor NA-151 variant in A(H1N1)pdm09 was associated with a reduced susceptibility to NA inhibitors. We conclude that two distinct NA-151 variants can be identified in patient specimens and that such variants may increase viral replication and NA activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carrying the D614G mutation on the spike protein is the predominant circulating variant and is associated with enhanced infectivity. However, whether this dominant variant can potentially spread through the cold chain and whether the spike protein affects virus stability after cold storage remain unclear. To compare the infectivity of two SARS-CoV-2 variants, namely, SARS-CoV-2 variants with spike protein with the D614 mutation (S-D614) and G614 mutation (S-G614), after different periods of refrigeration (4°C) and freezing (−20°C). We also determined the integrity of the viral RNA and the ability of the spike protein to bind angiotensin-converting enzyme 2 (ACE2) after storage at these conditions. The results showed that SARS-CoV-2 was more stable and infectious after storage at −20°C than at 4°C. Particularly, the S-G614 variant was found to be more stable than the S-D614 variant. The spike protein of the S-G614 variant had better binding ability with the ACE2 receptor than that of the S-D614 variant after storage at −20°C for up to 30 days. Our findings revealed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, and their stability and infectivity up to 30 days depends on the spike variant. Stability and infectivity are related to each other, and the higher stability of S-G614 compared to that of S-D614 may contribute to rapid viral spread of the S-G614 variant. IMPORTANCE It has been observed that variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are more stable and infectious after storage at −20°C than at 4°C. A SARS-CoV-2 S-D614G variant is currently the most dominant variant in circulation and is associated with enhanced infectivity. We compared the stability of two SARS-CoV-2 variants: the early S-D614 variant carrying the D614 spike protein and the new S-G614 variant carrying the G614 spike protein, stored at both 4°C and −20°C for different periods. We observed that SARS-CoV-2 remains stable and infectious after refrigeration or freezing, which further depends on the spike variant, that is, the ability of the spike protein to bind with the ACE2 receptor with higher efficiency. The high stability of the S-G614 variant also explains its rapid spread and infectivity. Therefore, precautions should be taken during and after handling food preserved under cold conditions.

Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens., Real-time reverse transcription-PCR (RT-PCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes coronavirus disease 2019 (COVID-19). However, the correlation between detectable viral RNA and culturable virus in clinical specimens remains unclear. Here, we performed virus culture for 60 specimens that were confirmed to be positive for SARS-CoV-2 RNA by real-time RT-PCR. The virus could be successfully isolated from 12 throat and nine nasopharyngeal swabs and two sputum specimens. The lowest copy number required for virus isolation was determined to be 5.4, 6.0, and 5.7 log10 genome copies/ml sample for detecting the nsp12, E, and N genes, respectively. We further examined the correlation of genome copy number and virus isolation in different regions of the viral genome, demonstrating that culturable specimens are characterized by high copy numbers with a linear correlation observed between copy numbers of amplicons targeting structural and nonstructural regions. Overall, these results indicate that in addition to the copy number, the integrity of the viral genome should be considered when evaluating the infectivity of clinical SARS-CoV-2 specimens.

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.

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. Keywords: Influenza, Pandemic, Genome evolution, Surveillance

Enterovirus A71 (EV-A71) is an important nonpolio enterovirus that causes severe neurological complications. In 1998, Taiwan experienced an EV-A71 outbreak that caused 78 deaths. Since then, periodic epidemics of EV-A71 associated with newly emerging strains have occurred. Several of these strains are known to be recombinant; however, how these strains arose within such a short period of time remains unknown. Here, we sequenced 64 full-length genomes from clinical isolates collected from 2005 to 2016 and incorporated all 91 Taiwanese genomes downloaded from the Virus Pathogen Resource to extensively analyze EV-A71 recombination in Taiwan. We found that the B3 subgenotype was a potential recombinant parent of the EV-A71 C2-like and C4 strains by intratypic recombination. Such B3-similar regions were also found in many cocirculating coxsackieviruses belonging to Enterovirus A species (EV-A) through a series of intertypic recombinations. Therefore, locally enriched outbreaks of cocirculating viruses from different genotypes/serotypes may facilitate recombination. Most recombination breakpoints we found had nonrandom distributions and were located within the region spanning from the boundary of P1 (structural gene) and P2 (nonstructural) to the cis-Acting replication element at P2, indicating that specific genome reassembly of structural and nonstructural genes may be subject to natural selection. Through intensive recombination, 11 EV-A71-like signatures (including one in 3A, two in 3C, and eight in 3D) were found to be present in a variety of recently cocirculating EV-A viruses worldwide, suggesting that these viruses may be targets for wide-spectrum antiviral development.

There is conclusive evidential support for the existence of virus-derived small RNA (vsRNA) in mammals. Two types of vsRNA have been reported from picornaviruses. The first is virus-derived short-interfering RNA (vsiRNA) that is processed from viral double-stranded RNA intermediates during RNA replication. The other is small RNA derived from the highly base-paired single-stranded genomic region, e.g. the internal ribosome entry site (IRES) of picornaviruses. vsiRNA interacts with the Argonaute protein to control viral RNA replication through the process of RNA interference. However, the function of structure-based vsRNA is largely unknown. We previously identified vsRNA1 generated from the enterovirus-A71 (EV-A71) IRES region by the endogenous enzyme Dicer. Exogenous vsRNA1 can inhibit IRES activity both in vivo and in vitro, hence viral replication is inhibited. Here we describe key methods used to characterize vsRNA, including annotation by next-generation sequencing, abundance measurement by Northern blotting, determination of Dicer-dependence by gel-shift assay and in vitro cleavage assay, and the inhibitory effect on IRES activity via in vitro translation assay.

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease. IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.

Objectives We studied paediatric patients with human adenovirus (HAdV) infection during the 2011 outbreak in northern Taiwan to define the clinical features of different HAdV genotypes in children. Methods Between January and December 2011, 637 patients 3 years; p 100 mg/L, whereas leucocytosis (white blood cell count >15 000/mm3) was more common in HAdV-2-infected patients (p 0.007). HAdV-7 infections were significantly associated with a longer duration of fever, leucopenia (white blood cell count

A novel pandemic influenza A(H1N1)pdm09 virus emerged in 2009 globally, and it continues to circulate in humans. The National Influenza Surveillance Network in Taiwan identified five A(H1N1)pdm09-predominant seasons, representing the 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 outbreaks from 2009 to 2016. Independently, a retrospective cohort study (which enrolled 639 infected patients during the five seasons) was conducted at Chang Gung Memorial Hospital to explore the risk factors associated with influenza A(H1N1)pdm09-related complications. A phylogenetic analysis of hemagglutinin (HA) sequences showed that the circulating A(H1N1)pdm09 virus belonged to clades 1, 2, and 8 in 2009/2010; clades 3, 4, 5, and 7 in 2010/2011; clades 7 and 6C in 2012/2013; clades 6B in 2013/2014; and 6B/6B.1/6B.2 in 2015/2016. Compared to individuals infected in non-6B/6B.1/6B.2 seasons (2009/2010, 2010/2011, and 2012/2013), those infected in 6B/6B.1/6B.2 seasons (2013/2014 and 2015/2016) were at higher risk for influenza-related complications (adjusted odds ratio [aOR]: 1.6, 95% confidence interval [CI]: 1.0–2.8), pneumonia (aOR: 1.78, 95% CI: 1.04–3.04), mechanical ventilation (aOR: 2.6, 95% CI: 1.2–5.6), and acute respiratory distress syndrome (aOR: 5.5, 95% CI: 1.9–15.9). For the increased severity of infection during the influenza A(H1N1)pdm09 clade 6B/6B.1/6B.2 seasons, aspects related to the antigenic change of A(H1N1)pdm09 virus, immune response of the host, and environmental factors required further investigation.

Background/Purpose: Influenza A/H3N2 viruses are characterized by highly mutated RNA genomes. In this study, we focused on tracing the phylodynamics of Taiwanese strains over the past four decades. Methods: All Taiwanese H3N2 HA1 sequences and references were downloaded from public database. A Bayesian skyline plot (BSP) and phylogenetic tree were used to analyze the evolutionary history, and Bayesian phylogeographic analysis was applied to predict the spatiotemporal migrations of influenza outbreaks. Results: Genetic diversity was found to have peaked near the summer of 2009 in BSP, in addition to the two earlier reported ones in summer of 2005 and 2007. We predicted their spatiotemporal migrations and found the summer epidemic of 2005 from Korea, and 2007 and 2009 from the Western United States. BSP also predicted an elevated genetic diversity in 2015–2017. Quasispecies were found over approximately 20% of the strains included in this time span. In addition, a first-time seen N31S mutation was noted in Taiwan in 2016–2017. Conclusion: We comprehensively investigated the evolutionary history of Taiwanese strains in 1979–2017. An epidemic caution could thus be raised if genetic diversity was found to have peaked. An example showed a newly-discovered cluster in 2016–2017 strains featuring a mutation N31S together with HA-160 quasispecies. Phylogeographic analysis, moreover, provided useful insights in tracing the possible source and migrations of these epidemics around the world. We demonstrated that Asian destinations including Taiwan were the immediate followers, while U.S. continent was predicted the origin of two summer epidemics in 2007 and 2009. Keywords: Influenza A/H3N2 viruses, Phylodynamics, Phylogeography, Summer epidemics, Viral quasispecies

Neuraminidase (NA) is a homotetramer viral surface glycoprotein that is essential for virus release during influenza virus infections. Previous studies have not explored why influenza NA forms a tetramer when the bacterial monomer NA already exhibits excellent NA enzymatic activity levels. In this study, we focused on 28 highly conserved residues among all NA subtypes, identifying 21 of 28 positions as crucial residues for viral survival by using reverse genetics. Maintaining NA enzymatic activity levels is critical and numerous conserved residues were located at the oligomerization interface; however, these mutations did not affect NA enzymatic activity levels or NA cellular localization, but rather affected the stability of NA oligomerization, suggesting that the oligomerization of NA is essential for viral viability. An increased understanding of the biological functions of NA, in particular NA oligomerization, could facilitate an alternative design for antivirals to combat influenza virus infections.

Human enterovirus D68 (EV-D68) was first reported in the United States in 1962; thereafter, a few cases were reported from 1970 to 2005, but 2 outbreaks occurred in the Philippines (2008) and the United States (2014). However, little is known regarding the molecular evolution of this globally reemerging virus due to a lack of whole-genome sequences and analyses. Here, all publically available sequences including 147 full and 1248 partial genomes from GenBank were collected and compared at the clade and subclade level; 11 whole genomes isolated in Taiwan (TW) in 2014 were also added to the database. Phylogenetic trees were constructed to identify a new subclade, B3, and represent clade circulations among strains. Nucleotide sequence identities of the VP1 gene were 94% to 95% based on a comparison of subclade B3 to B1 and B2 and 87% to 91% when comparing A, C, and D. The patterns of clade circulation need to be clarified to improve global monitoring of EV-D68, even though this virus showed lower diversity among clades compared with the common enterovirus EV-71. Notably, severe cases isolated from Taiwan and China in 2014 were found in subclade B3. One severe case from Taiwan occurred in a female patient with underlying angioimmunoblastic T-cell lymphoma, from whom a bronchoalveolar lavage specimen was obtained. Although host factors play a key role in disease severity, we cannot exclude the possibility that EV-D68 may trigger clinical symptoms or death. To further investigate the genetic diversity of EV-D68, we reported 34 amino acid (aa) polymorphisms identified by comparing subclade B3 to B1 and B2. Clade D strains had a 1-aa deletion and a 2-aa insertion in the VP1 gene, and 1 of our TW/2014 strains had a shorter deletion in the 5′ untranslated region than a previously reported deletion. In summary, a new subclade, genetic indels, and polymorphisms in global strains were discovered elucidating evolutionary and epidemiological trends of EV-D68, and 11 genomes were added to the database. Virus variants may contribute to disease severity and clinical manifestations, and further studies are needed to investigate the associations between genetic diversity and clinical outcomes.

The NS1 protein encoded by influenza A virus antagonizes the interferon response through various mechanisms, including blocking cellular mRNA maturation by binding the cellular CPSF30 3′ end processing factor and/or suppressing the activation of interferon regulatory factor 3 (IRF3). In the present study, we identified two truncated NS1 proteins that are translated from internal AUGs at positions 235 and 241 of the NS1 open reading frame. We analyzed the cellular localization and function of the N-truncated NS1 proteins encoded by two influenza A virus strains, Udorn/72/H3N2 (Ud) and Puerto Rico/8/34/H1N1 (PR8). The NS1 protein of PR8, but not Ud, inhibits the activation of IRF3, whereas the NS1 protein of Ud, but not PR8, binds CPSF30. The truncated PR8 NS1 proteins are localized in the cytoplasm, whereas the full-length PR8 NS1 protein is localized in the nucleus. The infection of cells with a PR8 virus expressing an NS1 protein containing mutations of the two in-frame AUGs results in both the absence of truncated NS1 proteins and the reduced inhibition of activation of IRF3 and beta interferon (IFN-β) transcription. The expression of the truncated PR8 NS1 protein by itself enhances the inhibition of the activation of IRF3 and IFN-β transcription in Ud virus-infected cells. These results demonstrate that truncated PR8 NS1 proteins contribute to the inhibition of activation of this innate immune response. In contrast, the N-truncated NS1 proteins of the Ud strain, like the full-length NS1 protein, are localized in the nucleus, and mutation of the two in-frame AUGs has no effect on the activation of IRF3 and IFN-β transcription. IMPORTANCE Influenza A virus causes pandemics and annual epidemics in the human population. The viral NS1 protein plays a critical role in suppressing type I interferon expression. In the present study, we identified two novel truncated NS1 proteins that are translated from the second and third in-frame AUG codons in the NS1 open reading frame. The N-terminally truncated NS1 encoded by the H1N1 PR8 strain of influenza virus that suppresses IRF3 activation is localized primarily in the cytoplasm. We demonstrate that this truncated NS1 protein by itself enhances this suppression, demonstrating that some strains of influenza A virus express truncated forms of the NS1 protein that function in the inhibition of cytoplasmic antiviral events.

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 The influenza A virus has evolved into 16 hemagglutinin (HA) subtypes with different antigenic properties. Thus far typing has been primarily assay based, but the many sequences available from the US National Center for Biotechnology Information (NCBI) offer alternative ways of characterizing the HA gene. Methods All available HA sequences from the NCBI were analyzed. The software package HMMER was used to score how a training sequence fitted a profile hidden Markov model (profile HMM) constructed from the consensus sequence of one particular HA subtype, H x , where x =1 to 16. Scores from sequences of the same subtype and from other subtypes were then compared to see if they were separable. This approach was implemented in a stepwise manner, utilizing a sliding window of 100 amino acids with 10-amino-acid increments to build many subtype-specific models, and then assessing which 100-amino acid segments yielded the desired differentiability. Results Segment-based analysis revealed domains that correlate to HA sequence heterogeneity from one subtype to the others. For example, we showed that H1 segments covering only the second half of HA are not statistically separable from H2, H5 and H6 within the same region, suggesting evolutionary relatedness for these subtypes. The HA1 domain was found to be mostly differentiable between subtypes, which is in line with wet-lab findings that the domain is antigenicity-rich. We also reported a couple of regions that can be conveniently used to characterize all HA subtypes. Conclusion We established an analysis framework for assessing sequence-subtype association to provide insights into HA subtypes with close evolutionary relationships.

Background & Aims Gut dysbiosis plays a role in hepatic encephalopathy (HE), while its relationship at the acute episode of overt HE (AHE), the disease progression and clinical outcomes remains unclear. We aimed to identify AHE-specific microbiome and its association to patients’ outcomes. Methods We profiled fecal microbiome changes for a cohort of 62 patients with cirrhosis and AHE i) before treatment, ii) 2-3 days after medication and iii) 2-3 months after recovery, and three control cohorts i) healthy individuals, patients with ii) compensated or iii) decompensated cirrhosis. Results Comparison of the microbiome shift from compensated, decompensated cirrhosis, AHE to recovery revealed the AHE-specific gut-dysbiosis. The gut microbiome diversity was decreased during AHE, further reduced after medication, and only partially reversed during the recovery. The relative abundance of Bacteroidetes phylum in the microbiome decreased, whereas that of Firmicute, Proteobacteria and Actinobacteria increased in patients during AHE compared with those with compensated cirrhosis. A total of 70 operational taxonomic units (OTUs) were significantly different between AHE and decompensated cirrhosis abundances. Of them, the abundance of Veillonella parvula increased the most during AHE via a metagenomics recovery of the genomes. Moreover, the relative abundances of three (Alistipes, Bacteroides, Phascolarctobacterium) and five OTUs (Clostridium-XI, Bacteroides, Bacteroides, Lactobacillus, Clostridium-sedis) at AHE were respectively associated with HE recurrence and overall survival during the subsequent one-year follow-up. Conclusions AHE-specific gut OTUs were identified that may be involved in HE development and able to predict clinical outcomes, providing new strategies for the prevention and treatment of HE recurrence in patients with cirrhosis., Graphical abstract

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.

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.

Background/purpose An influenza A pandemic occurred in 2009–2010. A novel H1N1 virus (hereafter H1N1pdm) was responsible for this outbreak. H1N1pdm viruses have been largely seen in recent human influenza A viruses. This virus was descended from a triple-reassorted swine virus consisting of human, avian, and swine origins. As a result, the previously established species-associated signatures could be in jeopardy. Methods We analyzed all influenza A sequences in the past 5 years after the inclusion of H1N1pdm into human viruses since 2009, and examined how human signatures may lose their distinctness by mixing with avian residues that H1N1pdm have brought in. In particular, we compared how those signatures were changed/shifted in the past 5 years for human-isolated avian influenza A viruses and discussed their implications. Results Only eight out of 47 signatures remained human-like for human influenza A viruses in the past 5 years. They are PB2 271A; PB1 336I; PA 356R and 409N; NP 33I, 305K, and 357K; and NS1 227R. Although most avian-like residues were preserved in human-isolated avian influenza A viruses, a number of them were found to have become or on the verge of becoming human-like, including PB2 627, PA 100, 356, 404, 409, NP 33, 61, 305, 357, M2 20, and NS1 81. Conclusion Analyzing how species-associated signatures are becoming human-like in human-isolated avian influenza A viruses helps in assessing their potential to go pandemic as well as providing insights into host adaptation.

Mutual information (MI) is an approach commonly used to estimate the evolutionary correlation of 2 amino acid sites. Although several MI methods exist, prior to our contribution no systematic method had been developed to assess their performance, or to establish numerical thresholds to detect co-evolving amino acid sites. The current study performed a Markov chain Monte Carlo (MCMC) algorithm on influenza viral sequences to capture their evolutionary characteristics. A consensus maximum clade credibility (MCC) tree was estimated from the samples, together with their amino acid substitution statistics, from which we generated synthetic sequences of known dependent and independent paired amino acid sites. A pair-to-pair and influenza-specific amino acid substitution matrix (P2PFLU) incorporated into Bayesian Evolutionary Analysis Sampling Trees (BEAST) enumerated these synthetic sequences. The sequences inherited evolutionary features and co-varying characteristics from the real viral sequences, rendering these synthetic data ideal for exploring their co-evolving features. For the MI measure, we proposed a novel metric called the empirical MI (MI"E"m), which outperformed other MI measures in analysis of receiver operating characteristics (ROC). We implemented our approach on 1086 all-time PB2 sequences of influenza A H5N1 viruses, in which we found 97 sites exhibiting co-evolutionary substitution of one or more amino acid sites. In particular, PB2 451, along with eight other PB2 sites of various MI"E"m scores, was found to co-evolve with PB2 627, a known species-associated amino acid residue which plays a critical role in influenza virus replication.

A swine-origin influenza A was detected in April 2009 and soon became the 2009 H1N1 pandemic strain (H1N1pdm). The current study revealed the genetic diversity of H1N1pdm, based on 77 and 70 isolates which we collected, respectively, during the 2009/2010 and 2010/2011 influenza seasons in Taiwan. We focused on tracking the amino acid transitioning of hemagglutinin (HA) and neuraminidase (NA) genes in the early diversification of the virus and compared them with H1N1pdm strains reported worldwide. We identified newly emerged mutation markers based on A/California/04/2009, described how these markers shifted from the first H1N1pdm season to the one that immediately followed, and discussed how these observations may relate to antigenicity, receptor-binding, and drug susceptibility. It was found that the amino acid mutation rates of H1N1pdm were elevated, from 9.29×10(-3) substitutions per site in the first season to 1.46×10(-2) in the second season in HA, and from 5.23×10(-3) to 1.10×10(-2) in NA. Many mutation markers were newly detected in the second season, including 11 in HA and 8 in NA, and some were found having statistical correlation to disease severity. There were five noticeable HA mutations made to antigenic sites. No significant titer changes, however, were detected based on hemagglutination inhibition tests. Only one isolate with H275Y mutation known to reduce susceptibility to NA inhibitors was detected. As limited Taiwanese H1N1pdm viruses were isolated after our sampling period, we gathered 8,876 HA and 6,017 NA H1N1pdm sequences up to April 2012 from NCBI to follow up the dynamics of mentioned HA mutations. While some mutations described in this study seemed to either settle in or die out in the 2011-2012 season, a number of them still showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.