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Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo human gene therapy. To ensure safety and efficacy of gene therapy products, a comprehensive analytical profile of the rAAVs is needed, which provides crucial information for therapeutic development and manufacturing. Besides information on rAAV quantities and possible contaminating DNA and protein species, assessing rAAV quality is of utmost importance. In vitro biopotency and methods to determine the full/empty ratio of rAAV capsids are commonly applied, but methods to assess the integrity of the viral genome are still rarely used. Here we describe an orthogonal approach to characterize rAAV quality. Two biologically different rAAV9s from different stages of the bioprocess, generated each with two different transfection reagents, were investigated. In vitro biopotency tests in all cases demonstrated that rAAV9s generated with transfection reagent FectoVIR® possessed a higher biological activity. Mass-based analytical methods, such as sedimentation velocity analytical ultracentrifugation (AUC) and mass photometry, showed a high share of full capsids (>80 %) at late process stages but did not detect any differences in the rAAV9s from the different transfection reagents. Multiplex dPCR and Nanopore long-read sequencing both demonstrated that, also in late-stage process samples, sample heterogeneity was relatively high with a rather small share of full-length transgenes of ∼10-40 %. Intriguingly, both methods detected a higher share of complete transgenes in rAAV9 generated with transfection reagent FectoVIR® instead of Polyethylenimine (PEI), and thereby explain the differences already observed in the biopotency assays. This study therefore emphasizes the necessity to utilize multiple, orthogonal methods to gain a better understanding of recombinantly manufactured AAVs., Competing Interests: Declaration of Competing Interest All authors are employees of Baxalta Innovations GmbH, a part of Takeda companies, which are involved in the development of biologics and gene therapy products and may be owners of stock options. Other than that, no conflict of interest is declared. Juan A. Hernandez Bort, PhD conducted the research presented in this manuscript while employed at Baxalta Innovations GmbH, a part of Takeda companies. Between the completion of the research and the publication of this manuscript, he has transitioned to an additional affiliation with the University of Vienna, which is now listed as his correspondence address., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: SARS-CoV-2 is responsible for the ongoing global pandemic, and the continuous emergence of novel variants threatens fragile populations, such as immunocompromised patients. This subgroup of patients seems to be seriously affected by intrahost viral changes, as the pathogens, which are keen to cause replication inefficiency, affect the impaired immune system, preventing efficient clearance of the virus. Therefore, these patients may represent an optimal reservoir for the development of new circulating SARS-CoV-2 variants. The following study aimed to investigate genomic changes in SARS-CoV-2-positive immunocompromised patients over time., Methods: SARS-CoV-2-positive nasopharyngeal swabs were collected at different time points for each patient (patient A and patient B), extracted and then analyzed through next-generation sequencing (NGS). The resulting sequences were examined to determine mutation frequencies, describing viral evolution over time., Case Presentation: Patient A was a 53-year-old patient with onco-hematological disease with prolonged infection lasting for 51 days from May 28th to July 18th, 2022. Three confirmed SARS-CoV-2-positive samples were collected on May 28th, June 15th and July 4th. Patient B was 75 years old and had onco-hematological disease with prolonged infection lasting for 146 days. Two confirmed positive SARS-CoV-2 samples were collected at the following time points: May 21st and August 18th., Conclusion: Heat map construction provided evidence of gain and/or loss of mutations over time for both patients, suggesting within-host genomic evolution of the virus. In addition, mutation polymorphisms and changes in the SARS-CoV-2 lineage were observed in Patient B. Sequence analysis revealed high mutational pattern variability, reflecting the high complexity of viral replication dynamics in fragile patients., (© 2024. The Author(s).)

The emergence and spread of the African swine fever virus (ASFV) posed a significant threat to the global swine breeding industry, calling for innovative approaches benefiting viral containment and control. A recent study (Z. Zheng, L. Xu, H. Dou, Y. Zhou, X., et al., Microbiol Spectr 12: e02164-23, 2024, https://doi.org/10.1128/spectrum.02164-23) established a multiplexed CRISPR-Cas system targeting the genome of ASFV and tested the consequent antiviral activity both in vitro and in vivo . Application of this system showed a significant reduction of viral replication in vitro , while the germline-edited pigs expressing this system exhibited normal growth with continuous guide RNA expression. Although no survival advantage was observed upon ASFV challenge compared with nonengineered pigs, this marks the first attempt of germline editing to pursue ASFV resistance and paves the way for future disease-resistant animal breeding approaches utilizing CRISPR-Cas technology., Competing Interests: The authors declare no conflict of interest.

A disease called "hydrosalpinx fluid and egg drop syndrome" (HFEDS) was observed in four flocks of Jinding ducks (Anas platyrhynchos domesticus) in Northeast China during the years 2022 to 2023. Here, we investigated the possible involvement of avian metapneumovirus (AMPV) infection. Full-length genome sequencing and sequence analysis of two AMPV strains showed that they belong to Eurasian lineage of AMPV subtype C. Based on surface glycoprotein (G) sequence comparisons, the Eurasian lineage can be divided into two sublineages (E1 and E2), and sublineage E2 is circulating in Jinding duck populations in Northeast China., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Mitoviruses are cryptic capsidless viruses belonging to the family Mitoviridae that replicate and are maintained in the mitochondria of fungi. Complete mitovirus-like sequences were recently assembled from plant transcriptome data and plant leaf tissue samples. Passion fruit (Passiflora spp.) is an economically important crop for numerous tropical and subtropical countries worldwide, and many virus-induced diseases impact its production. From a large-scale genomic study targeting viruses infecting Passiflora spp. in Brazil, we detected a de novo-assembled contig with similarity to other plant-associated mitoviruses. The contig is ∼2.6 kb long, with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRP). This contig has been named "passion fruit mitovirus-like 1" (PfMv1). An alignment of the predicted amino acid sequence of the RdRP of PfMv1 and those of other plant-associated mitoviruses revealed the presence of the six conserved motifs of mitovirus RdRPs. PfMv1 has 79% coverage and 50.14% identity to Humulus lupulus mitovirus 1. Phylogenetic analysis showed that PfMV1 clustered with other plant-associated mitoviruses in the genus Duamitovirus. Using RT-PCR, we detected a PfMv1-derived fragment, but no corresponding DNA was identified, thus excluding the possibility that this is an endogenized viral-like sequence. This is the first evidence of a replicating mitovirus associated with Passiflora edulis, and it should be classified as a member of a new species, for which we propose the name "Duamitovirus passiflorae"., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The whole-genome sequence (WGS) analysis of Aichivirus (AiV) identified in Korea was performed in this study. Using Sanger and Nanopore sequencing, the 8228-nucleotide-long genomic sequence of AiV (OQ121963) was determined and confirmed to belong to genotype A. The full-length genome of OQ121963 consisted of a 7296 nt open reading frame (ORF) that encodes a single polyprotein, and 5' UTR (676 nt) and 3' UTR (256 nt) at 5' and 3' ends, respectively. The ORF consisted of leader protein (L), structural protein P1 (VP0, VP1, and VP3), and nonstructural protein P2 (2A, 2B, and 2C) and P3 (3A, 3B, 3C, and 3D). The secondary structure analysis of the 5' UTR identified only stem-loop C (SL-C) and not SL-A and SL-B. The variable region of the AiV genome was analyzed by MegAlign Pro and reconfirmed by SimPlot analysis using 16 AiV whole genomes known to date. Among the entire regions, structural protein region P1 showed the lowest amino acid identity (96.07%) with reference sequence AB040749 (originated in Japan; genotype A), while the highest amino acid identity (98.26%) was confirmed in the 3D region among nonstructural protein region P2 and P3. Moreover, phylogenetic analysis of the WGS of OQ121963 showed the highest homology (96.96%) with JX564249 (originated in Taiwan; genotype A) and lowest homology (90.14%) with DQ028632 (originated in Brazil; genotype B). Therefore, the complete genome characterization of OQ121963 and phylogenetic analysis of the AiV conducted in this study provide useful information allowing to improve diagnostic tools and epidemiological studies of AiVs., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Integration of nucleic acid sequences of Reticuloendotheliosis virus (REV) in Avipoxvirus（APV） has become commonplace. In this study, 4 strains of suspected Fowlpox virus (FPV) and 1 strain of suspected Pigeonpox virus (PPV) collected in Taiyuan, Shanxi Province were cultured in chicken embryos, and the 4b core protein gene was amplified by PCR, and the identity and genome similarity were determined by sequence analysis. The sequences between the end of ORF201 and the beginning of ORF203 of FPV and PPV were then amplified, sequenced, and subjected to sequence comparison to determine genome similarity. The results showed that the isolates were 4 strains of FPV and 1 strain of PPV. The 4 isolated strains of FPV belong to type A1 virus, with 100 % identity to each other and to the FWPV-09-Jilin strain isolated in Jilin, China, and the lowest identity to the type B2 virus TNPV5/NZL/2009, which is only 74 %. PPV belongs to type A2 virus, and its identity with local strain of fowlpox virus was 90.1 %, with the highest identity of 100 % with PPLH and ROPI/W370/ON/2012 and ow_2017_3 strains, which also belong to type A2 pigeonpox virus, and the lowest identity of 73.7 % with TNPV5/NZL/2009, a type B2 virus. The complete genome of REV sequences integrated into FPV and PPV were amplified, and 5 REV nucleic acid sequences were obtained after sequencing and concatenation, with lengths ranging from 7942 to 8005 bp. The identity analysis results indicate that it has high identity with isolates from Northeast China, Guangdong, and Guangxi regions in China. Based on its gp90 protein gene, the REV integrated into the poxvirus belong to type III, with the highest identity of 99.9% with strains such as APC-566 and CY1111, and the lowest identity with REV-Anhui1, at 95.4 %. The length of the pol gene varies among different strains of REV, and its encoded amino acid changes significantly after position 675, with deletions and alterations. This study indicates that all fowlpox viruses isolated in Taiyuan, Shanxi Province have integrated the entire REV gene sequence, with high identity between them. At the same time, it indicates that the pigeonpox virus isolate has also integrated the entire REV gene sequence, and has the highest identity with the integrated REV gene sequence in fowlpox virus., 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.)

Bats contain a diverse spectrum of viral species in their bodies. The RNA virus family Paramyxoviridae tends to infect several vertebrate species, which are accountable for a variety of devastating infections in both humans and animals. Viruses of this kind include measles, mumps, and Hendra. Some synonymous codons are favoured over others in mRNAs during gene-to-protein synthesis process. Such phenomenon is termed as codon usage bias (CUB). Our research emphasized many aspects that shape the CUB of genes in the Paramyxoviridae family found in bats. Here, the nitrogenous base A occurred the most. AT was found to be abundant in the coding sequences of the Paramyxoviridae family. RSCU data revealed that A or T ending codons occurred more frequently than predicted. Furthermore, 3 overrepresented codons (CAT, AGA, and GCA) and 7 underrepresented codons (CCG, TCG, CGC, CGG, CGT, GCG and ACG) were detected in the viral genomes. Correspondence analysis, neutrality plot, and parity plots highlight the combined impact of mutational pressure and natural selection on CUB. The neutrality plot of GC12 against GC3 yielded a regression coefficient value of 0.366, indicating that natural selection had a significant (63.4 %) impact. Moreover, RNA editing analysis was done, which revealed the highest frequency of C to T mutations. The results of our research revealed the pattern of codon usage and RNA editing sites in Paramyxoviridae genomes., Competing Interests: Declaration of competing interest Authors declare no conflict of interests in the manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nucleocytoplasmic large DNA viruses (NCLDVs; also called giant viruses), constituting the phylum Nucleocytoviricota, can infect a wide range of eukaryotes and exchange genetic material with not only their hosts but also prokaryotes and phages. A few NCLDVs were reported to encode genes conferring resistance to beta‑lactam, trimethoprim, or pyrimethamine, suggesting that they are potential vehicles for the transmission of antibiotic resistance genes (ARGs) in the biome. However, the incidence of ARGs across the phylum Nucleocytoviricota, their evolutionary characteristics, their dissemination potential, and their association with virulence factors remain unexplored. Here, we systematically investigated ARGs of 1416 NCLDV genomes including those of almost all currently available cultured isolates and high-quality metagenome-assembled genomes from diverse habitats across the globe. We reveal that 39.5% of them carry ARGs, which is approximately 37 times higher than that for phage genomes. A total of 12 ARG types are encoded by NCLDVs. Phylogenies of the three most abundant NCLDV-encoded ARGs hint that NCLDVs acquire ARGs from not only eukaryotes but also prokaryotes and phages. Two NCLDV-encoded trimethoprim resistance genes are demonstrated to confer trimethoprim resistance in Escherichia coli. The presence of ARGs in NCLDV genomes is significantly correlated with mobile genetic elements and virulence factors., (© 2024. The Author(s).)

The global epidemic of Mpox virus (MPXV) continues, and a local outbreak has occurred in Shenzhen city since June 2023. Herein, the evolutionary trajectory and characteristics of MPXV in 2023 were analyzed using 92 MPXV sequences from the Shenzhen outbreak and the available genomes from GISAID and GenBank databases. Phylogenetic tracing of the 92 MPXVs suggests that MPXVs in Shenzhen may have multiple sources of importation, and two main transmission chains have been established. The combination of phylogenetic relationships, epidemiological features, and mutation characteristics supports the emergence of a new lineage C.1.1. Together with the B.1 lineage diverging from the A.1 lineage, C.1.1 lineage diverging from the C.1 lineage may serve as another significant evolutionary events of MPXV. Moreover, increasing apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) related mutations, higher rate of missense mutations, and less mutations in the non-coding regions have been shown during MPXV evolution. Host regulation proteins of MPXV have accumulated considerable amino acid mutations since the B.1 lineage, and a lineage-defining APOBEC3-related mutation that disrupts the N2L gene encoding a viral innate immune modulator has been identified in the C.1.1 lineage. In summary, our study provides compelling evidence for the ongoing evolution of MPXV with specific features., (© 2024. The Author(s).)

Bats (order: Chiroptera) are known to host a diverse range of viruses, some of which present a human public health risk. Thorough viral surveillance is therefore essential to predict and potentially mitigate zoonotic spillover. Astroviruses (family: Astroviridae) are an understudied group of viruses with a growing amount of indirect evidence for zoonotic transfer. Astroviruses have been detected in bats with significant prevalence and diversity, suggesting that bats may act as important astrovirus hosts. Most astrovirus surveillance in wild bat hosts has, to date, been restricted to single-gene PCR detection and concomitant Sanger sequencing; additionally, many bat species and many geographic regions have not yet been surveyed for astroviruses at all. Here, we use metagenomic Next Generation Sequencing (mNGS) to detect astroviruses in three species of Madagascar fruit bats, Eidolon dupreanum, Pteropus rufus, and Rousettus madagascariensis. We detect numerous partial sequences from all three species and one near-full length astrovirus sequence from Rousettus madagascariensis, which we use to characterize the evolutionary history of astroviruses both within bats and the broader mammalian clade, Mamastrovirus. Taken together, applications of mNGS implicate bats as important astrovirus hosts and demonstrate novel patterns of bat astrovirus evolutionary history, particularly in the Southwest Indian Ocean region., (© 2024. The Author(s).)

Previous studies have linked the evolution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic variants to persistent infections in people with immunocompromising conditions, but the processes responsible for these observations are incompletely understood. Here we use high-throughput, single-genome amplification and sequencing (HT-SGS) to sequence SARS-CoV-2 spike genes from people with HIV (PWH, n = 22) and people without HIV (PWOH, n = 25). In PWOH and PWH with CD4 T cell counts (i.e., CD4 counts) ≥ 200 cells/μL, we find that most SARS-CoV-2 genomes sampled in each person share one spike sequence. By contrast, in people with advanced HIV infection (i.e., CD4 counts < 200 cells/μL), HT-SGS reveals a median of 46 distinct linked groupings of spike mutations per person. Elevated intra-host spike diversity in people with advanced HIV infection is detected immediately after COVID-19 symptom onset, and early intra-host spike diversity predicts SARS-CoV-2 shedding duration among PWH. Analysis of longitudinal timepoints reveals rapid fluctuations in spike sequence populations, replacement of founder sequences by groups of new haplotypes, and positive selection at functionally important residues. These findings demonstrate remarkable intra-host genetic diversity of SARS-CoV-2 in advanced HIV infection and suggest that adaptive intra-host SARS-CoV-2 evolution in this setting may contribute to the emergence of new variants of concern., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Family Smacoviridae (order Cremevirales, class Arfiviricetes, phylum Cressdnaviricota) comprises viruses with small circular genomes of ~2300-3000 nt in length that encode at least two proteins, the rolling-circle replication associated protein (Rep) and the capsid protein (CP). Smacovirids have been discovered in fecal samples of various animals and display remarkable sequence diversity. Here, we provide an overview of the genomic properties of classified smacovirids and report on the latest taxonomy update in the family Smacoviridae. The family has been expanded by 59 new species in the genera Porprismacovirus (n = 25), Inpeasmacovirus (n = 1), Felismacovirus (n = 22), Drosmacovirus (n = 4), Dragsmacovirus (n = 2), Bovismacovirus (n = 4), and Bonzesmacovirus (n = 1) and currently includes 12 genera with 143 species officially recognized by the International Committee on Taxonomy of Viruses (ICTV)., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Porcine transmissible gastroenteritis virus (TGEV) is a major pathogen that causes viral enteritis and severe diarrhea in newborn piglets. TGEV strains have been isolated in the USA, Europe, and China, and their molecular characteristics are well known. However, there have been few reports of molecular analysis of TGEV strains isolated in Southeast Asia. In 2016, we isolated TGEV strain VET-16 from fecal samples collected from piglets in Vietnam and determined its complete genome sequence by Sanger sequencing. We found that, while the full genome of the VET-16 strain was 92.4-99.9% identical to those of other TGEV strains, the ORF3 gene showed very little sequence similarity. Phylogenetic analysis suggested that the VET-16 strain belongs to the Purdue subgroup. Comparison of the predicted amino acid (aa) sequence of the spike protein of strain VET-16 with those of other TGEV strains revealed three aa substitutions (V378L, S379T, and D380N) and a 3-aa insertion (F383&#95;F387insWEK) in antigenic site D of the VET-16 strain. Also, a single aa deletion (∆F1413) was found in the transmembrane domain of the spike gene of VET-16. Like the ORF3 gene from the TGEV Miller M60 vaccine strain, the VET-16 strain has a large deletion (∆725 nt) in the ORF3 gene. Previous studies have suggested that these mutations in the spike and ORF3 genes might be associated with a reduction in pathogenicity. The data from this study will facilitate further genetic analysis and research into the evolution of TGEV in pigs in Vietnam., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Vast amounts of pathogen genomic, demographic and spatial data are transforming our understanding of SARS-CoV-2 emergence and spread. We examined the drivers of molecular evolution and spread of 291,791 SARS-CoV-2 genomes from Denmark in 2021. With a sequencing rate consistently exceeding 60%, and up to 80% of PCR-positive samples between March and November, the viral genome set is broadly whole-epidemic representative. We identify a consistent rise in viral diversity over time, with notable spikes upon the importation of novel variants (e.g., Delta and Omicron). By linking genomic data with rich individual-level demographic data from national registers, we find that individuals aged  < 15 and  > 75 years had a lower contribution to molecular change (i.e., branch lengths) compared to other age groups, but similar molecular evolutionary rates, suggesting a lower likelihood of introducing novel variants. Similarly, we find greater molecular change among vaccinated individuals, suggestive of immune evasion. We also observe evidence of transmission in rural areas to follow predictable diffusion processes. Conversely, urban areas are expectedly more complex due to their high mobility, emphasising the role of population structure in driving virus spread. Our analyses highlight the added value of integrating genomic data with detailed demographic and spatial information, particularly in the absence of structured infection surveys., (© 2024. The Author(s).)

Here, we characterized a novel mitovirus from the fungus Nigrospora oryzae, which was named "Nigrospora oryzae mitovirus 3" (NoMV3). The NoMV3 genome is 2,492 nt in length with a G + C content of 33%, containing a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 775 amino acids with a molecular mass of 88.75 kDa. BLASTp analysis revealed that the RdRp of NoMV3 had 68.6%, 50.6%, and 48.6% sequence identity to those of Nigrospora oryzae mitovirus 2, Suillus luteus mitovirus 6, and Fusarium proliferatum mitovirus 3, respectively, which belong to the genus Unuamitovirus within the family Mitoviridae. Phylogenetic analysis based on amino acid sequences supported the classification of NoMV3 as a member of a new species in the genus Unuamitovirus within the family Mitoviridae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The family Convolvulaceae comprises approximately 50-60 genera with approximately 1600-1700 species, exhibiting a rich diversity of morphological characteristics and occupying a broad range of ecological habitats. High-throughput sequencing identified a tentative new virus in the family Geminiviridae infecting Calystegia sepium var. japonica in South Korea. The 2,706 nt long genome comprises six open reading frames (ORFs). The analysis of the nucleotide sequence of the genome and the putative amino acid sequences of ORFs indicated that the virus was closely related to the members of the family Geminiviridae. The genome organization of the virus was similar to that of members of the genus Topilevirus in terms of the number of ORFs and splicing signal. However, the virus, tentatively named bindweed mottle virus (BWMV), may not be assigned to current genera into the family Geminiviridae., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Introduction: SARS-CoV-2 variants are defined by specific genome-wide mutations compared to the Wuhan genome. However, non-clade-defining mutations may also impact protein structure and function, potentially leading to reduced vaccine effectiveness. Our objective is to identify mutations across the entire viral genome rather than focus on individual mutations that may be associated with vaccine failure and to examine the physicochemical properties of the resulting amino acid changes., Materials and Methods: Whole-genome consensus sequences of SARS-CoV-2 from COVID-19 patients were retrieved from the GISAID database. Analysis focused on Dataset&#95;1 (7,154 genomes from Italy) and Dataset&#95;2 (8,819 sequences from Spain). Bioinformatic tools identified amino acid changes resulting from codon mutations with frequencies of 10% or higher, and sequences were organized into sets based on identical amino acid combinations., Results: Non-defining mutations in SARS-CoV-2 genomes belonging to clades 21 L (Omicron), 22B/22E (Omicron), 22F/23A (Omicron) and 21J (Delta) were associated with vaccine failure. Four sets of sequences from Dataset&#95;1 were significantly linked to low vaccine coverage: one from clade 21L with mutations L3201F (ORF1a), A27- (S) and G30- (N); two sets shared by clades 22B and 22E with changes A27- (S), I68- (S), R346T (S) and G30- (N); and one set shared by clades 22F and 23A containing changes A27- (S), F486P (S) and G30- (N). Booster doses showed a slight improvement in protection against Omicron clades. Regarding 21J (Delta) two sets of sequences from Dataset&#95;2 exhibited the combination of non-clade mutations P2046L (ORF1a), P2287S (ORF1a), L829I (ORF1b), T95I (S), Y145H (S), R158- (S) and Q9L (N), that was associated with vaccine failure., Discussion: Vaccine coverage associations appear to be influenced by the mutations harbored by marketed vaccines. An analysis of the physicochemical properties of amino acid revealed that primarily hydrophobic and polar amino acid substitutions occurred. Our results suggest that non-defining mutations across the proteome of SARS-CoV-2 variants could affect the extent of protection of the COVID-19 vaccine. In addition, alteration of the physicochemical characteristics of viral amino acids could potentially disrupt protein structure or function or both., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ulzurrun, Grande-Pérez, del Hoyo, Guevara, Gil, Sorzano and Campillo.)

The SARS-CoV-2 Omicron variant sparked the largest wave of infections worldwide. Mainland China eased its strict COVID-19 measures in late 2022 and experienced two nationwide Omicron waves in 2023. Here, we investigated lineage distribution and virus evolution in Guangdong, China, 2022-2023 by comparing 5813 local viral genomes with the datasets from other regions of China and worldwide. Additionally, we conducted three large-scale serological surveys involving 1696 participants to measure their immune response to the BA.5 and XBB.1.9 before and after the corresponding waves. Our findings revealed the Omicron variants, mainly the BA.5.2.48 lineage, causing infections in over 90% of individuals across different age groups within a month. This rapid spread led to the establishment of widespread immunity, limiting the virus's ability to further adaptive mutation and dissemination. While similar immune responses to BA.5 were observed across all age groups after the initial wave, children aged 3 to 11 developed a stronger cross immune response to the XBB.1.9 strain, possibly explaining their lower infection rates in the following XBB.1 wave. Reinfection with Omicron XBB.1 variant triggered a more potent neutralizing immune response among older adults. These findings highlight the impact of age-specific immune responses on viral spread in potential future waves., (© 2024. The Author(s).)

Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus , BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome., Competing Interests: The authors declare no conflict of interest.

Viruses as the prevailing biological entities are poorly understood in underground realms. Here, we establish the first metagenomic Groundwater Virome Catalogue (GWVC) comprising 280,420 viral species ( ≥ 5 kb) detected from 607 monitored wells in seven geo-environmental zones throughout China. In expanding ~10-fold the global portfolio of known groundwater viruses, we uncover over 99% novel viruses and about 95% novel viral clusters. By linking viruses to hosts from 119 prokaryotic phyla, we double the number of microbial phyla known to be virus-infected in groundwater. As keystone ultrasmall symbionts in aquifers, CPR bacteria and DPANN archaea are susceptible to virulent viruses. Certain complete CPR viruses even likely infect non-CPR bacteria, while partial CPR/DPANN viruses harbor cell-surface modification genes that assist symbiont cell adhesion to free-living microbes. This study reveals the unknown viral world and auxiliary metabolism associated with methane, nitrogen, sulfur, and phosphorus cycling in groundwater, and highlights the importance of subsurface virosphere in viral ecology., (© 2024. The Author(s).)

Oil reservoirs, being one of the significant subsurface repositories of energy and carbon, host diverse microbial communities affecting energy production and carbon emissions. Viruses play crucial roles in the ecology of microbiomes, however, their distribution and ecological significance in oil reservoirs remain undetermined. Here, we assemble a catalogue encompassing viral and prokaryotic genomes sourced from oil reservoirs. The catalogue comprises 7229 prokaryotic genomes and 3,886 viral Operational Taxonomic Units (vOTUs) from 182 oil reservoir metagenomes. The results show that viruses are widely distributed in oil reservoirs, and 85% vOTUs in oil reservoir are detected in less than 10% of the samples, highlighting the heterogeneous nature of viral communities within oil reservoirs. Through combined microcosm enrichment experiments and bioinformatics analysis, we validate the ecological roles of viruses in regulating the community structure of sulfate reducing microorganisms, primarily through a virulent lifestyle. Taken together, this study uncovers a rich diversity of viruses and their ecological functions within oil reservoirs, offering a comprehensive understanding of the role of viral communities in the biogeochemical cycles of the deep biosphere., (© 2024. The Author(s).)

Outbreaks of influenza A viruses are generally seasonal and cause annual epidemics worldwide. Due to their frequent reassortment and evolution, annual surveillance is of paramount importance to guide vaccine strategies. The aim of this study was to explore the molecular epidemiology of influenza A virus and nasopharyngeal microbiota composition in infected patients in Saudi Arabia. A total of 103 nasopharyngeal samples from 2015 and 12 samples from 2022 were collected from patients positive for influenza A. Sequencing of influenza A as well as metatranscriptomic analysis of the nasopharyngeal microbiota was conducted using Oxford Nanopore sequencing. Phylogenetic analysis of hemagglutinin, neuraminidase segments, and concatenated influenza A genomes was performed using MEGA7. Whole-genome sequencing analysis revealed changing clades of influenza A virus: from 6B.1 in 2015 to 5a.2a in 2022. One sample containing the antiviral resistance-mediating mutation S247N toward oseltamivir and zanamivir was found. Phylogenetic analysis showed the clustering of influenza A strains with the corresponding vaccine strains in each period, thus suggesting vaccine effectiveness. Principal component analysis and alpha diversity revealed the absence of a relationship between hospital admission status, age, or gender of infected patients and the nasopharyngeal microbial composition, except for the infecting clade 5a.2a. The opportunistic pathogens Staphylococcus aureus , Streptococcus pneumoniae , Haemophilus influenzae, and Moraxella catarrhalis were the most common species detected. The molecular epidemiology appears to be changing in Saudi Arabia after the COVID-19 pandemic. Antiviral resistance should be carefully monitored in future studies. In addition, the disease severity of patients as well as the composition of the nasopharyngeal microbiota in patients infected with different clades should also be assessed.IMPORTANCEIn this work, we have found that the clade of influenza A virus circulating in Riyadh, KSA, has changed over the last few years from 6B.1 to 5a.2a. Influenza strains clustered with the corresponding vaccine strains in our population, thus emphasizing vaccine effectiveness. Metatranscriptomic analysis showed no correlation between the nasopharyngeal microbiome and the clinical and/or demographic characteristics of infected patients. This is except for the 5a.2a strains isolated post-COVID-19 pandemic. The influenza virus is among the continuously evolving viruses that can cause severe respiratory infections. Continuous surveillance of its molecular diversity and the monitoring of anti-viral-resistant strains are thus of vital importance. Furthermore, exploring potential microbial markers and/or dysbiosis of the nasopharyngeal microbiota during infection could assist in the better management of patients in severe cases., Competing Interests: The authors declare no conflict of interest.

From May to July of 2023, one pig farm in Heyuan city, Guangdong Province of China, suffered severe piglet death and sow reproductive disorders. The common pig viruses and bacteria tested negative. To uncover the possible cause of the disease, a metagenomic analysis was performed in the pooled small intestine samples from three 8-day-old diseased piglets. The results showed that Getah virus (GETV), an RNA virus, might be the potential pathogen that affects pig health. Subsequently, GETV nucleotide was detected in all of the 15 samples collected from three diseased piglets using quantitative reverse transcription PCR, suggesting GETV as the main pathogen of the disease. A GETV strain, designated as GDHYLC23, was successfully isolated using the swine testicle cell line. Sequence analysis showed that the epidemic strain had a unique 32-nucleotide repeat insertion in the 3' noncoding region. Phylogenetic analysis showed that GDHYLC23 belonged to the pandemic group III. The identification of GETV with new variations implies the continuous evolution of the virus, which poses potential threats to the swine industry.IMPORTANCEPig farms are faced with emerging and re-emerging viruses that may cause substantial economic loss. The identification of potentially pathogenic viruses helps to prevent and control the spread of diseases. In this study, by using metagenomic analysis, we found that a neglected virus, GETV with a unique insertion in the genome, was the main pathogen in one pig farm that suffered severe piglet death and sow reproductive disorders. Although the potential impact of such an insertion on viral pathogenicity is unknown, the surveillance of the continuing evolution of GETV in pig farms cannot be ignored., Competing Interests: The authors declare no conflict of interest.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus that undergoes rapid mutation. Based on viral whole genome sequencing analysis in Hebei Province, China, we identified several essential single nucleotide variants (SNVs) on primer-probe regions accumulating within some Omicron variants' genomes. In this study, we focused on three SNVs, C28290T, T28297C, and C28311T emerging on 2019-nCoV-N1 (CDC-N1) primer-probe regions, recommended by CDC in 2020, and two SNVs, C26270T, A26275G emerging on E (Charité-E) primer-probe regions recommended by Charité, Germany. Our findings revealed that the presence of one or two SNVs in the primer or probe region affected the sensitivity of reverse transcription-quantitative polymerase chain reaction and droplet digital PCR to varying extents. This discovery underscores the importance of continuously monitoring the whole genome sequences of SARS-CoV-2 variants, especially the primer-probe targeting regions, and correspondingly updating commercial test kits or recommended primer-probe sequence sets., Importance: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has resulted in a growing number of mutations in its genome, presenting new challenges for the diagnosis of SARS-CoV-2 using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital PCR (RT-ddPCR) methods. There is an urgent need to develop refined methods for modifying primers and probes to improve the detection of these emerging variants. In this study, our focus was on the SNVs that have emerged in the CDC-N1 and Charité-E primer-probe regions. Our research has confirmed that the presence of these SNVs in the primer or probe region can significantly affect the results of coronavirus disease 2019 tests. we have developed and validated a modified detection method that can provide higher sensitivity and specificity. This study emphasizes the importance of refining the primer-probe sets to ensure the diagnostic accuracy of RT-qPCR and RT-ddPCR detection., Competing Interests: The authors declare no conflict of interest.

Infectious bronchitis virus (IBV) is caused by avian coronavirus and poses a global economic threat to the poultry industry. In 2023, a highly pathogenic IBV strain, IBV/CN/GD20230501, was isolated and identified from chickens vaccinated with IBV-M41 in Guangdong, China. This study comprehensively investigated the biological characteristics of the isolated IBV strain, including its genotype, whole genome sequence analysis of its S1 gene, pathogenicity, host immune response, and serum non-targeted metabolomics. Through the analysis of the S1 gene sequence, serum neutralization tests, and comparative genomics, it was proven that IBV/CN/GD20230501 belongs to the GI-I type of strain and is serotype II. One alanine residue in the S1 subunit of the isolated strain was mutated into serine, and some mutations were observed in the ORF1ab gene and the terminal region of the genome. Animal challenge experiments using the EID 50 and TCID 50 calculations showed that IBV/CN/GD20230501 possesses strong respiratory pathogenicity, with early and long-term shedding of viruses and rapid viral spread. Antibody detection indicated that chickens infected with IBV/CN/GD20230501 exhibited delayed expression of early innate immune genes, while those infected with M41 showed rapid gene induction and effective viral control. Metabolomics analysis demonstrated that this virus infection led to differential expression of 291 ions in chicken serum, mainly affecting the citric acid cycle (tricarboxylic acid cycle).IMPORTANCEThis study identified an infectious bronchitis virus (IBV) strain isolated from vaccinated chickens in an immunized population that had certain sequence differences compared to IBV-M41, resulting in significantly enhanced pathogenicity and host defense. This strain has the potential to replace M41 as a more suitable challenge model for drug research. The non-targeted metabolomics analysis highlighting the citric acid cycle provides a new avenue for studying this highly virulent strain., Competing Interests: The authors declare no conflict of interest.

SARS-CoV-2 molecular testing coupled with whole-genome sequencing is instrumental for real-time genomic surveillance. Genomic surveillance is critical for monitoring the spread of variants of concern (VOCs) as well as discovery of novel variants. Since the beginning of the pandemic, millions of SARS-CoV-2 genomes have been deposited into public sequence databases. This is the result of efforts of both national and regional diagnostic laboratories. In this study, we describe the results of SARS-CoV-2 genomic surveillance from February 2021 to June 2022 at a metropolitan hospital in the United States. We demonstrate that consistent daily sampling is sufficient to track the regional prevalence and emergence of VOCs and recapitulate national trends. Similar sampling efforts should be considered a viable option for local SARS-CoV-2 genomic surveillance at other regional laboratories., Importance: In our manuscript, we describe the results of SARS-CoV-2 genomic surveillance from February 2021 to June 2022 at a metropolitan hospital in the United States. We demonstrate that consistent daily sampling is sufficient to track the regional prevalence and emergence of variants of concern (VOCs). Similar sampling efforts should be considered a viable option for local SARS-CoV-2 genomic surveillance at other regional laboratories. While the SARS-CoV-2 pandemic has evolved into a more endemic form, we still believe that additional real-world information about sampling, procedures, and data interpretation is valuable for ongoing as well as future genomic surveillance efforts. Our study should be of substantial interest to clinical virologists., Competing Interests: The authors declare no conflict of interest.

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to identify depolymerase sequences and their enzymatic domains precisely. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which is subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can greatly enhance our understanding of phage-host interactions at the level of depolymerases., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Concha-Eloko 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.)

Equine rotavirus species A (ERVA) G3P[12] and G14P[12] are two dominant genotypes that cause foal diarrhoea with a significant economic impact on the global equine industry. ERVA can also serve as a source of novel (equine-like) rotavirus species A (RVA) reassortants with zoonotic potential as those identified previously in 2013-2019 when equine G3-like RVA was responsible for worldwide outbreaks of severe gastroenteritis and hospitalizations in children. One hurdle to ERVA research is that the standard cell culture system optimized for human rotavirus replication is not efficient for isolating ERVA. Here, using an engineered cell line defective in antiviral innate immunity, we showed that both equine G3P[12] and G14P[12] strains can be rapidly isolated from diarrhoeic foals. The genome sequence analysis revealed that both G3P[12] and G14P[12] strains share the identical genotypic constellation except for VP7 and VP6 segments in which G3P[12] possessed VP7 of genotype G3 and VP6 of genotype I6 and G14P[12] had the combination of VP7 of genotype G14 and VP6 of genotype I2. Further characterization demonstrated that two ERVA genotypes have a limited cross-neutralization. The lack of an in vitro broad cross-protection between both genotypes supported the increased recent diarrhoea outbreaks due to equine G14P[12] in foals born to dams immunized with the inactivated monovalent equine G3P[12] vaccine. Finally, using the structural modelling approach, we provided the genetic basis of the antigenic divergence between ERVA G3P[12] and G14P[12] strains. The results of this study will provide a framework for further investigation of infection biology, pathogenesis and cross-protection of equine rotaviruses.

Lumpy skin disease virus (LSDV), a double-stranded DNA virus from the Capripoxvirus genus, primarily affects Bos indicus, Bos taurus breeds, and water buffalo. Arthropod vectors, including mosquitoes and biting flies, are the main LSDV transmitters. Although LSDV is not zoonotic, this study unexpectedly detected LSDV reads in the upper respiratory tract microbiome of humans from rural and urban areas in Maharashtra, India. Nasopharyngeal and oropharyngeal swab samples collected for SARS-CoV-2 surveillance underwent whole-genome metagenomics sequencing, revealing LSDV reads in 25% of samples. Split kmer analysis provided insights into sample relatedness despite the low coverage of LSDV reads with the reference genome. Our findings, which include the detection of LSDV contigs aligning to specific locations on the reference genome, suggest a common source for LSDV reads, potentially shared water sources, or milk/milk products. Further investigation is needed to ascertain the mode of transmission and reason for the detection of LSDV reads in human upper respiratory tract., (© 2024 Wiley Periodicals LLC.)

Background: This study aimed to demonstrate that the genomic material of SARS-CoV-2 can be isolated from strips of COVID-19 rapid diagnostic test cassettes., Method: It was a prospective cross-sectional study involving patients admitted to treatment centers and sampling sites in the city of Conakry, Guinea. A total of 121 patients were double sampled, and 9 more patients were tested only for RDT. PCR was conducted according to the protocol of the RunMei kit. Sequencing was performed by using the illumina COVIDSeq protocol. Nine COVID-19 RDTs without nasopharyngeal swabs were in addition tested., Result: Among the 130 COVID-19 RDTs, forty-seven were macroscopically positive, whereas seventy-two were positive according to PCR using RDT strip, while among the 121 VTM swabs, sixty-four were positive. Among eighty-three negative COVID-19 RDTs, twenty-seven were positive by PCR using RDT strip with a geometric mean Ct value of 32.49 cycles. Compared to those of PCR using VTM, the sensitivity and specificity of PCR using RDT strip were estimated to be 100% and 85.96%, respectively, with 93.39% test accuracy. Among the fifteen COVID-19 RDT extracts eligible for sequencing, eleven had sequences identical to those obtained via the standard method, with coverage between 75 and 99.6%., Conclusion: These results show that COVID-19 RDTs can be used as biological material for the genomic surveillance of SARS-CoV-2., (© 2024. The Author(s).)

Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses., (© 2024. The Author(s).)

Bats are the natural reservoir hosts for SARS-related coronavirus (SARSr-CoV) and other highly pathogenic microorganisms. Therefore, it is conceivable that an individual bat may harbor multiple microbes. However, there is limited knowledge on the overall co-circulation of microorganisms in bats. Here, we conducted a 16-year monitoring of bat viruses in south and central China and identified 238 SARSr-CoV positive samples across nine bat species from ten provinces or administrative districts. Among these, 76 individual samples were selected for further metagenomics analysis. We found a complex microenvironment characterized by the general co-circulation of microbes from two different sources: mammal-associated viruses or environment-associated microbes. The later includes commensal bacteria, enterobacteria-related phages, and insect or fungal viruses of food origin. Results showed that 25% (19/76) of the samples contained at least one another mammal-associated virus, notably alphacoronaviruses (13/76) such as AlphaCoV/YN2012, HKU2-related CoV and AlphaCoV/Rf-HuB2013, along with viruses from other families. Notably, we observed three viruses co-circulating within a single bat, comprising two coronavirus species and one picornavirus. Our analysis also revealed the potential presence of pathogenic bacteria or fungi in bats. Furthermore, we obtained 25 viral genomes from the 76 bat SARSr-CoV positive samples, some of which formed new evolutionary lineages. Collectively, our study reveals the complex microenvironment of bat microbiome, facilitating deeper investigations into their pathogenic potential and the likelihood of cross-species transmission., Competing Interests: Conflict of interest Prof. Zheng-Li Shi is the Editor-in-Chief for Virologica Sinica and Prof. Peng Zhou is an editorial board member for Virologica Sinica. They were not involved in the editorial review or the decision to publish this article. The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses., Competing Interests: Declaration of interests The authors have no conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: We declare no competing interests. MFB and GW conceptualised and wrote the manuscript. There was no funding source for this study.

Rotaviruses belong to genotype VP4-P[8] are a significant cause of severe loose diarrhea in infants and young children. In the present study, we characterised the complete genome of three of the Pakistani P[8]b RVA strains by Illumina HiSeq sequencing technology to determine the complete genotype constellation providing insight into the evolutionary dynamics of their genes using maximum likelihood analysis. The maximum genomic sequences of our study strains were similar to more recent human Wa-Like G1P[8]a, G3P[8]a, G4P[6], G4P[8], G9P[4], G9P[8]a, G11P[25],G12P[8]a and G12P[6] strains circulating around the world. Therefore, strains PAK274, PAK439 and PAK624 carry natively distinctive VP4 gene with universally common human Wa-Like genetic backbone. Comparing our study P[8]b strains with vaccines strains Rotarix TM and RotaTeq TM , multiple amino acid differences were examined between vaccine virus antigenic epitopes and Pakistani isolates. Over time, these differences may result in the selection for strains that will escape the vaccine-induced RVA-neutralizing-antibody effect., Competing Interests: Declaration of competing interest Author and co-author declare no potential conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Newcastle disease virus (NDV) affects wild and domesticated bird species, including commercial poultry. Although the diversity of NDV in domestic chickens is well documented, limited information is available about Newcastle disease (ND) outbreaks in other bird species. We report an annotated sequence of NDV/Vulture/Borjuri/01/22, an avirulent strain of NDV reported from Borjuri, Northeast India, in Himalayan Griffon vulture. The complete genome is 15,186 bases long with a fusion protein (F) cleavage site 112 GRQGR↓L 117 . The phylogenetic analysis based on the F protein gene and the whole genome sequence revealed that the isolate from the vulture belongs to genotype II, sharing significant homology with vaccine strain LaSota. The study highlights the possible spillover of the virus from domestic to wild species through the food chain., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Avian encephalomyelitis (AE) is an important infectious poultry disease worldwide that is caused by avian encephalomyelitis virus (AEV). The causative virus can be transmitted both horizontally and vertically. In the present study, an AEV suspected outbreak with typical neurological signs occurred in broilers. Histopathological examination, RT-PCR assay and full genome sequencing were applied to confirm the presence of AEV. Phylogenetic analysis of the full genome sequence showed that the detected AEV strain at 7055 nucleotide length is classified in cluster I and is closely related to vaccinal USA and China originated isolates. Although, the outbreaks of AE in progeny of vaccinated breeders have been reported previously, the source of infection was unknown. Based on the results obtained in this study, the outbreaks are vaccine-originated. This study provides the first whole genome analysis of AEV from Iran and reveals that the AEV possesses a hepatitis C virus-like internal ribosome entry site., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Application of whole genome sequencing (WGS) has allowed monitoring of the emergence of variants of concern (VOC) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) globally. Genomic investigation of emerging variants and surveillance of clinical progress has reduced the public health impact of infection during the COVID-19 pandemic. These steps required developing and implementing a proficiency testing program (PTP), as WGS has been incorporated into routine reference laboratory practice. In this study, we describe how the PTP evaluated the capacity and capability of one New Zealand and 14 Australian public health laboratories to perform WGS of SARS-CoV-2 in 2022. The participants' performances in characterising a specimen panel of known SARS-CoV-2 isolates in the PTP were assessed based on: (1) genome coverage, (2) Pango lineage, and (3) sequence quality, with the choice of assessment metrics refined based on a previously reported assessment conducted in 2021. The participants' performances in 2021 and 2022 were also compared after reassessing the 2021 results using the more stringent metrics adopted in 2022. We found that more participants would have failed the 2021 assessment for all survey samples and a significantly higher fail rate per sample in 2021 compared to 2022. This study highlights the importance of choosing appropriate performance metrics to reflect better the laboratories' capacity to perform SARS-CoV-2 WGS, as was done in the 2022 PTP. It also displays the need for a PTP for WGS of SARS-CoV-2 to be available to public health laboratories ongoing, with continuous refinements in the design and provision of the PTP to account for the dynamic nature of the COVID-19 pandemic as SARS-CoV-2 continues to evolve., (Copyright © 2024 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Human cytomegalovirus has a linear DNA genome with a total length of approximately 235 kb. This large genome is divided into two domains, "Long" and "Short". There are four isomers of the cytomegalovirus genome with different orientations of each domain. To confirm the presence of four types of isomers, it is necessary to identify the sequence of the junction between the domains. However, due to the presence of repeat sequences, it is difficult to determine the junction sequences by next-generation sequencing analysis. To solve this problem, long-read sequencing was performed using the Oxford Nanopore sequencer and the junctions were successfully identified in four isomers in strain Merin and ATCC-2011-3. Nanopore sequencing also revealed the presence of multiple copies of the "a" sequence (a-seq) in the junctions, indicating the diversity of the junction sequences. These results strongly suggest that long-read sequencing using the nanopore sequencer would be beneficial for identifying the complex structure of the cytomegalovirus genome., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

White yam (Dioscorea rotundata) plants collected from farmers' fields and planted at the Areka Agricultural Research Center, Southern Ethiopia, displayed mosaic, mottling, and chlorosis symptoms. To determine the presence of viral pathogens, an investigation for virome characterization was conducted by Illumina high-throughput sequencing. The bioinformatics analysis allowed the assembly of five viral genomes, which according to the ICTV criteria were assigned to a novel potyvirus (3 genome sequences) and a novel crinivirus (2 genome sequences). The potyvirus showed ~ 66% nucleotide (nt) identity in the polyprotein sequence to yam mosaic virus (NC004752), clearly below the demarcation criteria of 76% identity. For the crinivirus, the RNA 1 and RNA 2 shared the highest sequence identity to lettuce chlorosis virus, and alignment of the aa sequence of the RdRp, CP and HSP70h (~ 49%, 45% and 76% identity), considered for the demarcation criteria, revealed the finding of a novel virus species. The names Ethiopian yam virus (EYV) and Yam virus 1 (YV-1) are proposed for the two tentative new virus species., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Identifying and characterizing mobile genetic elements in sequencing data is essential for understanding their diversity, ecology, biotechnological applications and impact on public health. Here we introduce geNomad, a classification and annotation framework that combines information from gene content and a deep neural network to identify sequences of plasmids and viruses. geNomad uses a dataset of more than 200,000 marker protein profiles to provide functional gene annotation and taxonomic assignment of viral genomes. Using a conditional random field model, geNomad also detects proviruses integrated into host genomes with high precision. In benchmarks, geNomad achieved high classification performance for diverse plasmids and viruses (Matthews correlation coefficient of 77.8% and 95.3%, respectively), substantially outperforming other tools. Leveraging geNomad's speed and scalability, we processed over 2.7 trillion base pairs of sequencing data, leading to the discovery of millions of viruses and plasmids that are available through the IMG/VR and IMG/PR databases. geNomad is available at https://portal.nersc.gov/genomad ., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

During the summer of 2022, a cluster of Madagascar periwinkle plants with white and mauve flowers were observed with foliar mild yellow mosaic symptoms on a private property in Harlingen, Cameron County, Texas. The symptoms were reproduced on mechanically inoculated periwinkle and Nicotiana benthamiana plants. Virions of 776 to 849 nm in length and 11.7 to 14.8 nm in width were observed in transmission electron microscopy of leaf dip preparations made from symptomatic periwinkle leaves. High-throughput sequencing (HTS) analysis of total RNA extracts from symptomatic leaves revealed the occurrence of two highly divergent variants of a novel Potyvirus species as the only virus-like sequences present in the sample. The complete genomes of both variants were independently amplified via reverse transcriptase PCR, cloned, and Sanger sequenced. The 5' and 3' of the genomes were acquired using random amplification of cDNA ends methodology. The assembled virus genomes were 9,936 and 9,944 nucleotides (nt) long, and they shared 99.9 to 100% identities with the respective HTS-derived genomes. Each genome encoded hypothetical polyprotein of 3,171 amino acids (aa) (362.6 kilodaltons [kDa]) and 3,173 aa (362.7 kDa), respectively, and they shared 77.3/84.4% nt/aa polyprotein identities, indicating that they represent highly divergent variants of the same Potyvirus species. Both genomes also shared below-species-threshold polyprotein identity levels with the most closely phylogenetically related known potyviruses, thus indicating that they belong to a novel species. The name periwinkle mild yellow mosaic virus (PwMYMV) is given to the potyvirus with complete genomes of 9,936 nt for variant 1 (PwMYMV-1) and 9,944 nt for variant 2 (PwMYMV-2). We propose that PwMYMV be assigned into the genus Potyvirus (family Potyviridae )., Competing Interests: The author(s) declare no conflict of interest.

In China, the proportion of HIV-1 infections due to men who have sex with men (MSM) has increased rapidly. More and more new subtypes are found among the MSM population besides known CRF01&#95;AE, CRF07&#95;BC, and B. The co-circulation of several HIV subtypes in the same population provides the opportunity to develop a new circulating recombinant form (CRF) and unique recombinant form (URF). Here we reported two new URFs from two HIV-1 positive subjects infected through homosexual contact in Hebei, China. Phylogenetic and recombinant analyses based on the near full-length genome (NFLG) of the two URFs are the second-generation recombinant strains that originated from B, CRF01&#95;AE, and CRF07&#95;BC. The CRF01&#95;AE segments in the genome of two URFs originated from cluster 4 of CRF01&#95;AE strains, while the CRF07&#95;BC segments were clustered with 07BC&#95;N in the phylogenetic tree. The emergence of the novel CRF01&#95;AE/CRF07&#95;BC and CRF01&#95;AE/B recombinant forms indicated the importance of the continuous monitoring of the HIV-1 epidemic and new URFs among the MSM population.

Since its initial detection in Africa, the West Nile virus has disseminated widely across all continents, becoming endemic in numerous countries, including the Russian Federation. A substantial expansion of the West Nile virus range was observed in the European part of the Russian territory in 1999. In light of this epidemiological trend, research endeavours focusing on monitoring West Nile virus circulation activity in endemic regions of the country have gained paramount significance. A substantial dataset has been accrued from 2007 onwards regarding genomic variability and dissemination dynamics across the country throughout the entire monitoring period for the West Nile fever pathogen. The objective of this study was to characterise West Nile virus isolates that have been circulating in the Russian Federation and identify their molecular and genetic characteristics. A phylogenetic analysis of 55 complete genome sequences revealed that the West Nile virus population within the Russian Federation is genetically heterogeneous and is represented by four major clades. One of these clades is currently exhibiting extensive spread into new regions of the country., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: During the 2017-18 influenza season in the USA, there was a high incidence of influenza illness and mortality. However, no apparent antigenic change was identified in the dominant H3N2 viruses, and the severity of the season could not be solely attributed to a vaccine mismatch. We aimed to investigate whether the altered virus properties resulting from gene reassortment were underlying causes of the increased case number and disease severity associated with the 2017-18 influenza season., Methods: Samples included were collected from patients with influenza who were prospectively recruited during the 2016-17 and 2017-18 influenza seasons at the Johns Hopkins Hospital Emergency Departments in Baltimore, MD, USA, as well as from archived samples from Johns Hopkins Health System sites. Among 647 recruited patients with influenza A virus infection, 411 patients with whole-genome sequences were available in the Johns Hopkins Center of Excellence for Influenza Research and Surveillance network during the 2016-17 and 2017-18 seasons. Phylogenetic trees were constructed based on viral whole-genome sequences. Representative viral isolates of the two seasons were characterised in immortalised cell lines and human nasal epithelial cell cultures, and patients' demographic data and clinical outcomes were analysed., Findings: Unique H3N2 reassortment events were observed, resulting in two predominant strains in the 2017-18 season: HA clade 3C.2a2 and clade 3C.3a, which had novel gene segment constellations containing gene segments from HA clade 3C.2a1 viruses. The reassortant re3C.2a2 viruses replicated with faster kinetics and to a higher peak titre compared with the parental 3C.2a2 and 3C.2a1 viruses (48 h vs 72 h). Furthermore, patients infected with reassortant 3C.2a2 viruses had higher Influenza Severity Scores than patients infected with the parental 3C.2a2 viruses (median 3·00 [IQR 1·00-4·00] vs 1·50 [1·00-2·00]; p=0·018)., Interpretation: Our findings suggest that the increased severity of the 2017-18 influenza season was due in part to two intrasubtypes, cocirculating H3N2 reassortant viruses with fitness advantages over the parental viruses. This information could help inform future vaccine development and public health policies., Funding: The Center of Excellence for Influenza Research and Response in the US, National Science and Technology Council, and Chang Gung Memorial Hospital in Taiwan., Competing Interests: Declaration of interests SV owns stock in VIR Biotechnology as an employee of the company, unrelated to the submitted work. The other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In this study, a deep quantum neural network (DQNN) based on the Lion-based Coot algorithm (LBCA-based Deep QNN) is employed to predict COVID-19. Here, the genome sequences are subjected to feature extraction. The fusion of features is performed using the Bray-Curtis distance and the deep belief network (DBN). Lastly, a deep quantum neural network (Deep QNN) is used to predict COVID-19. The LBCA is obtained by integrating Coot algorithm and LOA. The COVID-19 predictions are done with mutation points. The LBCA-based Deep QNN outperformed with testing accuracy of 0.941, true positive rate of 0.931, and false positive rate of 0.869.

Ticks are a major parasite on the Qīnghăi-Tibet Plateau, western China, and represent an economic burden to agriculture and animal husbandry. Despite research on tick-borne pathogens that threaten humans and animals, the viromes of dominant tick species in this area remain unknown. In this study, we collected Dermacentor nuttalli ticks near Qīnghăi Lake and identified 13 viruses belonging to at least six families through metagenomic sequencing. Four viruses were of high abundance in pools, including Xīnjiāng tick-associated virus 1 (XJTAV1), and three novel viruses: Qīnghăi Lake virus 1, Qīnghăi Lake virus 2 (QHLV1, and QHLV2, unclassified), and Qīnghăi Lake virus 3 (QHLV3, genus Uukuvirus of family Phenuiviridae in order Bunyavirales), which lacks the M segment. The minimum infection rates of the four viruses in the tick groups were 8.2%, 49.5%, 6.2%, and 24.7%, respectively, suggesting the prevalence of these viruses in D. ​nuttalli ticks. A putative M segment of QHLV3 was identified from the next-generation sequencing data and further characterized for its signal peptide cleavage site, N-glycosylation, and transmembrane region. Furthermore, we probed the L, M, and S segments of other viruses from sequencing data of other tick pools by ​using the putative M segment sequence of QHLV3. By revealing the viromes of D. nuttalli ticks, this study enhances our understanding of tick-borne viral communities in highland regions. The putative M segment identified in a novel uukuvirus suggests that previously identified uukuviruses without M segments should have had the same genome organization as typical bunyaviruses. These findings will facilitate virus discovery and our understanding of the phylogeny of tick-borne uukuviruses., Competing Interests: Conflict of interest Prof. Fei Deng is an editorial board member for Virologica Sinica and was not involved in the editorial review or the decision to publish this article. The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. We present an experimental method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A unique gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. We demonstrate this method's efficacy by spiking two known viral genomes, Simian virus 40 (SV40, 5,243 bp) and Human Adenovirus 5 (HAd5, 35,938 bp), into a sewage sample with a final abundance in the droplets of around 0.1% and 0.015%, respectively. We achieve 100% recovery of the complete sequence of the spiked-in SV40 genome with uniform coverage distribution. For the larger HAd5 genome, we cover approximately 99.4% of its sequence. Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables single-genome whole-genome amplification and targeting characterizations of rare viral species and will facilitate our ability to access the mutational profile in single-virus genomes and contribute to an improved understanding of viral ecology., Competing Interests: Competing interests statement:The authors declare no competing interest.