Search

An extensive mosquito survey was carried out in Hong Kong from September to October 2022, employing a variety of collection methods. Specimens were identified using a combination of morphology and mitochondrial cytochrome C oxidase subunit 1 (COI) barcode sequences. Twenty-nine species, including three new records, i.e., Culex bicornutus (Theobald), Culex cinctellus Edwards, and Lutzia chiangmaiensis Somboon & Harbach, were collected. Phylogenetic analysis of COI sequences of Culex annulus Theobald and Culex vishnui Theobald collected in Hong Kong and elsewhere revealed that the sequences of the two nominal species are genetically very similar and are included in the same clade. Consequently, the synonymy of Cx. annulus with Cx. vishnui is reinstated. Lutzia halifaxii (Theobald) is removed from the list of species in Hong Kong and is replaced with Lutzia vorax Edwards, the identification of which is confirmed in the present study. The record of Culex spiculothorax Bram recorded in Hong Kong is replaced with the senior synonym Culex sasai Kano, Nitahara & Awaya. The occurrence of Anopheles fluviatilis James and Aedes aegypti (Linnaeus) is discussed. Finally, an updated checklist of the mosquitoes of Hong Kong, which now includes 76 species representing 14 genera, is provided, with notation of those species that vector pathogens of human diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: This study explored neutralizing IgG antibody levels against COVID-19 decline over time post-vaccination. We conducted this prospective cohort study to investigate the function of gut microbiota in the host immune response following three doses of BNT162b2., Methods: Subjects who received three doses of BNT162b2 were recruited from three centers in Hong Kong. Blood samples were obtained before the first dose and at the one-year timepoint for IgG ELISA to determine the level of neutralizing antibody (NAb). The primary outcome was a high immune response (NAb > 600 AU/mL). We performed shotgun DNA metagenomic sequencing on baseline fecal samples to identify bacterial species and metabolic pathways associated with high immune response using linear discriminant analysis effect size analysis., Results: A total of 125 subjects were recruited (median age: 52 years [IQR: 46.2-59.0]; male: 43 [34.4%]), and 20 were regarded as low responders at the one-year timepoint. Streptococcus parasanguinis (log 10 LDA score = 2.38, p = 0.003; relative abundance of 2.97 × 10 -5 vs. 0.03%, p = 0.001), Bacteroides stercoris (log 10 LDA score = 4.29, p = 0.024; relative abundance of 0.14% vs. 2.40%, p = 0.014) and Haemophilus parainfluenzae (log 10 LDA score = 2.15, p = 0.022; relative abundance of 0.01% vs. 0, p = 0.010) were enriched in low responders. Bifidobacterium pseudocatenulatum (log 10 LDA score = 2.99, p = 0.048; relative abundance of 0.09% vs. 0.36%, p = 0.049) and Clostridium leptum (log 10 LDA score = 2.38, p = 0.014; relative abundance of 1.2 × 10 -5 % vs. 0, p = 0.044) were enriched in high responders. S. parasanguinis was negatively correlated with the superpathway of pyrimidine ribonucleotides de novo biosynthesis (log 10 LDA score = 2.63), which contributes to inflammation and antibody production. H. parainfluenzae was positively correlated with pathways related to anti-inflammatory processes, including the superpathway of histidine, purine, and pyrimidine biosynthesis (log 10 LDA score = 2.14)., Conclusion: Among three-dose BNT162b2 recipients, S. parasanguinis , B. stercoris and H. parainfluenzae were associated with poorer immunogenicity at one year, while B. pseudocatenulatum and C. leptum was associated with a better response.

Influenza A/H9 viruses circulate worldwide in wild and domestic avian species, continuing to evolve and posing a zoonotic risk. A substantial increase in human infections with A/H9N2 subtype avian influenza viruses (AIVs) and the emergence of novel reassortants carrying A/H9N2-origin internal genes has occurred in recent years. Different names have been used to describe the circulating and emerging A/H9 lineages. To address this issue, an international group of experts from animal and public health laboratories, endorsed by the WOAH/FAO Network of Expertise on Animal Influenza, has created a practical lineage classification and nomenclature system based on the analysis of 10,638 hemagglutinin sequences from A/H9 AIVs sampled worldwide. This system incorporates phylogenetic relationships and epidemiologic characteristics designed to trace emerging and circulating lineages and clades. To aid in lineage and clade assignment, an online tool has been created. This proposed classification enables rapid comprehension of the global spread and evolution of A/H9 AIVs.

Background: Ticks, as critical vectors of a variety of pathogens, pose a significant public health challenge globally. In Southeast Asia (SEA), ticks are responsible for transmitting a diverse array of pathogens affecting humans and animals. The geographical and ecological diversity of SEA provides a unique environment that supports a wide range of tick species, which complicates the management and study of tick-borne diseases (TBDs)., Methodology/principal Findings: This article synthesizes findings from the first international symposium on ticks and TBDs in Southeast Asia, held in Phnom Penh on June 22 and 23, 2023. It highlights regional efforts to understand tick ecology and pathogen transmission. This paper proposes to present a summary of the various presentations given during the symposium following 3 main parts. The first one is devoted to the state of knowledge regarding ticks and TBDs in SEA countries, with presentations from 6 different countries, namely Cambodia, Indonesia, Laos, Malaysia, Thailand, and Vietnam. The second part focuses on the development of new research approaches on tick-borne pathogens (TBPs) and TBDs. The last part is a summary of the round table discussion held on the final day, with the aim of defining the most important challenges and recommendations for researches on TBP and TBD in the SEA region., Conclusions/significance: Key topics discussed include advancements in diagnostic tools, such as MALDI-TOF MS and proteomics, and the development of sustainable strategies for tick management and disease prevention. The symposium facilitated the exchange of knowledge and collaborative networks among experts from various disciplines, promoting a unified approach to tackling TBDs in the region. The symposium underscored the need for enhanced surveillance, diagnostics, and inter-regional cooperation to manage the threat of TBDs effectively. Recommendations include the establishment of a regional database for tick identification and the expansion of vector competence studies. These initiatives are crucial for developing targeted interventions and understanding the broader implications of climate change and urbanization on the prevalence of TBDs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yean et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

Background: Neutralizing antibody level wanes with time after COVID-19 vaccination. We aimed to study the relationship between baseline gut microbiota and immunogenicity after three doses of CoronaVac., Methods: This was a prospective cohort study recruiting three-dose CoronaVac recipients from two centers in Hong Kong. Blood samples were collected at baseline and one year post-first dose for virus microneutralization (vMN) assays to determine neutralization titers. The primary outcome was high immune response (defined as with vMN titer ≥ 40). Shotgun DNA metagenomic sequencing of baseline fecal samples identified potential bacterial species and metabolic pathways using Linear Discriminant Analysis Effect Size (LEfSe) analysis. Univariate and multivariable logistic regression models were used to identify high response predictors., Results: In total, 36 subjects were recruited (median age: 52.7 years [IQR: 47.9-56.4]; male: 14 [38.9%]), and 18 had low immune response at one year post-first dose vaccination. Eubacterium rectale (log 10 LDA score = 4.15, p = 0.001; relative abundance of 1.4% vs. 0, p = 0.002), Collinsella aerofaciens (log 10 LDA score = 3.31, p = 0.037; 0.39% vs. 0.18%, p = 0.038), and Streptococcus salivarius (log 10 LDA score = 2.79, p = 0.021; 0.05% vs. 0.02%, p = 0.022) were enriched in low responders. The aOR of high immune response with E. rectale, C. aerofaciens , and S. salivarius was 0.03 (95% CI: 9.56 × 10 -4 -0.32), 0.03 (95% CI: 4.47 × 10 -4 -0.59), and 10.19 (95% CI: 0.81-323.88), respectively. S. salivarius had a positive correlation with pathways enriched in high responders like incomplete reductive TCA cycle (log 10 LDA score = 2.23). C. aerofaciens similarly correlated with amino acid biosynthesis-related pathways. These pathways all showed anti-inflammation functions., Conclusion: E. rectale, C. aerofaciens , and S. salivarius correlated with poorer long-term immunogenicity following three doses of CoronaVac.

Massive sequencing of SARS-CoV-2 genomes has urged novel methods that employ existing phylogenies to add new samples efficiently instead of de novo inference. 'TIPars' was developed for such challenge integrating parsimony analysis with pre-computed ancestral sequences. It took about 21 seconds to insert 100 SARS-CoV-2 genomes into a 100k-taxa reference tree using 1.4 gigabytes. Benchmarking on four datasets, TIPars achieved the highest accuracy for phylogenies of moderately similar sequences. For highly similar and divergent scenarios, fully parsimony-based and likelihood-based phylogenetic placement methods performed the best respectively while TIPars was the second best. TIPars accomplished efficient and accurate expansion of phylogenies of both similar and divergent sequences, which would have broad biological applications beyond SARS-CoV-2. TIPars is accessible from https://tipars.hku.hk/ and source codes are available at https://github.com/id-bioinfo/TIPars., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Coronaviruses (CoVs) pose a major risk to global public health due to their ability to infect diverse animal species and potential for emergence in humans. The CoV spike protein mediates viral entry into the cell and plays a crucial role in determining the binding affinity to host cell receptors. With particular emphasis on α- and β-coronaviruses that infect humans and domestic animals, current research on CoV receptor use suggests that the exploitation of the angiotensin-converting enzyme 2 (ACE2) receptor poses a significant threat for viral emergence with pandemic potential. This review summarizes the approaches used to study binding interactions between CoV spike proteins and the human ACE2 (hACE2) receptor. Solid-phase enzyme immunoassays and cell binding assays allow qualitative assessment of binding but lack quantitative evaluation of affinity. Surface plasmon resonance, Bio-layer interferometry, and Microscale Thermophoresis on the other hand, provide accurate affinity measurement through equilibrium dissociation constants (K D ). In silico modeling predicts affinity through binding structure modeling, protein-protein docking simulations, and binding energy calculations but reveals inconsistent results due to the lack of a standardized approach. Machine learning and deep learning models utilize simulated and experimental protein-protein interaction data to elucidate the critical residues associated with CoV binding affinity to hACE2. Further optimization and standardization of existing approaches for studying binding affinity could aid pandemic preparedness. Specifically, prioritizing surveillance of CoVs that can bind to human receptors stands to mitigate the risk of zoonotic spillover., Competing Interests: We declare that all authors have read and approved the final version of the manuscript. We declare that the manuscript has not been published before and is not under consideration for publication elsewhere. We declare no conflicts of interest., (© 2024 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.)

Spillovers of viruses from animals to humans occur more frequently under warmer conditions, particularly arboviruses. The invasive tick species Haemaphysalis longicornis, the Asian longhorned tick, poses a significant public health threat due to its global expansion and its potential to carry a wide range of pathogens. We analyzed meta-transcriptomic data from 3595 adult H. longicornis ticks collected between 2016 and 2019 in 22 provinces across China encompassing diverse ecological conditions. Generalized additive modeling revealed that climate factors exerted a stronger influence on the virome of H. longicornis than other ecological factors, such as ecotypes, distance to coastline, animal host, tick gender, and antiviral immunity. To understand how climate changes drive the tick virome, we performed a mechanistic investigation using causality inference with emphasis on the significance of this process for public health. Our findings demonstrated that higher temperatures and lower relative humidity/precipitation contribute to variations in animal host diversity, leading to increased diversity of the tick virome, particularly the evenness of vertebrate-associated viruses. These findings may explain the evolution of tick-borne viruses into generalists across multiple hosts, thereby increasing the probability of spillover events involving tick-borne pathogens. Deep learning projections have indicated that the diversity of the H. longicornis virome is expected to increase in 81.9% of regions under the SSP8.5 scenario from 2019 to 2030. Extension of surveillance should be implemented to avert the spread of tick-borne diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Between January 2015 and October 2022, 38 patients with culture-confirmed melioidosis were identified in the Kowloon West (KW) Region, Hong Kong. Notably, 30 of them were clustered in the Sham Shui Po (SSP) district, which covers an estimated area of 2.5 km 2 . Between August and October 2022, 18 patients were identified in this district after heavy rainfall and typhoons. The sudden upsurge in cases prompted an environmental investigation, which involved collecting 20 air samples and 72 soil samples from residential areas near the patients. A viable isolate of Burkholderia pseudomallei was obtained from an air sample collected at a building site five days after a typhoon. B. pseudomallei DNA was also detected in 21 soil samples collected from the building site and adjacent gardening areas using full-length 16S rRNA gene sequencing, suggesting that B. psuedomallei is widely distributed in the soil environment surrounding the district. Core genome-multilocus sequence typing showed that the air sample isolate was phylogenetically clustered with the outbreak isolates in KW Region. Multispectral satellite imagery revealed a continuous reduction in vegetation region in SSP district by 162,255 m 2 from 2016 to 2022, supporting the hypothesis of inhalation of aerosols from the contaminated soil as the transmission route of melioidosis during extreme weather events. This is because the bacteria in unvegetated soil are more easily spread by winds. In consistent with inhalational melioidosis, 24 (63.2%) patients had pneumonia. Clinicians should be aware of melioidosis during typhoon season and initiate appropriate investigation and treatment for patients with compatible symptoms.

Although influenza A viruses of several subtypes have occasionally infected humans, to date only those of the H1, H2, and H3 subtypes have led to pandemics and become established in humans. The detection of two human infections by avian H3N8 viruses in April and May of 2022 raised pandemic concerns. Recent studies have shown the H3N8 viruses were introduced into humans from poultry, although their genesis, prevalence, and transmissibility in mammals have not been fully elucidated. Findings generated from our systematic influenza surveillance showed that this H3N8 influenza virus was first detected in chickens in July 2021 and then disseminated and became established in chickens over wider regions of China. Phylogenetic analyses revealed that the H3 HA and N8 NA were derived from avian viruses prevalent in domestic ducks in the Guangxi-Guangdong region, while all internal genes were from enzootic poultry H9N2 viruses. The novel H3N8 viruses form independent lineages in the glycoprotein gene trees, but their internal genes are mixed with those of H9N2 viruses, indicating continuous gene exchange among these viruses. Experimental infection of ferrets with three chicken H3N8 viruses showed transmission through direct contact and inefficient transmission by airborne exposure. Examination of contemporary human sera detected only very limited antibody cross-reaction to these viruses. The continuing evolution of these viruses in poultry could pose an ongoing pandemic threat. IMPORTANCE A novel H3N8 virus with demonstrated zoonotic potential has emerged and disseminated in chickens in China. It was generated by reassortment between avian H3 and N8 virus(es) and long-term enzootic H9N2 viruses present in southern China. This H3N8 virus has maintained independent H3 and N8 gene lineages but continues to exchange internal genes with other H9N2 viruses to form novel variants. Our experimental studies showed that these H3N8 viruses were transmissible in ferrets, and serological data suggest that the human population lacks effective immunological protection against it. With its wide geographical distribution and continuing evolution in chickens, other spillovers to humans can be expected and might lead to more efficient transmission in humans., Competing Interests: The authors declare no conflict of interest.

The widely distributed antibiotic resistance genes (ARGs) were unevenly proliferated in various habitats. Great endeavors are needed to resolve the resistome features that can differentiate or connect different habitats. This study retrieved a broad spectrum of resistome profiles from 1723 metagenomes categorized into 13 habitats, encompassing industrial, urban, agricultural, and natural environments, and spanning most continents and oceans. The resistome features (ARG types, subtypes, indicator ARGs, and emerging mobilizable ARGs: mcr and tet(X)) in these habitats were benchmarked via a standardized workflow. We found that wastewater and wastewater treatment works were characterized to be reservoirs of more diverse genotypes of ARGs than any other habitats including human and livestock fecal samples, while fecal samples were with higher ARG abundance. Bacterial taxonomy composition was significantly correlated with resistome composition across most habitats. Moreover, the source-sink connectivities were disentangled by developing the resistome-based microbial attribution prediction model. Environmental surveys with standardized bioinformatic workflow proposed in this study will help comprehensively understand the transfer of ARGs in the environment, thus prioritizing the critical environments with high risks for intervention to tackle the problem of ARGs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2023. Published by Elsevier Ltd. All rights reserved.)

Sensitive detection of Mycobacterium tuberculosis (TB) in small percentages in metagenomic samples is essential for microbial classification and drug resistance prediction. However, traditional methods, such as bacterial culture and microscopy, are time-consuming and sometimes have limited TB detection sensitivity. Oxford nanopore technologies (ONT) MinION sequencing allows rapid and simple sample preparation for sequencing. Its recently developed adaptive sequencing selects reads from targets while allowing real-time base-calling to achieve sequence enrichment or depletion during sequencing. Another common enrichment method is PCR amplification of the target TB genes. In this study, we compared both methods using ONT MinION sequencing for TB detection and variant calling in metagenomic samples using both simulation runs and those with synthetic and patient samples. We found that both methods effectively enrich TB reads from a high percentage of human (95%) and other microbial DNA. Adaptive sequencing with readfish and UNCALLDE achieved a 3.9-fold and 2.2-fold enrichment compared to the control run. We provide a simple automatic analysis framework to support the detection of TB for clinical use, openly available at https://github.com/HKU-BAL/ONT-TB-NF . Depending on the patient's medical condition and sample type, we recommend users evaluate and optimize their workflow for different clinical specimens to improve the detection limit., (© 2023. The Author(s).)

Background: The impact of host skin microbiome on horizontal transmission of tick-borne pathogens , and of pathogen associated transstadial and transovarial changes in tick microbiome are largely unknown, but are important to control increasingly emerging tick-borne diseases worldwide., Methods: Focusing on a rickettsiosis pathogen, Rickettsia raoultii, we used R. raoultii-positive and R. raoultii-negative Dermacentor spp. tick colonies to study the involvement of skin microbiota in cutaneous infection with rickettsiae in laboratory mice, and the function of the tick microbiome on maintenance of rickettsiae through all tick developmental stages (eggs, larvae, nymphs, adults) over two generations., Results: We observed changes in the skin bacteria community, such as Chlamydia, not only associated with rickettsial colonization but also with tick feeding on skin. The diversity of skin microbiome differed between paired tick-bitten and un-bitten sites. For vertical transmission, significant differences in the tick microbiota between pathogenic rickettsia-positive and -negative tick chorts was observed across all developmental stages at least over two generations, which appeared to be a common pattern not only for R. raoultii but also for another pathogenic species, Candidatus Rickettsia tarasevichiae. More importantly, bacterial differences were complemented by functional shifts primed for genetic information processing during blood feeding. Specifically, the differences in tick microbiome gene repertoire between pathogenic Rickettsia-positive and -negative progenies were enriched in pathways associated with metabolism and hormone signals during vertical transmission., Conclusions: We demonstrate that host skin microbiome might be a new factor determining the transmission of rickettsial pathogens through ticks. While pathogenic rickettsiae infect vertebrate hosts during blood-feeding by the tick, they may also manipulate the maturation of the tick through changing the functional potential of its microbiota over the tick's life stages. The findings here might spur the development of new-generation control methods for ticks and tick-borne pathogens. Video Abstract., (© 2023. The Author(s).)

Introduction: Ticks are the most important obligate blood-feeding vectors of human pathogens. With the advance of high-throughput sequencing, more and more bacterial community and virome in tick has been reported, which seems to pose a great threat to people., Methods: A total of 14 skin specimens collected from tick-bite patients with mild to severe symptoms were analyzed through meta-transcriptomic sequencings., Results: Four bacteria genera were both detected in the skins and ticks, including Pseudomonas, Acinetobacter, Corynebacterium and Propionibacterium, and three tick-associated viruses, Jingmen tick virus (JMTV), Bole tick virus 4 (BLTV4) and Deer tick mononegavirales-like virus (DTMV) were identified in the skin samples. Except of known pathogens such as pathogenic rickettsia, Coxiella burnetii and JMTV, we suggest Roseomonas cervicalis and BLTV4 as potential new agents amplified in the skins and then disseminated into the blood. As early as 1 day after a tick-bite, these pathogens can transmit to skins and at most four ones can co-infect in skins., Discussion: Advances in sequencing technologies have revealed that the diversity of tick microbiome and virome goes far beyond our previous understanding. This report not only identifies three new potential pathogens in humans but also shows that the skin barrier is vital in preventing horizontal transmissions of tick-associated bacteria or virus communities to the host. It is the first research on patients' skin infectome after a tick bite and demonstrates that more attention should be paid to the cutaneous response to prevent tick-borne illness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Zheng, Chu, Cui, Wei, Bian, Liu, Yao, Jiang, Huo, Yuan, Li, Zhao, Li, Wang, Wei, Zhu, Chen, Gao, Wang, Ye, Song, Jiang, Lam, Ni and Jia.)

The increasing prevalence and expanding distribution of tick-borne viruses globally have raised health concerns, but the full repertoire of the tick virome has not been assessed. We sequenced the meta-transcriptomes of 31 different tick species in the Ixodidae and Argasidae families from across mainland China, and identified 724 RNA viruses with distinctive virome compositions among genera. A total of 1,801 assembled and complete or nearly complete viral genomes revealed an extensive diversity of genome architectures of tick-associated viruses, highlighting ticks as a reservoir of RNA viruses. We examined the phylogenies of different virus families to investigate virome evolution and found that the most diverse tick-associated viruses are positive-strand RNA virus families that demonstrate more ancient divergence than other arboviruses. Tick-specific viruses are often associated with only a few tick species, whereas virus clades that can infect vertebrates are found in a wider range of tick species. We hypothesize that tick viruses can exhibit both 'specialist' and 'generalist' evolutionary trends. We hope that our virome dataset will enable much-needed research on vertebrate-pathogenic tick-associated viruses., (© 2023. The Author(s).)

Bacteria community provides essential ecological services to rice plants. The bacterial diversity of rice varies across host plant genotype and organs. This study employed 16S rDNA amplicon sequencing to characterise the bacterial community associated with three rice landraces using leaf blade and stem samples. The prokaryotic community found in these rice landraces comprised of two kingdoms, 12 phyla, 25 classes, 40 orders, 80 families, and 118 genera. Proteobacteria (53.9%) was the most abundant phylum. The most abundant genus was an undefined genus under Cyanobacteria (33.0%). Homogeneity of prokaryotic community was observed across the three rice landraces, which may suggest a high similarity in biological and genetical properties of the rice landraces. The difference in prokaryotic composition between leaf blade and stem was depicted based on principal coordinate analysis. This study observed that the prokaryotic inhabitants in rice plants is predominantly determined by rice plant organs., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: COVID-19 patients may experience "cytokine storm" when human immune system produces excessive cytokines/chemokines. However, it remains unclear whether early responses of inflammatory cytokines would lead to high or low titers of anti-SARS-CoV-2 antibodies., Methods: This retrospective study enrolled a cohort of 272 hospitalized patients with laboratory-confirmed SARS-CoV-2. Laboratory assessments of serum cytokines (IL-2R, IL-6, IL-8, IL-10, TNF-α), anti-SARS-CoV-2 IgG/IgM antibodies, and peripheral blood biomarkers were conducted during hospitalization., Results: At hospital admission, 36.4% patients were severely ill, 51.5% patients were ≥ 65 years, and 60.3% patients had comorbidities. Higher levels of IL-2R and IL-6 were observed in older patients (≥65 years). Significant differences of IL-2R (week 2 to week ≥5 from symptom onset), IL-6 (week 1 to week ≥5), IL-8 (week 2 to week ≥5), and IL-10 (week 1 to week 3) were observed between moderately-ill and severely ill patients. Anti-SARS-CoV-2 IgG titers were significantly higher in severely ill patients than in moderately ill patients, but such difference was not observed for IgM. High titers of early-stage IL-6, IL-8, and TNF-α (≤2 weeks after symptom onset) were positively correlated with high titers of late-stage IgG (≥5 weeks after symptom onset). Deaths were mostly observed in severely ill older patients (45.9%). Survival analyses revealed risk factors of patient age, baseline COVID-19 severity, and baseline IL-6 that affected survival time, especially in severely ill older patients., Conclusion: Early responses of elevated cytokines such as IL-6 reflect the active immune responses, leading to high titers of IgG antibodies against COVID-19., (© 2022. The Author(s).)

The CRF01_AE and CRF07_BC clades dominate the human immunodeficiency virus (HIV) epidemics in China. Both clades have been identified in the men who have sex with men (MSM) population in Guangdong province, raising a serious concern of possible complex recombination events ahead. Here, we report the first case of CRF01_AE/CRF07_BC recombinant sampled from a MSM patient in southern China. The genomic structure of this case is a mosaic with some regions resembling the CRF01_AE and CRF07_BC clades. Our phylogenetic analyses show that the two parental lineages of this recombinant virus were mainly found in the MSM population. This case has a different genomic composition compared with other recombinants descended from the same parental clades CRF01_AE and CRF07_BC. Our finding suggests that the MSM populations have become a hotspot for expanding viral diversity through the viral recombination mechanism. Therefore, further epidemiologic surveillance and monitoring should be conducted within the MSM populations to help advance our knowledge of viral transmission mechanisms. Additionally, these measures will serve to enhance the control and prevention of HIV/acquired immunodeficiency syndrome in China., (© 2021 Wiley Periodicals LLC.)

Enteroviruses (EVs) species A are a major public health issue in the Asia-Pacific region and cause frequent epidemics of hand, foot and mouth disease (HFMD) in China. Mild infections are common in children; however, HFMD can also cause severe illness that affects the central nervous system. To molecularly characterize EVs, a prospective HFMD virological surveillance program was performed in China between 2013 and 2016. Throat swabs, rectal swabs and stool samples were collected from suspected HFMD patients at participating hospitals. EVs were detected using generic real-time and nested reverse transcription-polymerase chain reactions (RT-PCRs). Then, the complete VP1 regions of enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16) and CVA6 were sequenced to analyze amino acid changes and construct a viral molecular phylogeny. Of the 2836 enrolled HFMD patients, 2,517 (89%) were EV positive. The most frequently detected EVs were CVA16 (32.5%, 819), CVA6 (31.2%, 785), and EV-A71 (20.4%, 514). The subgenogroups CVA16_B1b, CVA6_D3a and EV-A71_C4a were predominant in China and recombination was not observed in the VP1 region. Sequence analysis revealed amino acid variations at the 30, 29 and 44 positions in the VP1 region of EV-A71, CVA16 and CVA6 (compared to the respective prototype strains BrCr, G10 and Gdula), respectively. Furthermore, in 21 of 24 (87.5%) identified EV-A71 samples, a known amino acid substitution (D31N) that may enhance neurovirulence was detected. Our study provides insights about the genetic characteristics of common HFMD-associated EVs. However, the emergence and virulence of the described mutations require further investigation.

The Blueline Rasbora (Rasbora sarawakensis) is a small ray-finned fish categorized under the genus Rasbora in the Cyprinidae family. In this study, the complete mitogenome sequence of R. sarawakensis was sequenced using four primers targeting overlapping regions. The mitogenome is 16,709 bp in size, accommodating 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organisation was detected between this species and other genus counterparts. The heavy strand houses 28 genes while the light strand stores the other nine genes. Most protein-coding genes employ ATG as start codon, excluding COI gene, which utilizes GTG instead. The central conserved sequence blocks (CSB-F, CSB-E and CSB-D), variable sequence blocks (CSB-3, CSB-2 and CSB-1) as well as the terminal associated sequence (TAS) are conserved in the control region. The maximum likelihood phylogenetic tree revealed the divergence of R. sarawakensis from the basal region of the Rasbora clade, where its evolutionary relationships with R. maculatus and R. pauciperforata are poorly resolved as indicated by the low bootstrap values. This work acts as steppingstone towards further molecular evolution and population genetics studies of Rasbora genus in future., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

There are outstanding evolutionary questions on the recent emergence of human coronavirus SARS-CoV-2 including the role of reservoir species, the role of recombination and its time of divergence from animal viruses. We find that the sarbecoviruses-the viral subgenus containing SARS-CoV and SARS-CoV-2-undergo frequent recombination and exhibit spatially structured genetic diversity on a regional scale in China. SARS-CoV-2 itself is not a recombinant of any sarbecoviruses detected to date, and its receptor-binding motif, important for specificity to human ACE2 receptors, appears to be an ancestral trait shared with bat viruses and not one acquired recently via recombination. To employ phylogenetic dating methods, recombinant regions of a 68-genome sarbecovirus alignment were removed with three independent methods. Bayesian evolutionary rate and divergence date estimates were shown to be consistent for these three approaches and for two different prior specifications of evolutionary rates based on HCoV-OC43 and MERS-CoV. Divergence dates between SARS-CoV-2 and the bat sarbecovirus reservoir were estimated as 1948 (95% highest posterior density (HPD): 1879-1999), 1969 (95% HPD: 1930-2000) and 1982 (95% HPD: 1948-2009), indicating that the lineage giving rise to SARS-CoV-2 has been circulating unnoticed in bats for decades.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

There is considerable public and scientific interest in the origin, spread, and evolution of SARS-CoV-2. Lu et al. recently conducted genomic sequencing and analysis of SARS-CoV-2 in Guangdong, revealing its early transmission out of Hubei and shedding light on the effectiveness of controlling local transmission chains., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-2 1 . This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection 2 . Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.

The Trigonopoma pauciperforatum or the redstripe rasbora is a cyprinid commonly found in marshes and swampy areas with slight acidic tannin-stained water in the tropics. In this study, the complete mitogenome sequence of T. pauciperforatum was first amplified in two parts using two pairs of overlapping primers and then sequenced. The size of the mitogenome is 16,707 bp, encompassing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organisation was detected between this species and other family members. The heavy strand accommodates 28 genes while the light strand houses the remaining nine genes. Most protein-coding genes utilise ATG as start codon except for COI gene which uses GTG instead. The terminal associated sequence (TAS), central conserved sequence block (CSB-F, CSB-D and CSB-E) as well as variable sequence block (CSB-1, CSB-2 and CSB-3) are conserved in the control region. The maximum likelihood phylogenetic tree revealed the divergence of T. pauciperforatum from the basal region of the major clade, where its evolutionary relationships with Boraras maculatus , Rasbora cephalotaenia and R. daniconius are poorly resolved as suggested by the low bootstrap values. This work contributes towards the genetic resource enrichment for peat swamp conservation and comprehensive in-depth comparisons across other phylogenetic researches done on the Rasbora -related genus., (© Penerbit Universiti Sains Malaysia, 2020.)

Phylogenetic trees and data are often stored in incompatible and inconsistent formats. The outputs of software tools that contain trees with analysis findings are often not compatible with each other, making it hard to integrate the results of different analyses in a comparative study. The treeio package is designed to connect phylogenetic tree input and output. It supports extracting phylogenetic trees as well as the outputs of commonly used analytical software. It can link external data to phylogenies and merge tree data obtained from different sources, enabling analyses of phylogeny-associated data from different disciplines in an evolutionary context. Treeio also supports export of a phylogenetic tree with heterogeneous-associated data to a single tree file, including BEAST compatible NEXUS and jtree formats; these facilitate data sharing as well as file format conversion for downstream analysis. The treeio package is designed to work with the tidytree and ggtree packages. Tree data can be processed using the tidy interface with tidytree and visualized by ggtree. The treeio package is released within the Bioconductor and rOpenSci projects. It is available at https://www.bioconductor.org/packages/treeio/., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: A tick-borne segmented RNA virus called Jingmen tick virus (JMTV) was recently identified, variants of which were detected in a non-human primate host and fatal patients with Crimean-Congo haemorrhagic fever. We investigated its infectivity and pathogenicity for humans., Methods: We obtained skin-biopsy, blood and serum samples from patients with tick bites, and used high-throughput sequencing, in situ hybridisation, and serologic testing to diagnose and ascertain the cases of JMTV infection., Findings: A JMTV strain was isolated from the tick Amblyomma javanense into an embryo-derived tick cell line. We obtained sustained passage of JMTV, and revealed that it was able to accumulate in salivary glands of experimentally infected ticks. Four JMTV-infected patients were identified by high-throughput sequencing of skin biopsies and blood samples. The virus replication in skin tissue was visualised by in situ hybridisation. The four patients all had an itchy or painful eschar at the site of tick bite, with or without lymphadenopathy. Immunohistochemical examination revealed remarkable local inflammation manifested as infiltration by neutrophils. Eight patients were identified by serological testing and showed more severe clinical manifestations. Two Ixodes persulcatus ticks detached from patients were positive for JMTV. All JMTV strains identified in this study formed a well-supported sub-lineage, distinct from those previously reported in China. Interpretation The public significance of JMTV should be highly concerning due to its potential pathogenicity for humans and efficient transmission by potential ticks. FUND: China Natural Science Foundation, State Key Research Development Programme, and United Kingdom Biotechnology and Biological Sciences Research Council., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Ggtree is a comprehensive R package for visualizing and annotating phylogenetic trees with associated data. It can also map and visualize associated external data on phylogenies with two general methods. Method 1 allows external data to be mapped on the tree structure and used as visual characteristic in tree and data visualization. Method 2 plots the data with the tree side by side using different geometric functions after reordering the data based on the tree structure. These two methods integrate data with phylogeny for further exploration and comparison in the evolutionary biology context. Ggtree is available from http://www.bioconductor.org/packages/ggtree.

Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern United States. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the United States from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV strains in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the United States and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region. IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with a >90% mortality rate in horses, an ∼33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the United States, and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas., (Copyright © 2018 American Society for Microbiology.)

Detecting the temporally and spatially varying correlations is important to understand the biological and disease systems. Here we proposed a geographically weighted temporally correlated logistic regression (GWTCLR) model to identify such dynamic correlation of predictors on binomial outcome data, by incorporating spatial and temporal information for joint inference. The local likelihood method is adopted to estimate the spatial relationship, while the smoothing method is employed to estimate the temporal variation. We present the construction and implementation of GWTCLR and the study of the asymptotic properties of the proposed estimator. Simulation studies were conducted to evaluate the robustness of the proposed model. GWTCLR was applied on real epidemiologic data to study the climatic determinants of human seasonal influenza epidemics. Our method obtained results largely consistent with previous studies but also revealed certain spatial and temporal varying patterns that were unobservable by previous models and methods.

Background: A febrile man, who returned to China after a 9-day travel in Fiji and Samoa, was detected to be infected with Zika virus (ZIKV) at the port by Shenzhen Entry-Exit Inspection and Quarantine Bureau on 14 February 2016., Methods: The patient and his 32 travelling companions were traced for ZIKV infection. A standardised questionnaire was used to obtain the information on demographics, clinical manifestations and exposure history. Their samples were tested for ZIKV by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The positive samples were subjected to viral culture and genome sequencing., Findings: Four of the 33 travellers were confirmed to be infected by ZIKV through qRT-PCR and viral culture, with an overall infection rate of 12%. Interestingly, one case (Patient 3) where high viremia levels were tested 4 days prior to symptoms. In addition, a 7-year-old girl was identified to have ZIKV infection on 17 February, but never had any manifestation. ZIKV was isolated from the four imported cases. Phylogenetic analysis based on whole genome sequences revealed that these isolates were similar to each other and close to the strain causing the French Polynesia outbreak in 2013., Interpretation: The travellers should be informed of the high risk for ZIKV infection during their stay in areas with active transmission and measures to prevent mosquito bites.

Phylogenetic analysis of the highly pathogenic avian influenza (HPAI) H5 viruses causing recent outbreaks in Taiwan showed that they belonged to the Asian HPAI H5 lineage, clade 2.3.4.4 viruses, and were apparently introduced by migratory birds. These viruses reassorted with Eurasian influenza gene pool viruses and formed five genotypic variants. As Taiwan has a similar influenza ecosystem to southern China, the HPAI H5 lineage could become established and enzootic in the island., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The occurrence of human infections with avian H7N9 viruses since 2013 demonstrates the continuing pandemic threat posed by the current influenza ecosystem in China. Influenza surveillance and phylogenetic analyses showed that these viruses were generated by multiple interspecies transmissions and reassortments among the viruses resident in domestic ducks and the H9N2 viruses enzootic in chickens. A large population of domestic ducks hosting diverse influenza viruses provided the precondition for these events to occur, while acquiring internal genes from enzootic H9N2 influenza viruses in chickens promoted the spread of these viruses. Human infections effectively act as sentinels, reflecting the intensity of the activity of these viruses in poultry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: The H9N2 influenza viruses that are enzootic in terrestrial poultry in China pose a persistent pandemic threat to humans. To investigate whether the continuous circulation and adaptation of these viruses in terrestrial poultry increased their infectivity to pigs, we conducted a serological survey in pig herds with H9N2 viruses selected from the aquatic avian gene pool (Y439 lineage) and the enzootic terrestrial poultry viruses (G1 and Y280 lineages). We also compared the infectivity and transmissibility of these viruses in pigs. It was found that more than 15% of the pigs sampled from 2010 to 2012 in southern China were seropositive to either G1 or Y280 lineage viruses, but none of the sera were positive to the H9 viruses from the Y439 lineage. Viruses of the G1 and Y280 lineages were able to infect experimental pigs, with detectable nasal shedding of the viruses and seroconversion, whereas viruses of the Y439 lineage did not cause a productive infection in pigs. Thus, adaptation and prevalence in terrestrial poultry could lead to interspecies transmission of H9N2 viruses from birds to pigs. Although H9N2 viruses do not appear to be continuously transmissible among pigs, repeated introductions of H9 viruses to pigs naturally increase the risk of generating mammalian-adapted or reassorted variants that are potentially infectious to humans. This study highlights the importance of monitoring the activity of H9N2 viruses in terrestrial poultry and pigs., Importance: H9N2 subtype of influenza viruses has repeatedly been introduced into mammalian hosts, including humans and pigs, so awareness of their activity and evolution is important for influenza pandemic preparedness. However, since H9N2 viruses usually cause mild or even asymptomatic infections in mammalian hosts, they may be overlooked in influenza surveillance. Here, we found that the H9N2 viruses established in terrestrial poultry had higher infectivity in pigs than those from aquatic birds, which suggests that adaptation of the H9N2 viruses in terrestrial poultry might have increased the infectivity of the virus to mammals. Therefore, monitoring the prevalence and evolution of H9 viruses prevalent in terrestrial birds and conducting risk assessment of their threat to mammals are critical for evaluating the pandemic potential of this virus., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies., Importance: Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: The emergence in humans of the A(H1N1)pdm09 influenza virus, a complex reassortant virus of swine origin, highlighted the importance of worldwide influenza virus surveillance in swine. To date, large-scale surveillance studies have been reported for southern China and North America, but such data have not yet been described for Europe. We report the first large-scale genomic characterization of 290 swine influenza viruses collected from 14 European countries between 2009 and 2013. A total of 23 distinct genotypes were identified, with the 7 most common comprising 82% of the incidence. Contrasting epidemiological dynamics were observed for two of these genotypes, H1huN2 and H3N2, with the former showing multiple long-lived geographically isolated lineages, while the latter had short-lived geographically diffuse lineages. At least 32 human-swine transmission events have resulted in A(H1N1)pdm09 becoming established at a mean frequency of 8% across European countries. Notably, swine in the United Kingdom have largely had a replacement of the endemic Eurasian avian virus-like ("avian-like") genotypes with A(H1N1)pdm09-derived genotypes. The high number of reassortant genotypes observed in European swine, combined with the identification of a genotype similar to the A(H3N2)v genotype in North America, underlines the importance of continued swine surveillance in Europe for the purposes of maintaining public health. This report further reveals that the emergences and drivers of virus evolution in swine differ at the global level., Importance: The influenza A(H1N1)pdm09 virus contains a reassortant genome with segments derived from separate virus lineages that evolved in different regions of the world. In particular, its neuraminidase and matrix segments were derived from the Eurasian avian virus-like ("avian-like") lineage that emerged in European swine in the 1970s. However, while large-scale genomic characterization of swine has been reported for southern China and North America, no equivalent study has yet been reported for Europe. Surveillance of swine herds across Europe between 2009 and 2013 revealed that the A(H1N1)pdm09 virus is established in European swine, increasing the number of circulating lineages in the region and increasing the possibility of the emergence of a genotype with human pandemic potential. It also has implications for veterinary health, making prevention through vaccination more challenging. The identification of a genotype similar to the A(H3N2)v genotype, causing zoonoses at North American agricultural fairs, underlines the importance of continued genomic characterization in European swine., (Copyright © 2015 Watson et al.)

Epidemiological and evolutionary dynamics of influenza B Victoria and Yamagata lineages remained poorly understood in the tropical Southeast Asia region, despite causing seasonal outbreaks worldwide. From 2012-2014, nasopharyngeal swab samples collected from outpatients experiencing acute upper respiratory tract infection symptoms in Kuala Lumpur, Malaysia, were screened for influenza viruses using a multiplex RT-PCR assay. Among 2,010/3,935 (51.1%) patients infected with at least one respiratory virus, 287 (14.3%) and 183 (9.1%) samples were tested positive for influenza A and B viruses, respectively. Influenza-positive cases correlate significantly with meteorological factors-total amount of rainfall, relative humidity, number of rain days, ground temperature and particulate matter (PM10). Phylogenetic reconstruction of haemagglutinin (HA) gene from 168 influenza B viruses grouped them into Yamagata Clade 3 (65, 38.7%), Yamagata Clade 2 (48, 28.6%) and Victoria Clade 1 (55, 32.7%). With neuraminidase (NA) phylogeny, 30 intra-clade (29 within Yamagata Clade 3, 1 within Victoria Clade 1) and 1 inter-clade (Yamagata Clade 2-HA/Yamagata Clade 3-NA) reassortants were identified. Study of virus temporal dynamics revealed a lineage shift from Victoria to Yamagata (2012-2013), and a clade shift from Yamagata Clade 2 to Clade 3 (2013-2014). Yamagata Clade 3 predominating in 2014 consisted of intra-clade reassortants that were closely related to a recent WHO vaccine candidate strain (B/Phuket/3073/2013), with the reassortment event occurred approximately 2 years ago based on Bayesian molecular clock estimation. Malaysian Victoria Clade 1 viruses carried H274Y substitution in the active site of neuraminidase, which confers resistance to oseltamivir. Statistical analyses on clinical and demographic data showed Yamagata-infected patients were older and more likely to experience headache while Victoria-infected patients were more likely to experience nasal congestion and sore throat. This study describes the evolution of influenza B viruses in Malaysia and highlights the importance of continuous surveillance for better vaccination policy in this region.

Since 2013 the occurrence of human infections by a novel avian H7N9 influenza virus in China has demonstrated the continuing threat posed by zoonotic pathogens. Although the first outbreak wave that was centred on eastern China was seemingly averted, human infections recurred in October 2013 (refs 3-7). It is unclear how the H7N9 virus re-emerged and how it will develop further; potentially it may become a long-term threat to public health. Here we show that H7N9 viruses have spread from eastern to southern China and become persistent in chickens, which has led to the establishment of multiple regionally distinct lineages with different reassortant genotypes. Repeated introductions of viruses from Zhejiang to other provinces and the presence of H7N9 viruses at live poultry markets have fuelled the recurrence of human infections. This rapid expansion of the geographical distribution and genetic diversity of the H7N9 viruses poses a direct challenge to current disease control systems. Our results also suggest that H7N9 viruses have become enzootic in China and may spread beyond the region, following the pattern previously observed with H5N1 and H9N2 influenza viruses.

Unlabelled: The cases of human infections with H10N8 viruses identified in late 2013 and early 2014 in Jiangxi, China, have raised concerns over the origin, prevalence, and development of these viruses in this region. Our long-term influenza surveillance of poultry and migratory birds in southern China in the past 12 years showed that H10 influenza viruses have been introduced from migratory to domestic ducks over several winter seasons at sentinel duck farms at Poyang Lake, where domestic ducks share their water body with overwintering migratory birds. H10 viruses were never detected in terrestrial poultry in our survey areas until August 2013, when they were identified at live-poultry markets in Jiangxi. Since then, we have isolated 124 H10N8 or H10N6 viruses from chickens at local markets, revealing an ongoing outbreak. Phylogenetic analysis of H10 and related viruses showed that the chicken H10N8 viruses were generated through multiple reassortments between H10 and N8 viruses from domestic ducks and the enzootic chicken H9N2 viruses. These chicken reassortant viruses were highly similar to the human isolate, indicating that market chickens were the source of human infection. Recently, the H10 viruses further reassorted, apparently with H5N6 viruses, and generated an H10N6 variant. The emergence and prevalence of H10 viruses in chickens and the occurrence of human infections provide direct evidence of the threat from the current influenza ecosystem in China., Importance: After the outbreak of avian-origin H7N9 influenza viruses in China, fatal human infections with a novel H10N8 virus were reported. Utilizing data from 12 years of influenza surveillance in southern China, we showed that H10 viruses were regularly introduced by migratory ducks to domestic ducks on Poyang Lake, a major aggregative site of migratory birds in Asia. The H10 viruses were maintained and amplified in domestic ducks and then transmitted to chickens and reassorted with enzootic H9N2 viruses, leading to an outbreak and human infections at live-poultry markets. The emergence of the H10N8 virus, following a pathway similar to that of the recent H7N9 virus, highlights the role of domestic ducks and the current influenza ecosystem in China that facilitates influenza viruses moving from their reservoir hosts through the live-poultry system to cause severe consequences for public health., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Over the course of two waves of infection, H7N9 avian influenza A virus has caused 436 human infections and claimed 170 lives in China as of July 2014. To investigate the prevalence and genetic diversity of H7N9, we surveyed avian influenza viruses in poultry in Jiangsu province within the outbreak epicenter. We found frequent occurrence of H7N9/H9N2 coinfection in chickens. Molecular clock phylogenetic analysis confirms coinfection by H7N9/H9N2 viruses and also reveals that the identity of the H7N9 outbreak lineage is confounded by ongoing reassortment between outbreak viruses and diverse H9N2 viruses in domestic birds. Experimental inoculation of a coinfected sample in cell culture yielded two reassortant H7N9 strains with polymerase segments from the original H9N2 strain. Ongoing reassortment between the H7N9 outbreak lineage and diverse H9N2 viruses may generate new strains with the potential to infect humans, highlighting the need for continued viral surveillance in poultry and humans., Importance: We found frequent occurrence of H7N9/H9N2 coinfection in chickens. The H7N9 outbreak lineage is confounded by ongoing reassortment between H7N9 and H9N2 viruses. The importance of H9N2 viruses as the source of novel avian influenza virus infections in humans requires continuous attention., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Two-way transmission of influenza viruses between humans and swine has been frequently observed, and the occurrence of the 2009 H1N1 pandemic influenza virus (pdm/09) demonstrated that swine-origin viruses could facilitate the genesis of a pandemic strain. Although multiple introductions to and reassortment in swine of the pdm/09 virus have been repeatedly reported in both Eurasia and the Americas, its long-term impact on the development of swine influenza viruses (SIVs) has not been systematically explored. Our comprehensive evolutionary studies of the complete genomes of 387 SIVs obtained from 2009 to 2012 by influenza virus surveillance in China revealed 17 reassortant genotypes with pdm/09-origin genes. Even though the entire 2009 pandemic virus and its surface genes cannot persist, its internal genes have become established and are now the predominant lineages in pigs in the region. The main persistent pdm/09-origin reassortant forms had at least five pdm/09-origin internal genes, and their surface genes were primarily of European avian-like (EA) or human H3N2-like SIV origin. These findings represent a marked change in the evolutionary patterns and ecosystem of SIVs in China. It is possible that the pdm/09-origin internal genes are in the process of replacing EA or triple-reassortant-like internal genes. These alterations in the SIV gene pool need to be continually monitored to assess changes in the potential for SIV transmission to humans., Importance: Shortly after the emergence of the 2009 pandemic H1N1 (pdm/09) influenza virus, it was transmitted from humans to pigs and this continues to occur around the world. Many reassortants between pdm/09-origin viruses and enzootic swine influenza viruses (SIVs) have been detected. However, the long-term impact of pdm/09-origin viruses on the SIV gene pool, which could lead to the generation of influenza viruses with the potential to infect humans, has not been systematically examined. From extensive surveillance of SIVs over a 38-month period in southern China, it was found that although neither complete pdm/09 viruses nor their surface genes could persist in pigs, their internal genes did persist. Over the survey period, these internal genes became predominant, potentially replacing those of the enzootic SIV lineages. The altered diversity of the SIV gene pool needs to be closely monitored for changes in the potential for SIV transmission to humans., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The Southwest United States, including Arizona and New Mexico, has a diverse climate and is home to many different avian species. We sequenced the hemagglutinin (HA) gene of twenty influenza specimens for the years 2007-2009. This included four from Arizona, and sixteen from New Mexico. We analyzed the sequences and determined the following HA subtypes: H3, H4, H6, H8, and H11. For each subtype, we combined our virus sequences with those from a public database, and inferred phylogeographic models of influenza diffusion. Statistical phylogeography indicated that overall evolutionary diffusion of avian influenza viruses is geographically structured (p<0.05). In addition, we found that diffusion to the Southwest was often from nearby states including California. For H3, H4 and H6, the intra-flyway gene flow rates were significantly (p<0.001) higher than those of inter-flyway. Such rate difference was also observed in H8 and H11, yet, without statistical significance (p=0.132, p=0.190, respectively). Excluding any one flyway from the calculation generated similar results, suggesting that such barrier effect on gene flow rates is not exclusively produced by any single flyway. We also calculated the Bayes factor test for the significant non-zero rates between states and identified significant routes both within and across flyways. Such inter-flyway spread of influenza was probably the result of birds from four flyways co-mingling on breeding grounds in northern regions or marshaling on staging areas post breeding in Canada or Alaska, before moving south each fall. This study provides an initial analysis of evolutionary diffusion of avian influenza virus to and from the Southwest United States. However, more sequences from this region need to be generated to determine the role of host migration and other factors on influenza diffusion., (Copyright © 2014 Elsevier B.V. All rights reserved.)