ARGNet: using deep neural networks for robust identification and classification of antibiotic resistance genes from sequences.

BACKGROUND: Emergence of antibiotic resistance in bacteria is an important threat to global health. Antibiotic resistance genes (ARGs) are some of the key components to define bacterial resistance and their spread in different environments. Identification of ARGs, particularly from high-throughput sequencing data of the specimens, is the state-of-the-art method for comprehensively monitoring their spread and evolution. Current computational methods to identify ARGs mainly rely on alignment-based sequence similarities with known ARGs. Such approaches are limited by choice of reference databases and may potentially miss novel ARGs. The similarity thresholds are usually simple and could not accommodate variations across different gene families and regions. It is also difficult to scale up when sequence data are increasing. RESULTS: In this study, we developed ARGNet, a deep neural network that incorporates an unsupervised learning autoencoder model to identify ARGs and a multiclass classification convolutional neural network to classify ARGs that do not depend on sequence alignment. This approach enables a more efficient discovery of both known and novel ARGs. ARGNet accepts both amino acid and nucleotide sequences of variable lengths, from partial (30-50 aa; 100-150 nt) sequences to full-length protein or genes, allowing its application in both target sequencing and metagenomic sequencing. Our performance evaluation showed that ARGNet outperformed other deep learning models including DeepARG and HMD-ARG in most of the application scenarios especially quasi-negative test and the analysis of prediction consistency with phylogenetic tree. ARGNet has a reduced inference runtime by up to 57% relative to DeepARG. CONCLUSIONS: ARGNet is flexible, efficient, and accurate at predicting a broad range of ARGs from the sequencing data. ARGNet is freely available at https://github.com/id-bioinfo/ARGNet , with an online service provided at https://ARGNet.hku.hk . Video Abstract.

Effect of human H3N2 influenza virus reassortment on influenza incidence and severity during the 2017-18 influenza season in the USA: a retrospective observational genomic analysis.

BACKGROUND: During the 2017-18 influenza season in the USA, there was a high incidence of influenza illness and mortality. However, no apparent antigenic change was identified in the dominant H3N2 viruses, and the severity of the season could not be solely attributed to a vaccine mismatch. We aimed to investigate whether the altered virus properties resulting from gene reassortment were underlying causes of the increased case number and disease severity associated with the 2017-18 influenza season. METHODS: Samples included were collected from patients with influenza who were prospectively recruited during the 2016-17 and 2017-18 influenza seasons at the Johns Hopkins Hospital Emergency Departments in Baltimore, MD, USA, as well as from archived samples from Johns Hopkins Health System sites. Among 647 recruited patients with influenza A virus infection, 411 patients with whole-genome sequences were available in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance network during the 2016-17 and 2017-18 seasons. Phylogenetic trees were constructed based on viral whole-genome sequences. Representative viral isolates of the two seasons were characterised in immortalised cell lines and human nasal epithelial cell cultures, and patients' demographic data and clinical outcomes were analysed. FINDINGS: Unique H3N2 reassortment events were observed, resulting in two predominant strains in the 2017-18 season: HA clade 3C.2a2 and clade 3C.3a, which had novel gene segment constellations containing gene segments from HA clade 3C.2a1 viruses. The reassortant re3C.2a2 viruses replicated with faster kinetics and to a higher peak titre compared with the parental 3C.2a2 and 3C.2a1 viruses (48 h vs 72 h). Furthermore, patients infected with reassortant 3C.2a2 viruses had higher Influenza Severity Scores than patients infected with the parental 3C.2a2 viruses (median 3·00 [IQR 1·00-4·00] vs 1·50 [1·00-2·00]; p=0·018). INTERPRETATION: Our findings suggest that the increased severity of the 2017-18 influenza season was due in part to two intrasubtypes, cocirculating H3N2 reassortant viruses with fitness advantages over the parental viruses. This information could help inform future vaccine development and public health policies. FUNDING: The Center of Excellence for Influenza Research and Response in the US, National Science and Technology Council, and Chang Gung Memorial Hospital in Taiwan.

Novel intertypic recombination of Echovirus 11 in the Enterovirus species B.

Enteroviruses (EVs), single-stranded, positive-sense RNA viruses, can be classified into four species (A-D), which have previously been linked to a diverse range of disease manifestations and infections affecting the central nervous system. In the Enterovirus species B (EV-B), Echovirus type 11 (E11) has been observed to occasionally circulate in Taiwan, which was responsible for an epidemic of enterovirus infections in 2018. Here, 48 clinical specimens isolated in 2003, 2004, 2009, and 2018 were collected for the high-throughput sequencing. Notably, we identified 2018 Taiwanese strains having potential recombinations in the 3D gene, as well as one 2003 strain having a double recombination with E6 and Coxsackievirus B5 in the P2 and P3 regions, respectively. Additionally, one amino acid signature mutated from the Histidine (H) in throat swab specimens to the Tyrosine (Y) in cerebral spinal fluid specimens was detected at position 1496 (or 57) of the genomic coordinate (or 3A gene) to further demonstrate intra-host evolution in different organs. In conclusion, this study identifies potential intertypic recombination events and an intra-host signature mutation in E11 strains, isolated during a 2018 neurological disease outbreak in Taiwan, contributing to our understanding of its evolution and pathogenesis.

Evolution of Influenza A(H3N2) Viruses in 2 Consecutive Seasons of Genomic Surveillance, 2021-2023.

Background: The circulation and the genomic evolution of influenza A(H3N2) viruses during the 2021/2022 and 2022/2023 seasons were studied and associated with infection outcomes. Methods: Remnant influenza A-positive samples following standard-of-care testing from patients across the Johns Hopkins Health System (JHHS) were used for the study. Samples were randomly selected for whole viral genome sequencing. The sequence-based pEpitope model was used to estimate the predicted vaccine efficacy (pVE) for circulating H3N2 viruses. Clinical data were collected and associated with viral genomic data. Results: A total of 121 683 respiratory specimens were tested for influenza at JHHS between 1 September 2021 and 31 December 2022. Among them, 6071 (4.99%) tested positive for influenza A. Of these, 805 samples were randomly selected for sequencing, with hemagglutinin (HA) segments characterized for 610 samples. Among the characterized samples, 581 were H3N2 (95.2%). Phylogenetic analysis of HA segments revealed the exclusive circulation of H3N2 viruses with HA segments of the 3C.2a1b.2a.2 clade. Analysis of a total of 445 complete H3N2 genomes revealed reassortments; 200 of 227 of the 2022/2023 season genomes (88.1%) were found to have reassorted with clade 3C.2a1b.1a. The pVE was estimated to be -42.53% for the 2021/2022 season and 30.27% for the 2022/2023 season. No differences in clinical presentations or admissions were observed between the 2 seasons. Conclusions: The increased numbers of cases and genomic diversity of influenza A(H3N2) during the 2022/2023 season were not associated with a change in disease severity compared to the previous influenza season.

The emergence and successful elimination of SARS-CoV-2 dominant strains with increasing epidemic potential in Taiwan's 2021 outbreak.

Taiwan's experience with severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 guided its development of strategies to defend against SARS-CoV-2 in 2020, which enabled the successful control of Coronavirus disease 2019 (COVID-19) cases from 2020 through March 2021. However, in late-April 2021, the imported Alpha variant began to cause COVID-19 outbreaks at an exceptional rate in Taiwan. In this study, we aimed to determine what epidemiological conditions enabled the SARS-CoV-2 Alpha variant strains to become dominant and decline later during a surge in the outbreak. In conjunction with contact-tracing investigations, we used our bioinformatics software, CoVConvert and IniCoV, to analyze whole-genome sequences of 101 Taiwan Alpha strains. Univariate and multivariable regression analyses revealed the epidemiological factors associated with viral dominance. Univariate analysis showed the dominant Alpha strains were preferentially selected in the surge's epicenter (p = 0.0024) through intensive human-to-human contact and maintained their dominance for 1.5 months until the Zero-COVID Policy was implemented. Multivariable regression found that the epidemic periods (p = 0.007) and epicenter (p = 0.001) were two significant factors associated with the dominant virus strains spread in the community. These dominant virus strains emerged at the outbreak's epicenter with frequent human-to-human contact and low vaccination coverage. The Level 3 Restrictions and Zero-COVID policy successfully controlled the outbreak in the community without city lockdowns. Our integrated method can identify the epidemiological conditions for emerging dominant virus with increasing epidemiological potential and support decision makers in rapidly containing outbreaks using public health measures that target fast-spreading virus strains.

Emergence and Persistent Dominance of SARS-CoV-2 Omicron BA.2.3.7 Variant, Taiwan.

Since April 2022, waves of SARS-CoV-2 Omicron variant cases have surfaced in Taiwan and spread throughout the island. Using high-throughput sequencing of the SARS-CoV-2 genome, we analyzed 2,405 PCR-positive swab samples from 2,339 persons and identified the Omicron BA.2.3.7 variant as a major lineage within recent community outbreaks in Taiwan.

Targeting influenza A virus by splicing inhibitor herboxidiene reveals the importance of subtype-specific signatures around splice sites.

BACKGROUND: The association between M segment splicing and pathogenicity remains ambiguous in human influenza A viruses. In this study, we aimed to investigate M splicing in various human influenza A viruses and characterize its physiological roles by applying the splicing inhibitor, herboxidiene. METHODS: We examined the M splicing of human H1N1 and H3N2 viruses by comparing three H1N1 and H3N2 strains, respectively, through reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. We randomly selected M sequences of human H1N1, H2N2, and H3N2 viruses isolated from 1933 to 2020 and examined their phylogenetic relationships. Next, we determined the effects of single nucleotide variations on M splicing by generating mutant viruses harboring the 55C/T variant through reverse genetics. To confirm the importance of M2 splicing in the replication of H1N1 and H3N2, we treated infected cells with splicing inhibitor herboxidiene and analyzed the viral growth using plaque assay. To explore the physiological role of the various levels of M2 protein in pathogenicity, we challenged C57BL/6 mice with the H1N1 WSN wild-type strain, mutant H1N1 (55T), and chimeric viruses including H1N1 + H3wt and H1N1 + H3mut. One-tailed paired t-test was used for virus titer calculation and multiple comparisons between groups were performed using two-way analysis of variance. RESULTS: M sequence splice site analysis revealed an evolutionarily conserved single nucleotide variant C55T in H3N2, which impaired M2 expression and was accompanied by collinear M1 and mRNA3 production. Aberrant M2 splicing resulted from splice-site selection rather than a general defect in the splicing process. The C55T substitution significantly reduced both M2 mRNA and protein levels regardless of the virus subtype. Consequently, herboxidiene treatment dramatically decreased both the H1N1 and H3N2 virus titers. However, a lower M2 expression only attenuated H1N1 virus replication and in vivo pathogenicity. This attenuated phenotype was restored by M replacement of H3N2 M in a chimeric H1N1 virus, despite low M2 levels. CONCLUSIONS: The discrepancy in M2-dependence emphasizes the importance of M2 in human influenza A virus pathogenicity, which leads to subtype-specific evolution. Our findings provide insights into virus adaptation processes in humans and highlights splicing regulation as a potential antiviral target.

Outbreak investigation in a COVID-19 designated hospital: The combination of phylogenetic analysis and field epidemiology study suggesting airborne transmission.

BACKGROUND: Healthcare-associated COVID-19 infections caused by SARS-CoV-2 have increased morbidity and mortality. Hospitals and skilled nursing facilities (SNFs) have been challenged by infection control and management. METHODS: This case study presents an outbreak investigation in a COVID-19-designated hospital and a hospital-based SNF. Real-time polymerase chain reaction (PCR) and other studies were performed on samples obtained from SNF residents, hospital patients, and healthcare workers (HCWs). The results of the laboratory tests and field epidemiological data were analyzed. Genome sequencing and phylogenetic analysis of SARS-CoV-2 were performed to identify the associations between cases. The tracer gas was released and recorded by a thermal imaging camera to investigate the spatial relations within clusters. RESULTS: During the outbreak, 29 COVID-19 infections in 3 clusters were identified through hospital-wide, risk-guided, and symptom-driven PCR tests. This included 12 HCWs, 5 patients, and 12 SNF residents who had been hospitalized for at least 14 days. Serology tests did not identify any cases among the PCR-negative individuals. The phylogenetic analysis revealed that viral strains from the 3 clusters shared a common mutation of G3994T and were phylogenetically related, which suggested that this outbreak had a common source rather than multiple introductions from the community. Linked cases exhibited vertical spatial distribution, and the sulfur hexafluoride release test confirmed a potential airborne transmission. CONCLUSIONS: This report addressed the advantage of a multi-disciplinary team in outbreak investigation. Identifying an airborne transmission within an outbreak highlighted the importance of regular maintenance of ventilation systems.

Gut microbiome profiles and associated metabolic pathways in patients of adult-onset immunodeficiency with anti-interferon-gamma autoantibodies.

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.

Visualizing Influenza A Virus vRNA Replication.

Influenza A virus (IAV) has caused recurrent epidemics and severe pandemics. In this study, we adapted an MS2-MCP live-cell imaging system to visualize IAV replication. A reporter plasmid, pHH-PB2-vMSL, was constructed by replacing a part of the PB2-coding sequence in pHH-PB2 with a sequence encoding 24 copies of a stem-loop structure from bacteriophage MS2 (MSL). Binding of MS2 coat protein (MCP) fused to green fluorescent protein (GFP) to MSL enabled the detection of vRNA as fluorescent punctate signals in live-cell imaging. The introduction of pHH-PB2-vMSL into A549 cells transduced to express an MCP-GFP fusion protein lacking the nuclear localization signal (MCP-GFPdN), subsequently allowed tracking of the distribution and replication of PB2-vMSL vRNA after IAV PR8 infection. Spatial and temporal measurements revealed exponential increases in vRNA punctate signal intensity, which was only observed after membrane blebbing in apoptotic cells. Similar signal intensity increases in apoptotic cells were also observed after MDCK cells, transduced to express MCP-GFPdN, were infected with IAV carrying PB2-vMSL vRNA. Notably, PB2-vMSL vRNA replication was observed to occur only in apoptotic cells, at a consistent time after apoptosis initiation. There was a lack of observable PB2-vMSL vRNA replication in non-apoptotic cells, and vRNA replication was suppressed in the presence of apoptosis inhibitors. These findings point to an important role for apoptosis in IAV vRNA replication. The utility of the MS2-imaging system for visualizing time-sensitive processes such as viral replication in live host cells is also demonstrated in this study.

Evolution and Epidemiology of SARS-CoV-2 Virus.

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.

Quantifying Neutralizing Antibodies in Patients with COVID-19 by a Two-Variable Generalized Additive Model.

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.

Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 Is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis.

Enterovirus infections can cause severe complications, such as poliomyelitis, encephalitis, myocarditis, meningitis, neurological pulmonary edema, and even death. Here, we used genome-wide CRISPR screens to gain new insight into the mechanism by which enteroviruses co-opt host pathways to potentiate replication and propagation. We found that acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) is involved in viral replication organelle formation. ACSL4 is a key component of ferroptosis, an iron-dependent, nonapoptotic programmed cell death. Our results indicated that enteroviruses and coronaviruses can induce ferroptosis via ACSL4. Most importantly, ferroptosis inhibitors, including two FDA-approved drugs, rosiglitazone (ROSI; ACSL4 inhibitor) and pioglitazone (PIO; ACSL4 inhibitor), decreased the viral load of human enteroviruses and coronaviruses, suggesting that ACSL4 is a target for counteracting viral infection. IMPORTANCE We provide the first evidence for the role of ACSL4 in enterovirus replication organelle formation. Moreover, both enteroviruses and coronaviruses induce ferroptosis via ACSL4. These findings establish a novel regulatory mechanism for viral replication. The inhibition of ACSL4 by ferroptosis inhibitors can reduce viral yields of enteroviruses and coronaviruses, including SARS-CoV-2, implying that ACSL4-mediated ferroptosis is a promising therapeutic target for viral diseases.

Enterovirus A71 Induces Neurological Diseases and Dynamic Variants in Oral Infection of Human SCARB2-Transgenic Weaned Mice.

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.

Bacterial factors required for Streptococcus pneumoniae coinfection with influenza A virus.

BACKGROUND: Streptococcus pneumoniae is a common cause of post-influenza secondary bacterial infection, which results in excessive morbidity and mortality. Although 13-valent pneumococcal conjugate vaccine (PCV13) vaccination programs have decreased the incidence of pneumococcal pneumonia, PCV13 failed to prevent serotype 3 pneumococcal disease as effectively as other vaccine serotypes. We aimed to investigate the mechanisms underlying the co-pathogenesis of influenza virus and serotype 3 pneumococci. METHODS: We carried out a genome-wide screening of a serotype 3 S. pneumoniae transposon insertion mutant library in a mouse model of coinfection with influenza A virus (IAV) to identify the bacterial factors required for this synergism. RESULTS: Direct, high-throughput sequencing of transposon insertion sites identified 24 genes required for both coinfection and bacterial infection alone. Targeted deletion of the putative aminotransferase (PA) gene decreased bacterial growth, which was restored by supplementation with methionine. The bacterial burden in a coinfection with the PA gene deletion mutant and IAV in the lung was lower than that in a coinfection with wild-type pneumococcus and IAV, but was significantly higher than that in an infection with the PA gene deletion mutant alone. These data suggest that IAV infection alters host metabolism to benefit pneumococcal fitness and confer higher susceptibility to pneumococcal infection. We further demonstrated that bacterial growth was increased by supplementation with methionine or IAV-infected mouse lung homogenates. CONCLUSIONS: The data indicates that modulation of host metabolism during IAV infection may serve as a potential therapeutic intervention against secondary bacterial infections caused by serotype 3 pneumococci during IAV outbreaks in the future.

Differential disease severity and whole-genome sequence analysis for human influenza A/H1N1pdm virus in 2015-2016 influenza season.

During the 2015-16 winter, the US experienced a relatively mild influenza season compared to Taiwan, which had a higher number of total and severe cases. While H1N1pdm viruses dominated global surveillance efforts that season, the global distribution of genetic variants and their contributions to disease severity have not been investigated. Samples collected from influenza A-positive patients by the Johns Hopkins Center of Excellence for Influenza Research and Surveillance active surveillance in the emergency rooms in Baltimore, Maryland, USA, and northern Taiwan between November 2015 and April 2016, were processed for influenza A virus whole-genome sequencing. In Baltimore, the majority of the viruses were the H1N1pdm clade 6B.1 and no H1N1pdm clade 6B.2 viruses were detected. In northern Taiwan, more than half of the H1N1pdm viruses were clade 6B.1 and 38% were clade 6B.2, consistent with the global observation that most 6B.2 viruses circulated in Asia and not North America. Whole virus genome sequence analysis identified two genetic subgroups present in each of the 6B.1 and 6B.2 clades and one 6B.1 interclade reassortant virus. Clinical data showed 6B.2 patients had more disease symptoms including higher crude and inverse probability weighted odds of pneumonia than 6B.1 patients, suggesting 6B.2 circulation may be one of the reasons for the severe flu season in Taiwan. Local surveillance efforts linking H1N1pdm virus sequences to patient clinical and demographic data improve our understanding of influenza circulation and disease potential.

Stability of SARS-CoV-2 Spike G614 Variant Surpasses That of the D614 Variant after Cold Storage.

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.

Culture-Based Virus Isolation To Evaluate Potential Infectivity of Clinical Specimens Tested for COVID-19.

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.

SARS-CoV-2 genomic surveillance in Taiwan revealed novel ORF8-deletion mutant and clade possibly associated with infections in Middle East.

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.