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Chronic hepatitis B is a major cause of liver disease worldwide and is currently incurable. Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is a key form of the virus responsible for its persistence and is the transcriptional template for all viral transcripts. The field is focussed on methods to clear HBV cccDNA but this been limited by technical difficulties in its quantification due to: identical sequence to other forms of HBV DNA; low copy number per cell; and high resistance to denaturation by heat, leading to difficulty using polymerase chain reaction or hybridization methods for detection. A number of assays have been developed in order to overcome these hurdles either directly or detecting cccDNA levels indirectly via its transcriptional products. In this review, we summarize the approaches to cccDNA quantification that are currently used, and outline key open questions in the cccDNA biology field which remain to be answered due to the limitations of current methods.

Most existing wastewater surveillance studies that focus on viruses have identified a large fraction of bacteriophages. Identifying bacteria by considering bacteriophage-host interactions is a novel method for detecting bacterial pathogens circulating in a community, using wastewater surveillance. This study aims to identify human-related bacterial pathogens in municipal wastewater collected in metro Detroit, using high-throughput sequencing and bioinformatics. Untreated municipal wastewater samples were collected on August 11, 2020, and bacteriophages were concentrated using the VIRus ADsorption-ELution (VIRADEL) method. Bacteriophage-related contigs in samples ranged from 15.53 % to 18.91 %, with 2477 classified and 8853 unclassified contigs. Most identified bacteriophages were from Caudoviricetes and Malgrandaviricetes classes belonging to 19 families. Hosts of bacteriophages were predicted with the PhaBOX (CHERRY) tool. The results indicated that out of the 2477 classified phages, 2373 were associated with known bacterial hosts. Also, out of 8853 unclassified bacteriophages, 8421 were associated with known bacterial hosts, and the remaining 432 were with unknown bacterial hosts. Among all bacteriophage-associated hosts, 399 were identified as pathogenic bacteria at the species level. Approximately, 85 % of the identified pathogenic bacteria are reported to be associated with human diseases. Genome quality assessments showed that 15 bacteriophages had nearly complete genomes, which were further analyzed to understand bacteriophage-bacteria interactions in wastewater. Identified hosts of these complete-genome phages included human pathogens such as Salmonella enterica, Bacillus cereus, Achromobacter xylosoxidans, and Escherichia coli. The S. enterica bacteriophage (k141_1005294) genomic map was annotated, and responsible open reading frames (ORFs) were characterized to illustrate bacteriophage behavior during infection of pathogenic bacteria in untreated wastewater. To the best of our knowledge, this is the first attempt to characterize human bacterial pathogens in wastewater through bacteriophage-pathogen interactions. Novel bioinformatic approaches enhance pathogen detection and improve the understanding of community wastewater microbiomes., 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 study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Herpes simplex virus 1 (HSV-1) latently infected neurons display diverse patterns in the distribution of the viral genomes within the nucleus. A key pattern involves quiescent HSV-1 genomes sequestered in promyelocytic leukemia nuclear bodies (PML NBs) forming viral DNA-containing PML-NBs (vDCP NBs). Using a cellular model that replicates vDCP NB formation, we previously demonstrated that these viral genomes are chromatinized with the H3.3 histone variant modified on its lysine 9 by trimethylation (H3.3K9me3), a mark associated with transcriptional repression. Here, we identify the HUSH complex and its effectors, SETDB1 and MORC2, as crucial for the acquisition of H3K9me3 on PML NB-associated HSV-1 and the maintenance of HSV-1 transcriptional repression. ChIP-seq analyses show H3K9me3 association with the entire viral genome. Inactivating the HUSH-SETDB1-MORC2 complex before infection significantly reduces H3K9me3 on the viral genome, with minimal impact on the cellular genome, aside from expected changes in LINE-1 retroelements. Depletion of HUSH, SETDB1, or MORC2 alleviates HSV-1 repression in infected primary human fibroblasts and human induced pluripotent stem cell-derived sensory neurons (hiPSDN). We found that the viral protein ICP0 induces MORC2 degradation via the proteasome machinery. This process is concurrent with ICP0 and MORC2 depletion capability to reactivate silenced HSV-1 in hiPSDN. Overall, our findings underscore the robust antiviral function of the HUSH-SETDB1-MORC2 repressor complex against a herpesvirus by modulating chromatin marks linked to repression, thus presenting promising avenues for anti-herpesvirus therapeutic strategies., Competing Interests: Competing interests statement:The authors declare no competing interest.

We identified a novel mastadenovirus, herein referred to as colobus adenovirus (CoAdV), as the likely cause of fatal respiratory and enteric diseases in multiple black-and-white colobuses (Colobus guereza) imported into Thailand in 2022. Among 9 affected colobuses, 4 died. Viral antigen was abundant in respiratory and enteric tissues, where prominent lesions and clinical signs were observed. We successfully cultivated CoAdV in Vero cells and characterized the complete viral genome, which indicated the virus is genetically distinct from other simian adenoviruses. We also conducted a retrospective study of archival samples from 7 other unrelated colobuses that had respiratory distress or diarrhea and found similar viral strains in 4 of those colobuses. Although we could not determine the potential harm to humans or other nonhuman primates from current information, the zoonotic and spillover potential of this virus to other related hosts should not be neglected. Veterinarians should consider CoAdV when pneumonia is diagnosed in colobuses.

The genetic analysis of the Dengue virus circulating in Ethiopia's Afar region, in 2023, identified three distinct introductions with spatiotemporal clustering linked to genomes from Asia and Italy. These findings are crucial for enhancing prevention and control strategies, reinforcing the necessity to provide sustainable tools for genomic epidemiology in Africa.

Double-stranded DNA viruses actively package their genomes into pre-assembled protein capsids using energy derived from virus-encoded ASCE ATPase ring motors. Single molecule experiments in the aughts and early 2010s demonstrated that these motors are some of the most powerful molecular motors in nature, and that the activities of individual subunits around the ATPase ring motor are highly coordinated to ensure efficient genome encapsidation. While these studies provided a comprehensive kinetic scheme describing the events that occur during packaging, the physical basis of force generation and subunit coordination remained elusive. This article reviews recent structural and computational results that have begun to illuminate the molecular basis of force generation and DNA translocation in these powerful molecular motors., 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., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Unlike Ff, the packaging signal (PS) and the mechanism of integrative filamentous phage assembly remains largely unknown. Here we revived two Inoviridae prophage sequences, ϕLf2 and ϕLf-UK, as infectious virions that lysogenize black rot pathogen Xanthomonas campestris pv. campestris. The genomes of ϕLf2 and ϕLf-UK consist of 6363 and 6062 nucleotides each, and share 85.8% and 98.7% identity with ϕLf, respectively. To explore integrative filamentous phage assembly, we first identified 20-26-nucleotide long PS sequences of 10 Xanthomonas phages. These PS consist of a DNA hairpin with the consensus GGX(A/-)CCG(C/T)G sequence in the stem and C/T nucleotides in the loop, both of which are conserved and essential for PS activity. In contrast to Ff, the 5' to 3' orientation of the PS sequence is not conserved or critical for viral competence. This is the first report to offer insights into the structure and function of the integrative phage PS, revealing the diversity of filamentous phage encapsidation., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Genomic data is growing exponentially, posing new challenges for sequence analysis and classification, particularly for managing and understanding harmful new viruses that may later cause pandemics. Recent genome sequence classification models yield promising performance. However, the majority of them do not consider the sequential arrangement of nucleotides and amino acids, a critical aspect for uncovering their inherent structure and function. To overcome this, we introduce GenoAnaCla, a novel approach for analyzing and classifying genome sequences, based on sequential pattern mining (SPM). The proposed approach first constructs and preprocesses datasets comprising RNA virus genome sequences in three formats: nucleotide, coding region, and protein. Then, to capture sequential features for the analysis and classification of viruses, GenoAnaCla extracts frequent sequential patterns and rules in three forms and in codons. Eight classifiers are utilized, and their effectiveness is assessed by employing a variety of evaluation metrics. A performance comparison demonstrates that the suggested approach surpasses the current state-of-the-art genome sequence classification and detection techniques with a 3.18% performance increase in accuracy on average., 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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Yellow fever virus (YFV) is endemic in the (sub)tropical regions of Africa and South America and is prone to cause epidemics. Molecular testing of YFV by reverse transcription-polymerase chain reaction (RT-PCR) was recently adopted by WHO using blood. Urine is a non-invasive diagnostic specimen which has been proven to be useful in diagnosing several flavivirus infections. Until now, systematic data on the usefulness of urine in YFV molecular diagnostics was lacking., Methods: We have carried out an extensive literature search using key words "yellow fever AND urine" in PubMed/Medline, Embase and Web of Science., Results: The search resulted initially in 113 publications. All titles and abstracts were screened and 15 were analyzed in detail. After natural infection (10 articles), the detection ratio of YFV in blood with RT-PCR was 61 % (105/171 samples) vs. 59 % (139/234) in urine from patients with mild/severe infections. YFV could be first detected at average 4.3 days in blood vs. 6.1 days in urine and last detected till 17.2 vs. 31.1 days respectively (significant difference p < 0.05). Viral load over time in blood was not statistically different from urine. Virus could be isolated from blood, urine and semen. Following vaccination, virus was detected longer in patients with vaccine adverse events (VAE) compared to healthy vaccinees (average 34 vs. 25 days, not significant p > 0.05)., Conclusion: YFV can be detected in urine later but longer. Thus, we see added value for YF molecular diagnostics and sequencing and recommend it besides blood as a standard specimen, especially for late samples post onset., 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., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes due to their superior safety, relatively low immunogenicity, and ability to target diverse tissues. AAV gene therapy products are typically formulated as frozen liquid and stored below - 60 °C, and therefore are subjected to multiple freeze/thaw cycles during manufacturing and administration. Recent studies have shown that genome DNA leakage could be induced by freeze/thaw stress. DNA leakage from AAV capsids has been reported to potentially impact product stability, induce immune responses, and compromise product efficacy. Thus, further characterization to improve the understanding of genome DNA leakage is necessary for mitigating the risks associated with genome DNA leakage during AAV product development. In this work, we developed an optimized size-exclusion chromatography (SEC) method for quantifying the leakage of genome DNA across multiple different AAV serotypes and demonstrated satisfactory assay performance in sensitivity, precision, and linearity. Furthermore, we showed that this method could also be applied to quantifying additional quality attributes of AAV, including the percentage of full capsids and quantification of AAV dimers. By using this optimized SEC method, we demonstrated that significantly increased free DNA was observed with increasing freeze/thaw cycles or at a temperature approaching the onset temperature for genome DNA ejection, which was effectively mitigated by the addition of 1.5% w/v sucrose in the AAV formulation. Thus, this optimized SEC method can serve as an invaluable tool for AAV formulation, product, and process development in ensuring the quality and stability of AAV gene therapy products., Competing Interests: Declarations. Competing interests: All authors are employees of Regeneron Pharmaceuticals, Inc. The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Dengue virus (DENV) has been expanding its range to temperate areas that are not usually affected, where the spread of vectors has been facilitated by global trade and climate change. In Europe, there have been many cases of DENV imported from other regions in the past few years, leading to local outbreaks of DENV among people without travel history. Here we describe the epidemiological and molecular investigations of three transmission events locally acquired DENV infections caused by serotypes 1, 2 and 3, respectively, in the Latium Region from August to November 2023. Next-generation or Sanger sequencing was used to obtain the whole genomes, or the complete E-gene of the viruses, respectively. The structure of the DENV-1 and DENV-3 sequences was analysed to identify amino acid changes that were not found in the closest related sequences. The major cluster was supported by DENV-1 (originated in South America), with 42 autochthonous infections almost occurring in the eastern area of Rome, probably due to a single introduction followed by local sustained transmission. Seven DENV-1 subclusters have been identified by mutational and phylogenetic analysis. Structural analysis indicated changes whose meaning can be explained by the adaptation of the virus to human hosts and vectors and their interactions with antibodies and cell receptors.

Methicillin resistance Staphylococcus aureus (MRSA) is currently threatening global public health, and a similar issue was encountered in Hainan. For establishing a promising alternative therapeutic option, a phage with the capacity of lysing 18 out of 35 MRSA clinical isolates was recovered from domestic natural sources, which was termed as vB&#95;SauP&#95;L1 due to its morphology and genomic similarity with Rountreeviridae. Satisfactory proliferation rate and environmental tolerance were demonstrated by subsequent infectious properties tests and stability assessments. Sequencing revealed that its genome consisted of a linear double-stranded DNA of 17,114 bp, in which neither virulent nor resistant gene was detected, indicating its potential in MRSA prevention and treatment., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Canine circovirus (CCV) has been detected globally, but its genotyping remains unevenly characterized. To comprehend the evolution and genotyping of CCV, 887 rectal swabs of dogs were collected during 2018-2023. According to p-distance/frequency histograms and phylogenetic trees based on complete genome sequences, the 21 newly obtained Chinese and 84 reference CCV strains were mainly divided into 7 subtypes (CCV-1a, CCV-1b, CCV-1c, CCV-1d, CCV-1e, CCV-2a, and CCV-2b). Among the 21 newly obtained CCVs, 9 strains belonged to CCV-1d, 2 strains belonged to CCV-1b, and the remaining strains belonged to CCV-1c. Recombination analysis indicated that recombination events occurred between CCV strains of different subtypes, hosts, and countries. The CCV capsid protein features 19 variable sites, with 2 sites (T58Q, P239A) displaying regional specificity and 3 (Q57T, E150Q, and T195Q) manifesting subtype specificity. Therefore, this genotyping analysis provides a reference for the molecular characteristics of CCV strains found globally., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Objectives: A large cohort of pediatric patients with various forms of childhood cancer was investigated for the presence of viruses using metagenomics. A total of 476 patient samples, collected between 1989 and 2018, were analyzed, representing various pediatric oncological diagnoses and a control group of non-malignant diagnoses., Study Design: The study was carried out using metagenomic sequencing of serum samples. Viruses were identified and analyzed using bioinformatics methods, followed by Polymerase chain reaction (PCR) confirmation RESULTS: The results indicate that a wide range of viruses can be detected in the bloodstream of children with newly diagnosed cancer. Nine viral genomes were identified: Human Pegivirus (HPgV), Hepatitis C virus, Parechovirus 1, Rhinovirus C, Human papillomavirus 116, Human polyomavirus 10, Parvovirus B19, and different variants of Torque Teno Virus (TTV). In this study, a previously unknown virus was found belonging to the Iflavirdae family in the order Picornavirales. HPGV was significantly more common in patients with leukemia compared to other conditions., Conclusions: These results highlight the abundance of systemic virus infections in children, and the value of metagenomic sequencing for hypothesis forming regarding the associations between virus infections and cancer., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Italy was significantly affected by the COVID-19 pandemic, experiencing multiple waves of infection following the sequential emergence of new variants. Understanding the transmission patterns and evolution of SARS-CoV-2 is vital for future preparedness., Methods: We conducted an analysis of viral genome sequences, integrating epidemiological and phylodynamic approaches, to characterize how SARS-CoV-2 variants have spread within the country., Results: Our findings indicate bidirectional international transmission, with Italy transitioning between importing and exporting the virus. Italy experienced four distinct epidemic waves, each associated with a significant reduction in fatalities from 2021 to 2023. These waves were primarily driven by the emergence of VOCs such as Alpha, Delta, and Omicron, which were reflected in observed transmission dynamics and effectiveness of public health measures., Conclusions: The changing patterns of viral spread and variant prevalence throughout Italy's pandemic response underscore the continued importance of flexible public health strategies and genomic surveillance, both of which are crucial for tracking the evolution of variants and adapting control measures effectively to ensure preparedness for future outbreaks., Competing Interests: Declarations 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., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Arecanut palm is a commercially important plantation crop valued for its nut. In this investigation, we report the discovery of a putative novel arepavirus, named areca palm necrotic ringspot virus 2 (ANRSV2), in necrotic ringspot diseased areca palms in Bantwal, Dakshina Kannada, Karnataka, India through RNA-sequencing and transmission electron microscopy. Further, the presence of ANRSV2 in the diseased samples was confirmed through reverse transcriptase-polymerase chain reaction assays. In addition, by mining public domain transcriptome data for arepaviral sequences, we identified a putative novel arepavirus in Psychotria rubra, a non-palm host. We recovered the genome sequences of the areca palm necrotic ringspot virus in honey bees, tomato, Onobrychis viciifolia, and Rhamnus heterophylla. These findings broaden our comprehension of arepaviral diversity and host range, and suggest an intriguing possibility of pollen-mediated arepaviral transmission that necessitates empirical validation. Further studies are needed to understand the biology of identified putative novel arepaviruses., 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., (Copyright © 2024. Published by Elsevier Inc.)

We biochemically simulated the mutation synthesis process of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex (nsp7/nsp8/nsp12) involving two sequential mechanistic steps that occur during genomic replication: misinsertion (incorporation of incorrect nucleotides) and mismatch extension. Then, we also simulated mismatch repair process catalyzed by the viral nsp10/nsp14 ExoN complex. In these mechanistic simulations, while SARS-CoV-2 RdRp displays efficient mutation synthesis capability, the viral ExoN complex was able to effectively repair the mismatch primers generated during the mutation synthesis. Also, we observed that the delayed RNA synthesis induced by mutation synthesis process was rescued by the viral ExoN activity. Collectively, our biochemical simulations suggest that SARS-CoV-2 ExoN complex may contribute to both maintenance of proper viral genetic diversity levels and successful completion of the viral large RNA genome replication by removing mismatches generated by the viral RdRp., 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., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Canine parvovirus type 2 (CPV-2) infection in dogs is considered as one of the most common cause of morbidity and mortality in young dogs and continues to occur with high incidence worldwide. Despite a single-stranded DNA virus, CPV-2 possesses a high mutation rate which has led to the development of new variants from time to time. These variants are classically classified based on the amino acid markers present in the VP2 gene. In this study, we examined 20 different cases of CPV-2 infection from seven different states of the North East region (NER) of India. The near-complete genome sequences of all these isolates were subjected to phylodynamic and phylogeographic analysis to evaluate the genetic diversity and geographical spread of CPV-2 variants. Analysis of the deduced amino acid sequences revealed residues characteristic of the 'Asian CPV-2c lineage' in all the 20 sequences confirming it as the dominant strain circulating in NER, India. The phylogenetic analysis based on the whole genome showed that all 20 sequences formed a monophyletic clade together with other Asian CPV-2c sequences. Furthermore, phylogeographic analysis based on the VP2 gene showed the likely introduction of Asian CPV-2c strain to India from China. This study marks the first comprehensive report elucidating the molecular epidemiology of CPV-2 in India.

Purpose: In November 2023, the National Reference Laboratory for Enteroviruses (Budapest, Hungary) received stool, pharyngeal swab and cerebrospinal fluid samples from five newborns suspected of having human parechovirus (PEV-A) infection. The neonates were born in the same hospital and presented with fever and sepsis-like symptoms at 8-9 days of age, and three of them showed symptoms consistent with central nervous system involvement. PEV-A positivity was confirmed by quantitative reverse transcription polymerase chain reaction., Methods: To determine the PEV-A genotype responsible for the infections, fecal samples of four neonates were subjected to metagenomic sequencing. For further analyses, amplicon-based whole genome sequencing was performed directly from the clinical samples., Results: On the basis of whole genome analysis, sequences were allocated to PEV-A genotype 3 (PEV-A3) and consensus sequences were identical. Two ambiguities were identified in the viral protein 1 (VP1) region of all sequences at a frequency of 17.7-53.7%, indicating the simultaneous presence of at least two quasispecies in the clinical samples. The phylogenetic analysis and similarity plotting showed that all sequences clustered without any topological inconsistencies between the P1 capsid and P2, P3 non-capsid regions, suggesting that recombination events during evolution were unlikely., Conclusion: Our findings suggest that the apparent cluster of cases were microbiologically related, and the results may also inform future investigations on the evolution and pathogenicity of PEV-A3 infections., Competing Interests: Declarations. Ethical approval: Ethics Committee approval was not required as the Hungarian legislation on handling of personal health information (Law no. 1997. XLVII.) empowers the National Center for Public Health and Pharmacy to analyse data and take necessary measures in the interest of public health. Personal data have been handled in accordance with legal regulations and the Center’s data protection rules. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Group A rotaviruses (RVAs) are a major cause of acute gastroenteritis in children under 5 years of age worldwide. Herein, the genetic sequences of 11 RNA segments from three uncommon G9P[4] RVA strains found in the stool samples of children under 5 years of age in Iran were analyzed using next-generation sequencing (NGS) technology. The genomic constellations of these three uncommon G9P[4] strains indicated the presence of the double and quadruple reassortants of two G9P[4] strains, containing the VP7/NSP2 and VP7/VP2/NSP2/NSP4 genes on a DS-1-like genetic background, respectively. The genome of one strain indicated a Wa-like genetic backbone in a single-reassortant with the VP4 of the DS1-like human strains. With the exception of VP1, VP2, VP7, NSP2, NSP3, and NSP4 genes, which clustered with RVA of human origins belonging to cognate gene sequences of genogroup 1/2 genotypes/lineages, the remaining five genes (VP8/VP4, VP3, VP6, NSP1, NSP5) displayed direct evidence of recombination. It is presumed that the presence of uncommon G9P[4] strains in Iran is not linked to vaccination pressure, but rather to the high prevalence of RVA co-infection or the direct import of these uncommon RVA reassortants strains from other countries (especially those that have implemented RV vaccination)., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Avian orthoreoviruses (ARV) are an emerging threat for the poultry industry, both in the United States (US) and globally. ARV infections in turkeys have been associated with arthritis, lameness and neurological disorders, and cost the US economy approximately USD 33 million per year. There is not a commercial vaccine available and the shortage of turkey ARV (TRV) genomic data hinders the efforts to explore the molecular epidemiology of this virus, although several studies suggest a close relationship between European TRVs and TRVs present in the US. This study shows a snapshot of the genomic diversity of Avian orthoreoviruses (ARV) circulating in Germany in the mid-2000s. Through a deep genomic analysis of 18 ARV isolates recovered from sick turkeys, we observed that co-infection was a common condition. 80% of the samples showed signs of a simultaneous infection with a TRV and a chicken ARV (CRV). We believe this is the first reported evidence of CRV and TRV naturally occurring co-infections in commercial turkeys. These co-infection events were identified due to the significant genomic diversity observed among ARV infecting various production bird species. Our phylogenetic analysis revealed a consistent host-associated ARV clustering, with three main clades: (i) a TRV clade, (ii) a CRV clade, and (iii) a Duck ARV (DRV)/Goose ARV (GRV) clade. Furthermore, our findings indicate that German TRVs have interacted with their European and American counterparts, suggesting active mobilization of the virus, likely through the commercial trading of live animals. However, we also consider the potential role of migratory birds in the international movement of ARV., 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., (Published by Elsevier Inc.)

The emergence of highly pathogenic avian influenza strain A (H5N1) in the USA is a high concern. Here, we illustrated the evolution, divergence, transmission pattern, infection pattern, entropy diversity, nucleotide diversity, and mutational landscape of HPAI(H5N1). We depicted three phylogenetic trees, i.e., from three perspectives: considering the HPAI H5N1 genome of the current outbreak in the USA (n = 971), considering the HPAI H5N1 spared in different hosts (cattle, hunan, avian, and nonhuman primates) and using the global genome sequences (n = 3154). We found that the clade 2.3.4.4b was responsible for the present infection. We noted that the USA's divergence rate is 3.43e- 3 subs per site per year, and the global divergence rate is 5.21e- 3 subs per site per year. We reported significant nucleotide changes to illustrate the genome. Similarly, we observe several point mutations in some proteins, such as PB2, PA, HA, NA, and NS1. Among point mutations, some common mutations are noted in PB2 (E362G, M631L) and PA (L219I, K497R). However, elimination strategies should be a high priority for dairy farm workers, domestic cattle, and poultry birds to limit future outbreaks., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The ongoing panzootic of highly pathogenic H5 clade 2.3.4.4b avian influenza (HPAI) spread to North America in late 2021, with detections of HPAI viruses in Alaska beginning in April 2022. HPAI viruses have since spread across the state, affecting many species of wild birds as well as domestic poultry and wild mammals. To better understand the dissemination of HPAI viruses spatiotemporally and among hosts in Alaska and adjacent regions, we compared the genomes of 177 confirmed HPAI viruses detected in Alaska during April-December 2022. Results suggest multiple viral introductions into Alaska between November 2021 and August or September 2022, as well as dissemination to areas within and outside of the state. Viral genotypes differed in their spatiotemporal spread, likely influenced by timing of introductions relative to population immunity. We found evidence for dissemination of HPAI viruses between wild bird species, wild birds and domestic poultry, as well as wild birds and wild mammals. Continued monitoring for and genomic characterization of HPAI viruses in Alaska can improve our understanding of the evolution and dispersal of these economically costly and ecologically relevant pathogens.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380&#95;Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.

In response to the problems associated with drug resistance resulting from the use of antibiotics, phages have become desirable options for the treatment of Vibrio alginolyticus disease in aquaculture. In this study, we isolated a novel double-stranded DNA (dsDNA) phage named vB&#95;ValC&#95;WD615 infecting V. alginolyticus; this phage belongs to the family Podoviridae and has a short noncontractile tail (13 ± 1.5 nm) and an icosahedral head (60.2 ± 2 nm); its genome is 50,522 bp and encodes 69 open reading frames (ORFs) and no lysogenic genes were annotated in the genome. Physiological results indicate that vB&#95;ValC&#95;WD615 infects V. alginolyticus SC1 with a burst size of 335 PFU/cell and can maintain stable infectivity within temperature and pH conditions ranging from 4 to 45 °C and 3 to 11, respectively. The results suggest that the vB&#95;ValC&#95;WD615 isolated from coastal waters could be a potential candidate for phage therapy targeting V. alginolyticus., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The maize streak virus belongs in the genus Mastrevirus, in the family Geminiviridae. The A-strain of the virus (MSV-A) is recognised as the principal causative agent of the most severe manifestation of maize streak disease (MSD). This disease continues to be a persistent limitation on maize output across sub-Saharan Africa and the nearby Indian Ocean islands. Irrespective of the causes behind the spread of MSV-A, we can determine the paths and speeds with which MSV-A spreads by analysing MSV genome sequence data along with information on when and where samples were taken. This information is valuable for identifying the geographical origins of viral strains that cause sporadic MSD epidemics in specific places and the geographical regions where viruses remain in reservoirs and contribute to prolonged epidemics during outbreaks. Our aim is to utilise these analyses to estimate the timing and origin of the MSV-A that arrived on the island of Madagascar in the Indian Ocean. Specifically, we employ model-based phylogeographic analyses on 524 complete MSV-A genome sequences, which consist of 56 newly obtained genomes from infected maize plants collected in Madagascar. These studies allow us to reconstruct the most likely paths of MSV-A to Madagascar. We found strong evidence for the existence of at least four separate movements of MSV-A variants from East and southern Africa to Madagascar. These movements took place between roughly 1979 (with a 95% highest probability density interval [HPD] ranging from 1976 to 1982) and 2003 (with a 95% HPD ranging from 2002 to 2003). While we inferred that MSV-A variants are spreading at an average rate of 38.9 km/year (with a 95% highest posterior density interval of 34.0-44.4) across their geographical range. Since their arrival in Madagascar, MSV-A variants have been migrating at an average rate of 47.6 km/year (with a 95% highest posterior density interval of 36.05-61.70). Human influences are likely significant contributors to both sporadic long-range movements of MSV-A between mainland Africa and Madagascar, as well as shorter to medium range movements within the island., 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., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) significantly impacts the global swine industry. Sichuan province, a key pig breeding center in China, has limited data on the molecular epidemiology of PRRS Virus (PRRSV). To address this, 1618 suspected PRRSV samples were collected from 2021 to 2023, with a prevalence rate of 39.74% (643/1618). Phylogenetic analysis showed PRRSV-2 as dominant (95.65%, 615/643), with PRRSV-1 at 4.35% (28/643). PRRSV-2 strains were further classified into NADC30-like (74.18%), NADC34-like (11.98%), C-PRRSV (5.44%), and HP-PRRSV (4.04%). The significant change in the proportions of different lineages indicates genomic divergence. NADC30-like strains exhibited significant amino acid mutations in ORF5, aiding immune evasion. Recombination analysis revealed complex patterns, primarily involving NADC30-like strains. This study highlights the genomic divergence of PRRSV in Sichuan, with NADC30-like strains becoming predominant and emerging strains like NADC34-like showing potential for further spread., 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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever (CHIKF), and its primary vectors are the mosquitoes Aedes aegypti and Aedes albopictus. CHIKV was initially endemic to Africa but has spread globally in recent years and affected millions of people. According to a risk assessment by the World Health Organization, CHIKV has the potential seriously impact public health. A growing body of research suggests that mutations in the CHIKV gene that enhance viral fitness in the host are contributing to the expansion of the global CHIKF epidemic. In this article, we review the host-adapted gene mutations in CHIKV under natural evolution and laboratory transmission conditions, which can help improve our understanding of the adaptive evolution of CHIKV and provide a basis for monitoring and early warning of future CHIKV outbreaks.

The emergence and spread of novel viral pathogens is a major threat to human health, particularly in the context of climate and human-induced change in land use. Alongshan virus (ALSV) is a tick-borne virus associated with human disease, which was first identified in northeast China. More recently, several studies reported the emergence of ALSV in mammalian and arthropod hosts in multiple different countries outside of Asia, and the first viral genome sequencing data has become available. ALSV is a member of the Jingmenvirus group closely related to the Flaviviridae family. Unusually, the positive-sense, single-stranded RNA genome of ALSV is segmented and consists of four distinct segments, two of which show homology with the NS3 and NS5 protein encoding regions of non-segmented flaviviruses. Transmission of arthropod-borne pathogens will likely increase in the future due to environmental change mediated by a variety of environmental and ecological factors and increasing human encroachment into wild animal habitats. In this review, we present current knowledge of global ALSV distribution and emergence patterns, highlight genetic diversity, evolution and susceptible species. Finally, we discuss the role of this emerging tick-borne virus in the context of urbanization and global health.

African swine fever virus (ASFV) recombinant strains pose new challenges for diagnosis and control. This study characterizes genotype I and II recombinant ASFV strains identified in northern Vietnam in 2023 through whole-genome sequencing and comparative genomic analysis. Seven ASFV-positive samples from six provinces were analyzed, with recombinant strains detected in Bac Giang, Phu Tho, and Vinh Phuc provinces. Isolates showed hemadsorption positivity despite having genotype I B646L, indicating their recombinant nature. Genome-wide analysis revealed 19 recombination breakpoints consistent with Chinese recombinant strains. Vietnamese isolates shared 99.86-99.98% nucleotide identity with Chinese recombinants, forming a distinct monophyletic group. Comparative analysis identified 50 SNPs and INDELs, with 39 variations found across Vietnamese strains, distinguishing them from Chinese isolates. Unique genetic markers in C962R, I329L, and MGF 505-11L genes distinguished Vietnamese recombinants from Chinese counterparts, while mutations in C122R and NP1450L differentiated all recombinants from parental genotypes. The central variable region (CVR) of the B602L gene showed diversity among Vietnamese isolates, while the I73R-I329L intergenic regions were recognized as in the IGR2 group. This study enhances understanding of recombinant ASFV evolution through homologous recombination and identifies new genetic markers for improved detection and characterization. The observed genetic diversity highlights challenges for existing diagnostic methods and vaccine development, emphasizing the need for continued surveillance and research into the functional implications of these genetic variations on ASFV pathogenicity and transmissibility.

The 2017/18 influenza season was characterized by unusual high numbers of severe infections and hospitalizations. Instead of influenza A viruses, this season was dominated by infections with influenza B viruses of the Yamagata lineage. While this IBV/Yam dominance was associated with a vaccine mismatch, a contribution of virus intrinsic features to the clinical severity of the infections was speculated. Here, we performed a molecular and phenotypic characterization of three IBV isolates from patients with severe flu symptoms in 2018 and compared it to an IBV/Yam isolate from 2016 using experimental models of increasing complexity, including human lung explants, lung organoids, and alveolar macrophages. Viral genome sequencing revealed the presence of clade but also isolate specific mutations in all viral genes, except NP, M1, and NEP. Comparative replication kinetics in different cell lines provided further evidence for improved replication fitness, tolerance towards higher temperatures, and the development of immune evasion mechanisms by the 2018 IBV isolates. Most importantly, immunohistochemistry of infected human lung explants revealed an impressively altered cell tropism, extending from AT2 to AT1 cells and macrophages. Finally, transcriptomics of infected human lung explants demonstrated significantly reduced amounts of type I and type III IFNs by the 2018 IBV isolate, supporting the existence of additional immune evasion mechanisms. Our results show that the severeness of the 2017/18 Flu season was not only the result of a vaccine mismatch but was also facilitated by improved adaptation of the circulating IBV strains to the environment of the human lower respiratory tract.

Porcine Circovirus Type 2 (PCV2), a significant pathogen in the global swine industry, causes Porcine Circovirus Associated Diseases (PCVAD), contributing to substantial economic losses. This study investigates the genetic diversity and evolutionary dynamics of PCV2 in Vietnam from 2007 to 2023. We sequenced and analyzed 47 PCV2 genomes isolated from swine farms across Vietnam between 2022 and 2023, revealing predominant circulation of PCV2d (80.85%) followed by PCV2b (19.15%). Phylogenetic analysis identified PCV2 genotypes PCV2a, PCV2b, PCV2d, PCV2g, and PCV2h circulating in Vietnam, with PCV2d emerging as the most prevalent genotype. Comparison with historical data highlighted genotype shifts from PCV2b to PCV2d in 2014. Interestingly, PCV2h genotype was mainly observed between 2008 and 2012 but have not been detected since 2014. Regional analysis indicated varied PCV2 epidemiological patterns between northern and southern Vietnam. Amino acid substitutions within the capsid protein were identified, predominantly in antigenic regions critical for immune recognition. Positive selection analysis identified multiple sites under evolutionary pressure, indicating ongoing adaptation of Vietnamese PCV2 strains. These findings enhance understanding of PCV2 dynamics in Vietnam and underscore the importance of continuous surveillance and adaptive management strategies in controlling PCV2-associated diseases in swine populations., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The evolution of SARS-CoV-2, the agent of COVID-19, has been remarkable for its high mutation potential, leading to the appearance of variants. Some mutations have never appeared in the published genomes, which represent consensus, or bona fide genomes. Here we tested the hypothesis that mutations that did not appear in consensus genomes were, in fact, as frequent as the mutations that appeared during the various epidemic episodes, but were not expressed because lethal. To identify these mutations, we analysed the genomes of 90 nasopharyngeal samples and the quasispecies determined by next-generation sequencing. Mutations observed in the quasispecies and not in the consensus genomes were considered to be lethal, what we called "outlaw" mutations. Among these mutations, we analysed the 21 most frequent. Eight of these "outlaws" were in the RNA polymerase and we were able to use a structural biology model and molecular dynamics simulations to demonstrate the functional incapacity of these mutated RNA polymerases. Three other mutations affected the spike, a major protein involved in the pathogenesis of COVID-19. Overall, by analysing the SARS-CoV-2 quasispecies obtained during sequencing, this method made it possible to identify "outlaws," showing areas that could potentially become the target of treatments.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

Peste des petits ruminants virus (PPRV) is an infectious pathogen; causing highly contagious, acute febrile, and economically important disease of small ruminants. The virus is known to have intrinsic ability to adapt new hosts and to cross the species barrier. The incidence of PPR has already been reported in unusual host species such as camels, bovines, and wild animals from spill-over or natural infection. Still, there are elementary gaps in our knowledge of the extent of susceptibility of camel to PPRV and the adaptability of PPRV to camel. The present study delineates the potential role of preferential codon usage patterns responsible for adaptation, host immune evasion, and transmission of PPRV to unusual hosts like old world camel species namely, dromedary and bactrian camel. The results indicate codon usage of the PPRV genome is functioned by an interplay of mutational pressure and natural selection to exhort the adaptation and fitness of PPRV in probable hosts. The indices of natural selection like the relative codon deoptimization index (RCDI) and codon adaptation index (CAI) predict the ability of PPRV to adapt and evolve in camel species. The analysis also depicts the potential role of the CpG depletion mechanism employed by PPRV to evade host adaptive immune response. The report emphasizes the need for a comprehensive national PPR surveillance plan in unusual hosts like camels for the successful implementation of the PPR Global Eradication Programme (PPR- GEP)., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Foot-and-mouth disease Virus (FMDV) serotype Asia1 is prevalent in the Indian subcontinent, with only G-III and G-VIII reported in India until 2020. However, in 2019, a novel genetic group within serotype Asia1, designated as G-IX, emerged in Bangladesh, followed by its detection in India in 2020. This report presents analyses of the complete coding region sequences of the G-IX lineage isolates. The length of the open reading frame (ORF) of the two G-IX isolates was 6990 nucleotides without any deletion or insertion. The G-IX isolates showed the highest sequence similarity with an isolate of G-III at the ORF, L, P2, and P3 regions, and with an isolate of G-VIII at the P1 region. Phylogenetic analysis based on the capsid region (P1) supports the hypothesis that G-VIII and G-IX originated from a common ancestor, as speculated earlier. Further, VP1 region-based phylogenetic analyses revealed the re-emergence of G-VIII after a gap of 3 years. One isolate of G-VIII collected during 2023 revealed a codon insertion in the G-H loop of VP1. The vaccine matching studies support the suitability of the currently used Indian vaccine strain IND63/1972 to contain outbreaks due to viruses belonging to G-IX.

In March 2024, the emergence of highly pathogenic avian influenza (HPAI) A (H5N1) infections in dairy cattle was detected in the United Sates for the first time. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production, as well as from two cats, six wild birds, and one skunk. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627 K mutation in a human case associated with cattle underscores the potential for rapid evolution post infection, highlighting the need for continued surveillance to monitor public health threats.

Seasonal influenza A virus subtype H3N2 (A/H3N2) circulates globally and has been linked to higher hospitalization rates and summer outbreaks in temperate regions. Here, A/H3N2 circulation in Shanghai, China was systematically studied using data and materials generated by the Shanghai influenza surveillance network from 2005 to 2023. Time-series analysis of incidence and subtyping data showed that A/H3N2 co-circulated with other (sub)types and dominated in multiple seasonal influenza peaks, preferentially in summer. Whole genomes of 528 representative strains were sequenced, and spatiotemporal phylodynamic analysis using these and GISAID-archived sequences demonstrated that in the years before the COVID-19 pandemic, phylogenetically similar strains were circulating locally and elsewhere. However, clade 1a.1 (within 3C.2a.1b.2a), circulated in and only in Shanghai and domestically in 2022, while the sibling clade 2 predominated in other regions. Interestingly, clade 1a.1 was swiftly and completely replaced by clade 2, mostly 2a.3a.1, at the start of 2023. In hemagglutination inhibition and neutralization assays, sera from healthy donors collected in 2022 displayed higher or similar reactivity against 2a.3a.1 compared to 1a.1. By contrast, transcription and replication competence of 2a.3a.1 in MDCK cells was higher than 1a.1. These results indicated that instead of antigenicity differences enabling evasion of pre-existing immunity, higher replicative capability more likely contributed to 2a.3a.1 viruses achieving dominance in China. In addition to summarizing patterns of A/H3N2 local circulation in Shanghai, this work revealed an unusual episode in A/H3N2 global circulation and evolution dynamics in connection to the COVID-19 pandemic and explored possible mechanistic explanations.

Chikungunya virus (CHIKV) has emerged as a significant public health concern due to its rapid spread and potential for causing debilitating epidemics. In Argentina, the virus has garnered attention since its introduction to the Americas in 2013, due to its growing incidence and impact in neighbouring countries. Here we present a comprehensive analysis of the spatiotemporal dynamics of CHIKV in Argentina, focusing on the evolutionary trajectory of its genetic variants. Through a combination of active surveillance, screening of historical and recent samples, and whole-genome sequencing, we traced the evolutionary history of CHIKV lineages circulating within the country. Our results reveal that two distinct genotypes circulated in Argentina: The Asian lineage during the 2016 epidemic and the ECSA lineage in 2023. This distribution reflects the dominance of particular variants across Latin America. Since 2023, the ECSA lineage has led to a surge in cases throughout the Americas, marking a significant shift. The replacement of lineages in the American region constitutes a major epidemiological event, potentially affecting the dynamics of virus transmission and the clinical outcomes in impacted populations. The spatiotemporal analysis highlights CHIKV's distribution across Argentina and underscores the significant role of human mobility, especially when considering recent epidemics in neighbouring countries such as Paraguay and Uruguay, which have facilitated the spread and introduction of the viral strain into different districts. By integrating epidemiological data with genomic insights, we elucidate the patterns of virus dissemination, highlighting key areas of transmission and potential factors contributing to its spread.

Research Highlights: Identified the role of the hatchery in astrovirus transmission.Sequenced the avian nephritis virus complete genome.Investigated tissue distribution of astrovirus from egg to chicks.Demonstrated co-infection of ANV/CAstV.

Reverse genetic systems are mainly used to rescue recombinant viral strains in cell culture. These tools have also been used to generate, by inoculating infectious clones, viral strains directly in living animals. We previously developed the "Infectious Subgenomic Amplicons" (ISA) method, which enables the rescue of single-stranded positive sense RNA viruses in vitro by transfecting overlapping subgenomic DNA fragments. Here, we provide proof-of-concept for direct in vivo generation of infectious particles following the inoculation of subgenomic amplicons. First, we rescued a strain of tick-borne encephalitis virus in mice to transpose the ISA method in vivo . Subgenomic DNA fragments were amplified using a 3-fragment reverse genetics system and inoculated intramuscularly. Almost all animals were infected when quantities of DNA inoculated were at least 20 µg. We then optimized our procedure in order to increase the animal infection rate. This was achieved by adding an electroporation step and/or using a simplified 2- fragment reverse genetics system. Under optimal conditions, a large majority of animals were infected with doses of 20 ng of DNA. Finally, we demonstrated the versatility of this method by applying it to Japanese encephalitis and Chikungunya viruses. This method provides an efficient strategy for in vivo rescue of arboviruses. Furthermore, in the context of the development of DNA-launched live attenuated vaccines, this new approach may facilitate the generation of attenuated strains in vivo . It also enables to deliver a substance free of any vector DNA, which seems to be an important criterion for the development of human vaccines.

H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.

Identification of the diverse animal hosts responsible for spill-over events from animals to humans is crucial for comprehending the transmission patterns of emerging infectious diseases, which pose significant public health risks. To better characterize potential animal hosts of Lassa virus (LASV), we assessed domestic and non-domestic animals from 2021-2022 in four locations in southern Nigeria with reported cases of Lassa fever (LF). Birds, lizards, and domestic mammals (dogs, pigs, cattle and goats) were screened using RT-qPCR, and whole genome sequencing was performed for lineage identification on selected LASV positive samples. Animals were also screened for exposure to LASV by enzyme-linked immunosorbent assay (ELISA). Among these animals, lizards had the highest positivity rate by PCR. Genomic sequencing of samples in most infected animals showed sub-lineage 2 g of LASV. Seropositivity was highest among cattle and lowest in pigs. Though the specific impact these additional hosts may have in the broader virus-host context are still unknown - specifically relating to pathogen diversity, evolution, and transmission - the detection of LASV in non-rodent hosts living in proximity to confirmed human LF cases suggests their involvement during transmission as potential reservoirs. Additional epidemiological data comparing viral genomes from humans and animals, as well as those circulating within the environment will be critical in understanding LASV transmission dynamics and will ultimately guide the development of countermeasures for this zoonotic health threat.

Lassa virus (LASV), a risk-group 4 pathogen, must be handled in biosafety level-4 (BSL-4) conditions, thereby limiting its research and antiviral development. Here, we developed a novel LASV reverse genetics system which, to our knowledge, is the first to study the complete LASV life cycle under BSL-2 conditions. Viral particles can be produced efficiently when LASV minigenomic RNA harbouring minimal viral cis -elements and reporter genes is transfected into a helper cell line stably expressing viral NP, GP, Z and L proteins. The resulting defective virions, named LASVmg, can propagate only in the helper cell line, providing a BSL-2 model to study the complete LASV life cycle. Using this model, we found that a previously reported cellular receptor α-dystroglycan is dispensable for LASVmg infection. Furthermore, we showed that ribavirin can inhibit LASVmg infection by inducing viral mutations. This new BSL-2 system should facilitate studying the LASV life cycle and screening antivirals.

Chikungunya virus has caused millions of cases worldwide over the past 20 years, with recent outbreaks in Kedougou region in the southeastern Senegal, West Africa. Genomic characterization highlights that an ongoing epidemic in Kedougou in 2023 is not due to an introduction event but caused by the re-emergence of an endemic strain evolving linearly in a sylvatic context.

The Hulunbuir region, known for its diverse terrain and rich wildlife, is a hotspot for various natural epidemic diseases. Between 2021 and 2023, we collected 885 wild rodent samples from this area, representing three families, seven genera, and eleven species. Metagenomic analysis identified three complete nucleic acid sequences from the S, M, and L segments of the Hantaviridae family, which were closely related to the Khabarovsk virus. The nucleotide coding sequences for S, M, and L (1392 nt, 3465 nt, and 6491 nt, respectively) exhibited similarities of 82.34%, 81.68%, and 81.94% to known sequences, respectively, while protein-level analysis indicated higher similarities of 94.92%, 94.41%, and 95.87%, respectively. Phylogenetic analysis placed these sequences within the same clade as the Khabarovsk, Puumala, Muju, Hokkaido, Topografov, and Tatenalense viruses, all of which are known to cause febrile diseases in humans. Immunofluorescence detection of nucleic acid-positive rodent kidney samples using sera from patients with hemorrhagic fever and renal syndrome confirmed the presence of viral particles. Based on these findings, we propose that this virus represents a new member of the Hantaviridae family, tentatively named the Amugulang virus, after its primary distribution area.

The discovery of alphacoronaviruses and betacoronaviruses in plateau pikas ( Ochotona curzoniae ) expanded the host range of mammalian coronavirus (CoV) to a new order - Lagomorpha. However, the diversity and evolutionary relationships of CoVs in these plateau-region-specific animal population remains uncertain. We conducted a five-year longitudinal surveillance of CoVs harboured by pikas around Qinghai Lake, China. CoVs were identified in 33 of 236 plateau pikas and 2 of 6 Gansu pikas ( Ochotona cansus ), with a total positivity rate of 14.5%, and exhibiting a wide spatiotemporal distribution across seven sampling sites and six time points. Through meta-transcriptomic sequencing and RT-PCR, we recovered 16 near-complete viral genome sequences. Phylogenetic analyses classified the viruses as variants of either pika alphacoronaviruses or betacoronaviruses endemic to plateau pikas from the Qinghai-Tibet Plateau region. Of particular note, the pika-associated betacoronaviruses may represent a novel subgenus within the genus Betacoronavirus . Tissue tropism, evaluated using quantitative real-time PCR, revealed the presence of CoV in the rectal and/or lung tissues, with the highest viral loads at 10 3.55 or 10 2.80 RNA copies/μL. Surface plasmon resonance (SPR) assays indicated that the newly identified betacoronavirus did not bind to human or pika Angiotensin-converting enzyme 2 (ACE2) or Dipeptidyl peptidase 4 (DPP4). The findings highlight the ongoing circulation and broadening host spectrum of CoVs among pikas, emphasizing the necessity for further investigation to evaluate their potential public health risks.

Salmonella enteritidis is one of the most common pathogens that cause foodborne disease outbreaks and food spoilage, which seriously threatens human health. Bacteriophages have shown broad application prospects in controlling harmful microorganisms during food processing and preservation due to their ability to specifically infect bacteria. In this study, Salmonella enteritidis bacteriophage Salmp-p7 was isolated and characterized from slaughterhouse wastewater. Transmission electron microscopy (TEM) analysis showed that Salmp-p7 belonged to the Siphoviridae family and was active against Salmonella enteritidis and Escherichia coli. Whole genome sequence analysis showed that Salmp-p7 was a lytic bacteriophage with a total length of 60,066 bp of sequence. Salmp-p7 has a short incubation period and a long burst duration, with a burst volume of 55 PFU/cell and a good lysis effect. It can maintain a stable state within the temperature range of 30-60℃ and pH range of 4-12 and has the potential for application in food. In vitro, antimicrobial curves and inhibition of biofilm removal experiments showed that Salmp-p7 could effectively inhibit and eliminate Salmonella enteritidis. The application of Salmp-p7 to the whole liquid of infected eggs resulted in a significant reduction of viable bacteria. And Salmp-p7 has high stability and lytic activity and has the potential to become a new biological control agent for Salmonella enteritidis in eggs., Competing Interests: Declarations. Conflict of Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Human adenoviruses (HAdVs) are highly contagious and have significant clinical implications in the pediatric population. In the present study, we employed a combination of long-read sequencing and short-read sequencing to accurately reconstruct 32 genomes of HAdVs. The phylogenetic analyses based on the whole genome and genes revealed distinct sub-clusters within HAdV-B and -E. For HAdV-C, the phylogenetic trees constructed from hexon, fiber, and E3 gene sequences consistently matched the whole-genome phylogeny, reflecting the high sequence diversity in these regions. Notably, in regions with high sequence diversity, we observed a higher number of recombination breakpoints and lower GC content. Additionally, the E4 gene region of HAdV-C exhibited a Ka/Ks ratio > 1, indicating that positive selection may be driving the fixation of advantageous mutations. These genetic characterization analyses are crucial for enhancing future surveillance of HAdVs, facilitating a more strategic and proactive approach to monitoring their evolution, diversity, and epidemiological trends., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics declarations: Approval for this study was obtained from the research ethics board of the Hubei Provincial Center for Disease Control and Prevention. The need for informed consent from participants was waived as the retrospective samples were collected from the influenza surveillance system of Hubei Province., (© 2024. The Author(s).)

Five lytic bacteriophages specific for Salmonella enterica and Escherichia coli were isolated from wastewater in Minnesota. These phages, designated vB&#95;Sal&#95;EH1, vB&#95;Sal&#95;EH2, vB&#95;Sal&#95;EH3, vB&#95;Sal&#95;EH4, and vB&#95;Sal&#95;EH7, were characterized, and their genomes were sequenced. Phylogenetic analysis showed that they grouped within the genus Epseptimavirus, with genome sizes ranging from 108,554 to 115,218 bp. All five phages exhibited lytic activity against both S. enterica and Shiga-toxin-producing E. coli O157:H7. Transposon mutagenesis of the host genome identified the outer membrane protein BtuB as essential for phage infection, suggesting that it is a putative receptor. Genome sequence comparisons revealed genetic loci that are variable among the isolated phages and potentially influence their host specificity and virulence., Competing Interests: Declarations. Conflict of interest: The authors declare no conflicts of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)