Viral Genome Sequences Research Articles

Host-virus interaction between tobacco mild green mosaic virus strain U2 and tropical soda apple resulting in systemic hypersensitive necrosis and the host range, survival, spread, and molecular characterization of the virus.

Tobacco mild green mosaic virus strain U2 (TMGMV-U2) is a registered active ingredient in a bioherbicide to control tropical soda apple (TSA), Solanum viarum, an invasive weed. As required for registration, we developed empirical data on the host-virus interaction and the virus's host range, survival, spread, and genomic sequence. TMGMV-U2 killed TSA plants by causing systemic hypersensitive necrosis (SHN). It elicited local lesions in inoculated leaves which was followed by the plant's wilting and death. It moved from inoculated terminal leaves through the vasculature to roots and then to newly developed leaves. Phloem death was implicated in wilting and plant death. The SHN response was attenuated in plants grown at constant 32 °C. TMGMV-U2 titer in TSA was low compared to a systemically susceptible tobacco. The virus remained infective for up to 6 months in infected dead TSA tissues and in soil in which infected plants had grown. Susceptible tobacco and pepper plants grown in soil that previously had infected dead TSA or in soil amended with the virus remained asymptomatic and virus-free. A susceptible pepper crop grown in a field block following two consecutive crops of TMGMV-U2-infected susceptible tobacco grew disease-free and virus-free and without yield loss. Purified TMGMV-U2 was infective for 1 year when stored at -20 °C or 5 °C and for 1 month at room temperature. No virus spread was found in the field. Genomic analyses confirmed the registered isolate to be a U2 strain and free of satellite TMV. The TMGMV-U2-susceptible species preponderantly belonged to the Solanaceae. A few hosts that were killed belonged to this family. Several new hosts to TMGMV-U2 were found. These data enabled registration of TMGMV-U2. TMGMV-U2 can be used safely as a bioherbicide without risks to nontarget plants and the environment. © 2023 Society of Chemical Industry.

SARS-CoV-2 Nucleocapsid Protein Mutations Found in Switzerland Disrupt N-Gene Amplification in Commonly Used Multiplex RT-PCR Assay.

At the end of 2021, we observed an increase in N-gene target failures (NGTF) with the TaqPathTM COVID-19 CE-IVD RT-PCR Kit from Thermo Fisher Scientific (TaqPath). We subsequently used whole-genome sequencing (Oxford Nanopore Technology) to identify potential issues with N-gene PCR efficacy. Among 168,101 positive samples with a cycle threshold (CT) value <30 from August 2021 to May 2022, 194 specimens without N-gene amplification by PCR were identified (0.12%). Most NGTF samples originated from a wave of infection attributable to the Delta variant (B.1.617.2) and its sublineages. Sequencing revealed the nucleotide substitution G28922T (A217S) in 151 samples (88.8%). The substitution G215C, a hallmark mutation for Delta lineages, was concurrently present in all of these samples. Ten samples (5.9%) carried the deletion 28,913-28,918 (del214/215), eight samples (4.7%) the deletion 28,913-28,915 (del214) and one sample (0.6%) the deletion 28,892-28,930 (del207-219). Samples showing intact N-gene amplification by PCR lacked these specific mutations, but delayed-type amplification (i.e., partial or pNGTF) was attributable to the exclusive presence of A217S. As the N gene is a common target in many RT-PCR methods for SARS-CoV-2, an in-depth analysis of single-target failures using a combination with viral whole genome sequencing may allow for the identification of diagnostic flaws and eventual new variants.

Genome analysis of viruses of Phenuiviridae, Betaflexiviridae and Bromoviridae, and apple scar skin viroid in pear by high-throughput sequencing revealing host expansion of a rubodvirus and an ilarvirus

Pear (Pyrus spp.) is an important perennial fruit crop of India. During May 2022, pear leaf samples were collected from the orchard of ICAR-Indian Agricultural Research Institute, Regional Station, Dhanda, Shimla, Himachal Pradesh, India. RNA sequencing of two pooled samples, Pe1 and Pe2, was done using NovaSeq 6000. Host unaligned reads were de novo assembled into contigs and annotated through BLAST using the virus database. Near complete genome sequences of apple rubbery wood virus 1 (ARWV-1), apple necrotic mosaic virus (ApNMV), citrus virus A (CiVA), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and a genetic variant of ASGV named as ASGV-Shimla, and a partial sequence of apple scar skin viroid (ASSVd) were assembled. ARWV-1 and ApNMV belong to the genus Rubodvirus and Ilarvirus having tripartite negative sense and positive sense single stranded RNA genomes, respectively, and their presence in pear trees was observed for the first time indicating the host expansion. Presence of CiVA, ASGV and ACLSV in pear in India was also recorded for the first time in India. The fragments per kilobase of transcript per million reads mapped estimation showed the dominance of ASPV and CiVA in Pe1 and Pe2, respectively. Single nucleotide variants (SNVs) analysis revealed that ASPV had the highest number of SNVs (477), while ACLSV had the lowest number of SNVs (1). The presence of ARWV-1, ApNMV, CiVA, ASPV, ASGV, ACLSV, ASGV-Shimla, and ASSVd was also confirmed in pear samples through Sanger sequencing. The present study widens our understanding of the pear virome and host range expansion of viruses in the genus Rubodvirus and Ilarvirus. This information will be useful in biological study of these viruses and virus indexing programs in pear.

NeTaGFT: A similarity network‐based method for trait analysis

Abstract With the determination of numerous viral and bacterial genome sequences, sequence‐trait relationships, such as the evolution of virulence and associations to geographic location or host, are now being studied. In these studies, phylogenetic trees were first reconstructed, and trait data were analysed based on the trees. However, in some cases, such as fast evolution sequences and gene‐sharing network data, reconstructing the phylogenetic tree is challenging. Even in such cases, it is possible to quantify the similarity between sequences and construct an similarity network. Here, we propose a novel approach, Network‐Trait association with Graph Fourier Transform (NeTaGFT), to analyse network‐trait associations. NeTaGFT is inspired by graph signalling process techniques. The graph in this study corresponds to a similarity network representing the similarities between virus samples, and the graph signal corresponds to trait data. By using graph Fourier transform, NeTaGFT aims to identify trait signals and associations of various traits from a similarity network. We validated that NeTaGFT can find signals associated with network structures and associations of traits with the simulation dataset. We applied NeTaGFT for influenza type A and virome gene‐sharing datasets. As a result, we identified several network structures and their associated traits. Our approach is expected to provide novel insights into network‐based approach not only for typical sequence‐trait relationships but also for various biological data, such as antibody evolution.

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

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

A Snapshot on the Genomic Epidemiology of Turkey Reovirus Infections, Hungary.

Reovirus infections in turkeys are associated with arthritis and lameness. Viral genome sequence data are scarce, which makes an accurate description of the viral evolution and epidemiology difficult. In this study, we isolated and characterized turkey reoviruses from Hungary. The isolates were identified in 2016; these isolates were compared with earlier Hungarian turkey reovirus strains and turkey reoviruses isolated in the 2010s in the United States. Gene-wise sequence and phylogenetic analyses identified the cell-receptor binding protein and the main neutralization antigen, σC, to be the most conserved. The most genetically diverse gene was another surface antigen coding gene, μB. This gene was shown to undergo frequent reassortment among chicken and turkey origin reoviruses. Additional reassortment events were found primarily within members of the homologous turkey reovirus clade. Our data showed evidence for low variability among strains isolated from independent outbreaks, a finding that suggests a common source of turkey reoviruses in Hungarian turkey flocks. Given that commercial vaccines are not available, identification of the source of these founder virus strains would permit a more efficient prevention of disease outbreaks before young birds are settled to fattening facilities.

Characterization of SARS-CoV-2 Variants in Military and Civilian Personnel of an Air Force Airport during Three Pandemic Waves in Italy.

We investigated SARS-CoV-2 variants circulating, from November 2020 to March 2022, among military and civilian personnel at an Air Force airport in Italy in order to classify viral isolates in a potential hotspot for virus spread. Positive samples were subjected to Next-Generation Sequencing (NGS) of the whole viral genome and Sanger sequencing of the spike coding region. Phylogenetic analysis classified viral isolates and traced their evolutionary relationships. Clusters were identified using 70% cut-off. Sequencing methods yielded comparable results in terms of variant classification. In 2020 and 2021, we identified several variants, including B.1.258 (4/67), B.1.177 (9/67), Alpha (B.1.1.7, 9/67), Gamma (P.1.1, 4/67), and Delta (4/67). In 2022, only Omicron and its sub-lineage variants were observed (37/67). SARS-CoV-2 isolates were screened to detect naturally occurring resistance in genomic regions, the target of new therapies, comparing them to the Wuhan Hu-1 reference strain. Interestingly, 2/30 non-Omicron isolates carried the G15S 3CLpro substitution responsible for reduced susceptibility to protease inhibitors. On the other hand, Omicron isolates carried unusual substitutions A1803V, D1809N, and A949T on PLpro, and the D216N on 3CLpro. Finally, the P323L substitution on RdRp coding regions was not associated with the mutational pattern related to polymerase inhibitor resistance. This study highlights the importance of continuous genomic surveillance to monitor SARS-CoV-2 evolution in the general population, as well as in restricted communities.

The fourth COVID-19 vaccine dose increased the neutralizing antibody response against the SARS-CoV-2 Omicron (B.1.1.529) variant in a diverse Brazilian population.

Most of the original immunization regimens against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were composed of two doses, followed by a subsequent third booster dose to control the Omicron variant and hence coronavirus disease 2019 (COVID-19). However, most data generated regarding the fourth dose were not based on the general population. Therefore, this study aimed to verify the effect of the fourth COVID-19 vaccine dose in a diverse Brazilian population. This retrospective observational study was conducted between May and September 2022. We gathered data on the vaccine regimens and COVID-19 serologic status from 266 healthy volunteers who received three or four vaccine doses, as well as COVID-19 diagnosis and viral genome sequencing from 457 patients with flu-like symptoms. In addition, we conducted immunoinformatic analysis to assess the conserved epitopes in the locally circulating viruses. We showed that the fourth dose did not increase the serum levels of antiviral antibodies, as measured by enzyme-linked immunosorbent assay. However, it significantly increased neutralizing antibody (NAb) titers against the Omicron variant. All viral sequences generated in this study were Omicron subvariants, mainly B.A.5.1. Notably, most NAb epitopes present in the wild-type SARS-CoV-2 were not detected among the circulating Omicron subvariants. None of the volunteers who received the third or fourth doses presented COVID-19 for at least 1 year before the study period. Altogether, these results indicate that the fourth vaccine dose increases the serum levels of NAbs that recognize highly conserved epitopes in Omicron subvariants.IMPORTANCESeveral additional COVID-19 vaccine doses were administered in the Brazilian population to prevent the disease caused by the B.1.1.529 (Omicron) variant. The efficacy of a third dose as a booster is already well described. However, it is important to clarify the humoral immune response gain induced by a fourth dose. In this study, we evaluate the effect of the fourth COVID-19 vaccine dose in a diverse Brazilian population, considering a real-life context. Our study reveals that the fourth dose of the COVID-19 vaccine increased the neutralizing antibody response against SARS-CoV-2 Omicron and significantly contributed in the reduction of the disease caused by this variant.

Whole genome sequencing reveals insights into hepatitis E virus genome diversity, and virus compartmentalization in chronic hepatitis E.

Hepatitis E virus (HEV) is a leading cause of acute hepatitis and can cause chronic infections in immunocompromised patients. Although HEV infections can be treated with ribavirin, antiviral efficacy is hampered by resistance mutations, normally detected by virus sequencing. High-throughput sequencing (HTS) allows for cost-effective complete viral genome sequencing. This enables the discovery and delineation of new subtypes, and revised the recognition of quasispecies and putative resistance mutations. However, HTS is challenged by factors including low viral load, sample degradation, high host background, and high viral diversity. We apply complete genome sequencing strategies for HEV, including a targeted enrichment approach. These approaches were used to investigate sequence diversity in HEV RNA-positive animal and human samples and intra-host diversity in a chronically infected patient. Here, we describe the identification of potential novel subtypes in a blood donation (genotype 3) and in an ancient livestock sample (genotype 7). In a chronically infected patient, we successfully investigated intra-host virus diversity, including the presence of ribavirin resistance mutations. Furthermore, we found convincing evidence for HEV compartmentalization, including the central nervous system, in this patient. Targeted enrichment of viral sequences enables the generation of complete genome sequences from a variety of difficult sample materials. Moreover, it enables the generation of greater sequence coverage allowing more advanced analyses. This is key for a better understanding of virus diversity. Investigation of existing ribavirin resistance, in the context of minorities or compartmentalization, may be critical in treatment strategies of HEV patients.

Evidence of SARS-CoV-2 infection and co-infections in stray cats in Brazil

The zoonotic virus SARS-CoV-2, which causes severe acute respiratory syndrome in humans (COVID-19), has been identified in cats. Notably, most positive cases were in cats that had close contact with infected humans, suggesting a role for humans in animal transmission routes. Previous studies have suggested that animals with immune depletion are more susceptible to SARS-CoV-2 infection. To date, there is limited evidence of SARS-CoV-2 infections in stray and free-range cats affected by other pathogens. In this study, we investigated infections caused by SARS-CoV-2, Leishmania spp., Toxoplasma gondii, Mycoplasma spp., Bartonella spp., Feline leukemia virus (FeLV), and Feline immunodeficiency virus (FIV) in stray cats from an urban park in Brazil during the COVID-19 pandemic. From February to September 2021, 78 mixed-breed cats were tested for SARS-CoV-2 and hemopathogens using molecular analysis at Américo Renné Giannetti Municipal Park, Belo Horizonte, Minas Gerais, Brazil. An enzyme-linked immunosorbent assay (ELISA) was used to detect IgG in T. gondii. None of the animals in this study showed any clinical signs of infections. The SARS-CoV-2 virus RNA was detected in 7.7 % of cats, and a whole virus genome sequence analysis revealed the SARS-CoV-2 Delta lineage (B.1.617.2). Phylogenetic analysis showed that SARS-CoV-2 isolated from cats was grouped into the sublineage AY.99.2, which matches the epidemiological scenario of COVID-19 in the urban area of our study. Leishmania infantum was detected and sequenced in 9 % of cats. The seroprevalence of T. gondii was 23.1 %. Hemotropic Mycoplasma spp. was detected in 7.7 % of the cats, with Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum being the most common. Bartonella henselae and Bartonella clarridgeiae were detected in 38.5 % of the cats, FeLV was detected in 17,9 %, and none of the cats studied tested positive for FIV. This study reports, for the first time, the SARS-CoV-2 infection with whole-genome sequencing in stray cats in southeastern Brazil and co-infection with other pathogens, including Bartonella spp. and Feline leukemia virus. Our study observed no correlation between SARS-CoV-2 and the other detected pathogens. Our results emphasize the importance of monitoring SARS-CoV-2 in stray cats to characterize their epidemiological role in SARS-CoV-2 infection and reinforce the importance of zoonotic disease surveillance.

Circulation of Dengue Virus Serotype 1 Genotype V and Dengue Virus Serotype 2 Genotype III in Tocantins State, Northern Brazil, 2021-2022.

In Brazil, the state of Tocantins, located in north-central Brazil, has experienced a significant number of cases of arboviral disease, particularly Dengue virus (DENV). This study aimed to deepen the knowledge on DENV circulation within that state by conducting full genome sequencing of viral genomes recovered from 61 patients between June 2021 and July 2022. There were a total of 8807 and 20,692 cases in 2021 and 2022, respectively, as reported by the state's Secretary of Health. Nucleotide sequencing confirmed the circulation of DENV serotype 1, genotype V and DENV serotype 2, genotype III in the State. Younger age groups (4 to 43 years old) were mostly affected; however, no significant differences were detected regarding the gender distribution of cases in humans. Phylogenetic analysis revealed that the circulating viruses belong to DENV-1 genotype V American and DENV-2 genotype III Southeast Asian/American. The Bayesian analysis of DENV-1 genotype V genomes sequenced here are closely related to genomes previously sequenced in the state of São Paulo. Regarding the DENV-2 genotype III genomes, these clustered in a distinct, well-supported subclade, along with previously reported isolates from the states of Goiás and São Paulo. The findings reported here suggest that multiple introductions of these genotypes occurred in the Tocantins state. This observation highlights the importance of major population centers in Brazil on virus dispersion, such as those observed in other Latin American and North American countries. In the SNP analysis, DENV-1 displayed 122 distinct missense mutations, while DENV-2 had 44, with significant mutations predominantly occurring in the envelope and NS5 proteins. The analyses performed here highlight the concomitant circulation of distinct DENV-1 and -2 genotypes in some Brazilian states, underscoring the dynamic evolution of DENV and the relevance of surveillance efforts in supporting public health policies.

Recovery of complete genome sequences of Crimean-Congo haemorrhagic fever virus (CCHFV) directly from clinical samples: A comparative study between targeted enrichment and metagenomic approaches

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, endemic to the Balkans, Africa, Middle East and Asia. There are currently no licensed vaccines or effective antivirals against CCHF. CCHF virus (CCHFV) has a negative sense segmented tripartite RNA genome consisting of the small (S), medium (M) and large (L) segments. Depending on the segment utilised for genetic affiliation, there are up to 7 circulating lineages of CCHFV. The current lack of geographical representation of CCHFV sequences in various repositories highlights a requirement for increased CCHFV sequencing capabilities in endemic regions. We have optimised and established a multiplex PCR tiling methodology for the targeted enrichment of complete genomes of Europe 1 CCHFV lineage directly from clinical samples and compared its performance to a non-targeted enrichment approach on both short-read and long-read sequencing platforms. We have found a statistically significant increase in mapped viral sequencing reads produced with our targeted enrichment approach. This has allowed us to recover near complete S segment sequences and above 90% of the M and L segment sequences for samples with Ct values as high as 31.3. This study demonstrates the superiority of a targeted enrichment approach for recovery of CCHFV genomic sequences from samples with low virus titre. CCHFV is an important vector-borne human pathogen with wide geographical distribution. The validated methodology reported here adds value to front-line public health laboratories employing genomic sequencing for CCHFV Europe 1 lineage surveillance, particularly in the Balkan and Middle Eastern territories currently monitoring the spread of the pathogen. Tracking the genomic evolution of the virus across regions improves risk assessment and directly informs the development of diagnostics, therapeutics, and vaccines.

Genetic Diversity of Domestic Cat Hepadnavirus in Southern Taiwan.

Domestic cat hepadnavirus (DCH) is an infectious disease associated with chronic hepatitis in cats, which suggests a similarity with hepatitis B virus infections in humans. Since its first identification in Australia in 2018, DCH has been reported in several countries with varying prevalence rates, but its presence in Taiwan has yet to be investigated. In this study, we aimed to identify the presence and genetic diversity of DCH infections in Taiwan. Among the 71 samples tested, eight (11.27%) were positive for DCH. Of these positive cases, three cats had elevated levels of alanine transaminase (ALT) and aspartate transaminase (AST), suggesting an association between DCH infection and chronic hepatitis. Four DCH-positive samples were also tested for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) coinfection. One sample (25%) was positive for FIV, whereas there was no positive sample for FeLV (0%). In addition, we performed whole genome sequencing on six samples to determine the viral genome sequences. Phylogenetic analyses identified a distinct lineage compared with previously reported sequences. This study highlights the importance of continuous surveillance of DCH and further research to elucidate the pathophysiology and transmission route of DCH.

Highly pathogenic avian influenza A(H5N1) virus infection in foxes with PB2-M535I identified as a novel mammalian adaptation, Northern Ireland, July 2023.

We report cases of mammalian infection with highly pathogenic avian influenza (HPAI) virus A(H5N1) clade 2.3.4.4b in Northern Ireland. Two common gulls (Larus canus) and two red fox kits (Vulpes vulpes), were found dead in close vicinity. Comparison of viral whole genome sequences obtained from the animals identified a novel mammalian adaptation, PB2-M535I. Analysis of genetic sequences from other recent mammalian infections shows that this mutation has arisen on at least five occasions in three European countries since April 2023.

An Ensemble Approach for Prioritizing Antivirals Against COVID-19 via Heterogeneous Network Inference-Based Inductive Matrix Completion

The global spread of COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originated in Wuhan in December 2019, created a massive health crisis, and disrupted the world economy. Much research has been conducted to discover drugs, develop vaccines, and find repurposable drugs against the disease. Computational drug repurposing, the process of determining new uses for approved drugs through computational techniques, becomes an effective solution to fight the COVID-19 pandemic. This study aims to investigate and prioritize potential drugs against SARS-CoV-2 through an integrated network-based approach. We propose an ensemble approach based on network inference and inductive matrix completion (NIMCVDA) for virus–drug association prediction to identify antivirals against COVID-19. We constructed a heterogeneous drug–virus network using intra-similarities of virus genomic sequences and drug chemical structures and existing associations between viruses and drugs. A network inference method is used to infer missing drug–virus edges. Based on this, existing drug–virus association matrix is reconstructed. Finally, more accurate association scores between drugs and viruses are computed using the inductive matrix completion algorithm. The proposed method achieved an AUC of 0.9020 on five-fold cross-validation and 0.8786 on leave-one-out cross-validation. We compared the performance of the model with related approaches. In addition, we carried out case studies on the top-predicted drugs and implemented our model with other datasets to verify prediction performance. Our work prioritized repurposable drugs to battle with COVID-19 epidemic. The cross-validation results and case studies illustrate that the top-predicted drugs are strong candidates for further biological tests.

Genome sequences of Phasi Charoen-like phasivirus and Fako virus from Aedes aegypti mosquitoes collected in coastal Kenya.

We report the sequencing of two viruses, Phasi Charoen-like phasivirus (PCLV) and Fako virus (FAKV), which were detected in a pool of Aedes aegypti from Kenya. Analysis showed a high similarity of PCLV to publicly available PCLV genomes from Kenya. FAKV showed a high genetic divergence from publicly available FAKV genomes.

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses.

Enteroviruses (EVs) are a group of viruses that cause various human illnesses. While the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) method can generate VP1 gene fragments for enterovirus genotyping, it is unable to detect recombinant strains. Recent advances in viral genome sequencing using next-generation sequencing technologies have enabled comprehensive analyses. However, the high cost poses a challenge for widespread adoption. To address this issue, this study proposes a cost-effective approach for generating complete enterovirus genome sequences using the Oxford Nanopore MinION sequencer. This protocol not only facilitates the generation of accurate genome sequences for various enterovirus strains but also allows for the differentiation of co-infections from viral isolates. In addition, the method can generate polyprotein sequences as well as peptide sequences of the upstream ORF (uORF) whose expression can impact virus infection. Through the analysis of complete enterovirus genomes, this study successfully identified seven enterovirus A71 isolates obtained during the 2018 enterovirus outbreak in Malaysia and Taiwan as recombinants between enterovirus A71 and coxsackievirus A2. Furthermore, our study has made a significant discovery by establishing a strong correlation between uORF trees and the epidemics of EVA71. This finding highlights the potential of uORF sequences as valuable indicators for monitoring and understanding the spread of EVA71 infections. We also identified notable amino acid changes in the transmembrane domain of the uORF protein within a newly identified lineage. These findings provide crucial insights into the molecular characteristics and evolutionary dynamics of EVA71, offering valuable information for future research and intervention strategies. IMPORTANCE By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

Genome sequencing of cucumber mosaic virus (CMV) isolates infecting chilli and its interaction with host ferredoxin protein of different host for causing mosaic symptoms.

The online version contains supplementary material available at 10.1007/s13205-023-03777-8.

Development and evaluation of a real-time quantitative PCR for the detection of equine infectious anemia virus.

Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based real-time quantitative PCR (TG-qPCR) for the detection of EIAV by targeting the fragment between the tat and gag genes, which was relatively conserved in all the known EIAV strains. The performance of the TG-qPCR was evaluated against that of the standard qPCR (recommended by WOAH) by testing viral RNA extracted from viral supernatants of EIAVDLV2-6 and EIAVUK3, proviral DNA from peripheral blood mononuclear cells of artificially immunized horses, and virus nucleic acid from EIAV positive serum samples. The TG-qPCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the TG-qPCR assay was 1 copy/reaction for both viral RNA and proviral DNA based on the Poisson distribution. Compared to the qPCR, the TG-qPCR has better inclusivity and can detect not only Asian EIAV strains but also almost all the representative EIAV strains from other continents. The above results show that the TG-qPCR assay could serve as an effective tool for the early diagnosis of clinical EIA disease.IMPORTANCEEquine infectious anemia (EIA) has a worldwide distribution and causes significant losses to the equine industry worldwide. A reliable detection method is necessary to control the transmission of EIA virus (EIAV). Currently, most of the available real-time PCR assays, including the qPCR of recommended by WOAH, are developed according to the sequences of European or American EIAV strains; however, the primers and probe sequences have low homology with Asian EIAV strains. To the best of our knowledge, no qPCR method capable of the well detection of Asian EIAV strains, especially Chinese EIAV strains, has been published to date. The development of a sensitive, specific, and rapid qPCR assay for the detection of the EIAV strains is therefore of great importance.

AN OUTBREAK OF FELINE INFECTIOUS PERITONITIS IN THREE RELATED SAND CATS (FELIS MARGARITA) IN HUMAN CARE.

Feline infectious peritonitis (FIP) is a systemic disease in felid species caused by infection with mutated forms of feline coronavirus (FCoV), and outbreaks can devastate exotic felid populations in human care. Feline infectious peritonitis was diagnosed in three of four related juvenile sand cats (Felis margarita) from a single institution over a 6-wk period. Case 1 was a 7-mon-old male found deceased with no premonitory signs. Case 2, an 8-mon-old male (littermate to Case 1), and Case 3, a 6-mon-old male (from a different litter with identical parentage), were evaluated for lethargy and anorexia 1 mon after Case 1. Both exhibited transient anisocoria and progressive lethargy, anorexia, and dehydration despite antibiotic and supportive treatment. Approximately 1 wk after initial presentation, Case 2 was humanely euthanized, and Case 3 was found deceased. Necropsy findings included intrathoracic and/or intra-abdominal lymphadenopathy (3/3 cases), bicavitary effusion (2/3), multifocal tan hepatic and intestinal nodules (1/3), and multifocal yellow renal nodules (1/3). Histologically, all cats had severe pyogranulomatous vasculitis in multiple organs, and the presence of FCoV antigen was confirmed using immunohistochemical staining. Next-generation sequencing of the virus from Case 3's affected kidney demonstrated ∼93% homology to the UG-FH8 virus, a serotype 1 feline alphacoronavirus isolated from Denmark. Future research will focus on comparative viral genomic sequencing with the goals of identifying potential sources of FCoV infection and identifying features that may have contributed to the development of FIP in this species.