Complete Viral Genome Research Articles

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.

Weighted hypergraph learning and adaptive inductive matrix completion for SARS-CoV-2 drug repositioning.

The outbreak of the human coronavirus (SARS-CoV-2) has placed a huge burden on public health and the world economy. Compared with de novo drug discovery, drug repurposing is a promising therapeutic strategy that facilitates rapid clinical treatment decisions, shortens the development process, and reduces costs. In this study, we propose a weighted hypergraph learning and adaptive inductive matrix completion method, WHAIMC, for predicting potential virus-drug associations. Firstly, we integrate multi-source data to describe viruses and drugs from multiple perspectives, including drug chemical structures, drug targets, virus complete genome sequences, and virus-drug associations. Then, WHAIMC establishes an adaptive inductive matrix completion model to improve performance through adaptive learning of similarity relations. Finally, WHAIMC introduces weighted hypergraph learning into adaptive inductive matrix completion to capture higher-order relationships of viruses (or drugs). The results showed that WHAIMC had a strong predictive performance for new virus-drug associations, new viruses, and new drugs. The case study further demonstrates that WHAIMC is highly effective for repositioning antiviral drugs against SARS-CoV-2 and provides a new perspective for virus-drug association prediction. The code and data in this study is freely available at https://github.com/Mayingjun20179/WHAIMC.

Long-Read-Based Genome Assembly Reveals Numerous Endogenous Viral Elements in the Green Algal Bacterivore Cymbomonas tetramitiformis.

The marine tetraflagellate Cymbomonas tetramitiformis has drawn attention as an early diverging green alga that uses a phago-mixotrophic mode of nutrition (i.e., the ability to derive nourishment from both photosynthesis and bacterial prey). The Cymbomonas nuclear genome was sequenced previously, but due to the exclusive use of short-read (Illumina) data, the assembly suffered from missing a large proportion of the genome's repeat regions. For this study, we generated Oxford Nanopore long-read and additional short-read Illumina data and performed a hybrid assembly that significantly improved the total assembly size and contiguity. Numerous endogenous viral elements were identified in the repeat regions of the new assembly. These include the complete genome of a giant Algavirales virus along with many genomes of integrated Polinton-like viruses (PLVs) from two groups: Gezel-like PLVs and a novel group of prasinophyte-specific PLVs. The integrated ∼400 kb genome of the giant Algavirales virus is the first account of the association of the uncultured viral family AG_03 with green algae. The complete PLV genomes from C. tetramitiformis ranged between 15 and 25 kb in length and showed a diverse gene content. In addition, heliorhodopsin gene-containing repeat elements of putative mirusvirus origin were identified. These results illustrate past (and possibly ongoing) multiple alga-virus interactions that accompanied the genome evolution of C. tetramitiformis.

Temporal dynamics of microbe-virus interactions in the Baltic Sea

Bacteria and archaea are key drivers of all major element cycles. Viruses that infect bacteria and archaea also play a fundamental role by altering the metabolic state of their host during infection and causing cell death. The CRISPR-Cas system is one of many strategies employed by bacteria and archaea to defend against viral infection. Invading viral DNA is incorporated into a CRISPR array as a short sequence (spacer) that is then recognised during the next viral encounter providing an adaptive immunity. The temporal dynamics of this system in the environment, however, is not well constrained. Using a meta-omic dataset spanning several years of sampling, we leveraged the CRISPR-Cas system to explore microbe-virus interactions in the Baltic Sea. Our goal was to understand how quickly microbes in the environment adapt to virus predation, and conversely how quickly viruses adapt to the microbial defence mechanism by developing mutations in the spacer-targeted region. To explore these interactions, we first generated a database consisting of thousands of complete and high-quality viral genomes recovered from viromes collected from the Baltic Sea. CRISPR arrays were then identified in microbial metagenome assembled genomes (MAGs), metagenomic contigs, and unassembled metagenomic reads from corresponding sampling time points. Virus-host dynamics were uncovered by matching quality-filtered spacers from CRISPR arrays to the viral database. The results show that spacer turnover over time can be captured in temporal meta-omic datasets. In the Baltic Sea, this has implications for the termination of microbial blooms, biogeochemical cycling, and resource turnover.

Viruses, Vesicles, and other Biological Nanoparticles: The Sub-cellular Biosphere of a Deeply Buried 2km-Deep, 20-Million-Year-Old Coalbed Community

Horizontal gene transfer is an important driver of adaptation and evolution in microorganisms. Transducing biological nanoparticles such as viral particles are believed to be key facilitators of horizontal gene transfer. In deep subseafloor sediments, energy can be highly limiting, supporting only extremely slow metabolisms. In such low-energy, isolated environments where communities may subsist for millions of years, the mechanisms of subsurface microbial adaptation and evolution remain a mystery. Virus particles have been found everywhere that life has been found, including deep subsurface environments. Although microorganisms are abundant and active in the Earth's subsurface, the role of viruses in shaping and influencing these slow-growing communities is only recently starting to be explored. Here, we analyzed the deeply buried microbial community from a lignite coalbed layer 2km below the seafloor offshore Shimokita, Japan (IODP Expedition 337) that had been buried for 20 million years. We harvested cells (>0.2µm) and biological nanoparticles (<0.2µm) from a bioreactor enrichment seeded by lignite core samples. We sequenced DNA from the cells and nanoparticles and subsequently analyzed the metagenomes. Within the nanoparticle metagenome, numerous complete novel virus genomes were reconstructed. Comparison of the virus genomes to the prokaryotic MAGs (metagenome assembled genomes) revealed that many of the virus genomes had been integrated prophage within bacterial genomes, suggesting the potential for virus-host interactions to occur in the deep subseafloor. Additionally, lysogeny may be an important survival mechanism for viruses in deeply buried, low-energy environments. Host genes were found to be packaged by viral particles, demonstrating the potential for specialized and general transduction by viruses. Not only viral particles, but there was also evidence that membrane vesicles and gene transfer agents may participate in transduction in this deep subsurface community. Horizontal gene transfer mediated by biological nanoparticles may be an important mechanism of adaptation for deep subsurface microbial communities and may provide insight into possible evolutionary processes shaping microbial communities in the deep subsurface. These results may also shed some light onto the nature of viral infection in the subsurface, potentially revealing insights about the long-term persistence of life under extreme energy limitation and how viruses may survive this over geological timescales.

Accumulated metagenomic studies reveal recent migration, whole genome evolution, and undiscovered diversity of orthomyxoviruses.

The number of known virus species has increased dramatically through metagenomic studies, which search genetic material sampled from a host for non-host genes. Here, we focus on an important viral family that includes influenza viruses, the Orthomyxoviridae, with over 100 recently discovered viruses infecting hosts from humans to fish. We find that one virus called Wǔhàn mosquito virus 6, discovered in mosquitoes in China, has spread across the globe very recently. Surface proteins used to enter cells show signs of rapid evolution in Wǔhàn mosquito virus 6 and its relatives which suggests an ability to infect vertebrate animals. We compute the rate at which new orthomyxovirus species discovered add evolutionary history to the tree of life, predict that many viruses remain to be discovered, and discuss what appropriately designed future studies can teach us about how diseases cross between continents and species.

Unlocking the Viral Universe: Metagenomic Analysis of Bat Samples Using Next-Generation Sequencing.

Next-generation sequencing technologies have revolutionized the field of virology by enabling the reading of complete viral genomes, extensive metagenomic studies, and the identification of novel viral pathogens. Although metagenomic sequencing has the advantage of not requiring specific probes or primers, it faces significant challenges in analyzing data and identifying novel viruses. Traditional bioinformatics tools for sequence identification mainly depend on homology-based strategies, which may not allow the detection of a virus significantly different from known variants due to the extensive genetic diversity and rapid evolution of viruses. In this work, we performed metagenomic analysis of bat feces from different Russian cities and identified a wide range of viral pathogens. We then selected sequences with minimal homology to a known picornavirus and used "Switching Mechanism at the 5' end of RNA Template" technology to obtain a longer genome fragment, allowing for more reliable identification. This study emphasizes the importance of integrating advanced computational methods with experimental strategies for identifying unknown viruses to better understand the viral universe.

Circulation patterns and molecular epidemiology of human respiratory syncytial virus over five consecutive seasons in Morocco.

Respiratory syncytial virus (HRSV) is the leading cause of respiratory tract infections in infants and young children. we investigated the prevalence and characteristics of HRSV in Morocco and explored trends in circulating genotypes through partial G gene analysis of HRSV strains prevalent from 2012 to 2017. Respiratory samples were gathered from both outpatients and inpatients meeting ILI or SARI case definitions. The patients' ages varied from 1 month to 99 years old. Nucleic acids were extracted and HRSV type/subtype was detected by RT-qPCR. A subset of positive samples was randomly selected in each epidemic year, the complete viral genome was sequenced, phylogenetic analysis was performed using the MEGA7 program and the genotypes were confirmed. The 3679 specimens were collected from 2012 to 2017, of which 726 (19.7%) were positive for HRSV. The 35% (257/726) of HRSV-positives were of the HRSV-A subtype, while the HRSV-B subtype accounted for 61% (442/726). The co-infection rate was 3.7% (27/726). The virus circulates in a periodic pattern, where epidemics occur during the fall months through early spring. HRSV genotype was confirmed in 127 specimens (56 HRSV-A and 71 HRSV-B). Based on phylogenetic analysis, all HRSV-A were ON1 genotype, and HRSV-B were mostly BA9 genotype. HRSV-B belonging to the BA10 genotype was detected in 2012 exclusively. BA9, BA10, and ON1 were the only HRSV genotypes detected between 2012 and 2017. Variations in the G gene amino acid chain were identified in local strains, which suggests an increased need for continuous genomic surveillance.

Zika virus disease: an alarming situation resurfacing on the radar - a short communication.

On the 13th of December 2022, a 5-year-old girl from Karnataka, India, tested positive for Zika virus. The first Zika virus was isolated from the serum of a rhesus monkey in the Zika Forest of Uganda in 1947. Zika virus was largely dormant for about 70 years before suddenly resurfacing across all of America, from Brazil to the Pacific Islands and is connected to a grouping of microcephaly phenotypes based on a complete virus genome analysis. All of the aforementioned research provides an overview of the migration of this virus from the Americas to continental Africa via mosquitoes. The current study, therefore, aims to evaluate the virologic characteristics, prophylaxis, transmitting mechanisms, diagnosis, clinical manifestations, and treatment of ZIKV infection in light of the virus's widespread dissemination and deadly nature. The investigation's findings aim to demonstrate that in order to prevent further outbreaks, there is a national requirement for active epidemiological and entomological observation of Zika. Data were extracted from academic journals of medicine published in MEDLINE, PubMed, ScienceDirect, Ovid, and Embase inventory databases with a predetermined search strategy. Articles considering the Zika virus and its clinical manifestations, especially neurological, were included. The Zika virus has been declared a public health emergency of global significance by the World Health Organization (WHO). It is of alarming concern that it is now one of the most prevalent infectious diseases associated with birth abnormalities discovered in the past five decades. The onset and accelerated spread of disease to other parts of the world is attributed to the migration of infected hosts and climate change. Rapid laboratory diagnosis, evaluation of serological techniques, and virus isolation are urgently needed, along with newer modalities such as mathematical modeling as prediction devices to curb this issue. Due to its grave neurological manifestations, it is mandated to engineer peptide therapies and a virus-specific vaccination to treat this neurotropic virus. There is currently no vaccination against Zika virus infection. If societies are not adequately prepared, the epidemiological wave will have an impact on the workforce and could pose a serious threat. To alleviate the significant cost on health systems and manage its promotion globally, improved investigation and response activities are needed.

Epidemiology, genetic diversity, and evolutionary dynamics of Tembusu virus.

Tembusu virus (TMUV) is an emerging pathogenic flavivirus associated with acute egg-drop and fatal encephalitis in domestic waterfowl. Since its initial identification in mosquitoes in 1955, TMUV has been confirmed to infect ducks, pigeons, sparrows, geese, and chickens, posing a significant threat to the poultry industry. Here, we sequenced two DTMUV strains isolated in 2019 and systematically investigated the possible origin, genetic relationships, evolutionary dynamics, and transmission patterns of TMUV based on complete virus genome sequences in the public database. We found that TMUV can be divided into four major clusters: TMUV, cluster 1, cluster 2, and cluster 3. Interestingly, we found that cluster 2.2 (within cluster 2) is the most commonly involved in interspecies transmission events, and subcluster 2.1.2 (within cluster 2.1) is currently the most prevalent cluster circulating in Asia. Notably, we also identified three positively selected sites in the E and NS1 proteins, which may be involved in virus replication, immune evasion, and host adaptation. Finally, phylogeographic analysis revealed that cluster dispersal originated in Southeast Asia and that short-distance transmission events have occurred frequently. Altogether, these data provide novel insights into the evolution and dispersal of TMUV, facilitating the development of rapid diagnostics, vaccines, and therapeutics against TMUV infection.

Characterization of the complete genome of oat sterile dwarf virus: a fijivirus occurring in the temperate climate zone of Europe.

Oat sterile dwarf virus (OSDV) is a fijivirus whose genome segments 7 to 10 were sequenced earlier. In the current study, the complete genome was sequenced. To confirm the genome ends, rapid amplification and sequencing of cDNA ends were performed. The complete OSDV genome consists of 10 double-stranded RNA (dsRNA) segments with a total size of 28,686 bp. The sense strand sequence of all segments has the terminal consensus sequence motif 5'-AACGA(5-7)… U(6-8)(A/U)GUC-3', in which the length of the stretches of A and U varies, being slightly shorter for segments 1-4 and longer for segments 5-10. The 3' end of segment 3 is …UGUC, not AGUC as in the other segments. Segments 5, 7, and 10 contain two small ORFs, while each of the other segments contains one long ORF. ORF7-2 and ORF9 are slightly longer than annotated before. Phylogenetic analysis based on amino acid sequences of the RNA-directed RNA polymerase (RdRP) placed OSDV between the plant fijiviruses and Nilaparvata lugens reovirus (NLRV), an insect fijivirus that does not replicate in plants. OSDV RdRP shares 48-49% sequence identity with other plant-infecting fijivirus RdRPs and 30% identity with that of NLRV. OSDV has earlier been reported in several Northern and Central European countries. The sequencing of the complete genome serves as a reference for identifying all segments in future high-throughput sequencing datasets, enabling the investigation of the molecular epidemiology and evolution of OSDV.

Cucurbit chlorotic yellows virus, a crinivirus infecting Cannabis sativa plants

AbstractHigh cannabidiol‐containing plants of Cannabis sativa (high‐CBD) growing in farms in Israel displayed foliar symptoms of interveinal chlorosis and yellowing, brittleness and occasionally necrosis. These symptoms, which were more apparent in older leaves, resembled those caused by the crinivirus lettuce chlorosis virus (LCV). However, this virus was not detected by reverse transcription (RT)‐PCR using specific primer sets. High‐throughput sequencing of viral RNA extracted from symptomatic leaves revealed the presence of cucurbit chlorotic yellows virus (CCYV), a crinivirus in the Closteroviridae family. The complete viral genome sequence was obtained using RT‐PCR followed by Sanger sequencing. The two CCYV RNA genomic segments shared 99.5%–99.85% nucleotide sequence identity with CCYV isolates from the GenBank. The virus was transmitted from symptomatic cannabis leaves to healthy plants of cannabis and Cucumis sativus ‘King Star’ (cucumber) by the whitefly Bemisia tabaci Middle Eastern Asia Minor 1 (MEAM1) species, causing disease symptoms identical to those of the donor plants. Cannabis‐CCYV was also transmitted between infected cucumber plants and cannabis seedlings of unknown genotype. Severe disease symptoms of yellowing and leaf‐edge necrosis were observed on high‐CBD and high Δ9‐tetrahydrocannabinol‐containing (high‐THC) flowering cannabis plants and were associated with mixed infections of LCV and CCYV. To the best of our knowledge, this is the first report of CCYV infecting C. sativa plants.

Spreading of the High-Pathogenicity Avian Influenza (H5N1) Virus of Clade 2.3.4.4b into Uruguay.

Avian influenza viruses (genus Alphainfluenzavirus, family Orthomyxoviridae) infect avian and mammal hosts. In 2022, the high pathogenicity avian influenza virus (H5N1) spread to South America, resulting in the loss of thousands of wild birds, including endangered species, and severely impacting the global poultry industry. We analyzed the complete genomes of influenza viruses obtained from wild birds and backyard poultry in Uruguay between February and May 2023. Twelve complete genomes were obtained in 2023 from cloacal swabs using Illumina sequencing. Genomes were phylogenetically analyzed with regional and global strains. The identified strains have multiple basic amino acids at the hemagglutinin cleavage sites, which is typical for highly pathogenic strains. The Uruguayan viruses belonged to hemagglutinin clade 2.3.4.4b of the H5N1 subtype. A reassortment in North America has resulted in some segments of South American strains being of Eurasian or North American origins. The Uruguayan viruses shared a common ancestor with South American strains from Argentina and Chile. The influenza viruses displayed a spatiotemporal divergence pattern rather than being host-specific. The arrival of the 2.3.4.4b clade in Uruguay may have been mediated by birds that acquired the virus from Argentine and Chilean waterfowl migrating in the Pacific Flyway.

Detection and isolation of infectious SARS-CoV-2 omicron subvariants collected from residential settings.

Airborne transmission of infectious (viable) SARS-CoV-2 is increasingly accepted as the primary manner by which the virus is spread from person to person. Risk of exposure to airborne virus is higher in enclosed and poorly ventilated spaces. We present a study focused on air sampling within residences occupied by individuals with COVID-19. Air samplers (BioSpot-VIVAS, VIVAS, and BC-251) were positioned in primary- and secondary-occupancy regions in seven homes. Swab samples were collected from high-touch surfaces. Isolation of SARS-CoV-2 was attempted for samples with virus detectable by RT-qPCR. Viable virus was quantified by plaque assay, and complete virus genome sequences were obtained for selected samples from each sampling day. SARS-CoV-2 was detected in 24 of 125 samples (19.2%) by RT-qPCR and isolated from 14 (11.2%) in cell cultures. It was detected in 80.9% (17/21) and cultured from 61.9% (13/21) of air samples collected using water condensation samplers, compared to swab samples which had a RT-qPCR detection rate of 10.5% (4/38) and virus isolation rate of 2.63% (1/38). No statistically significant differences existed in the likelihood of virus detection by RT-qPCR or amount of infectious virus in the air between areas of primary and secondary occupancy within residences. Our work provides information about the presence of SARS-CoV-2 in the air within homes of individuals with COVID-19. Information herein can help individuals make informed decisions about personal exposure risks when sharing indoor spaces with infected individuals isolating at home and further inform health departments and the public about SARS-CoV-2 exposure risks within residences.

Complete genome sequence of a divergent strain of cardamom mosaic virus.

Cardamom mosaic virus (CdMV; genus Macluravirus), which causes mosaic (katte) disease in cardamom, is a highly variable member of the family Potyviridae. So far, the complete genome sequence of one isolate from Karnataka (KS) has been reported. In the present study, we determined the complete genome sequence of a CdMV isolate from Kerala (KI) and the complete CP gene sequences of nine isolates of CdMV from Kerala, Karnataka, and Tamil Nadu, India. The complete genome of CdMV (KI) consists of 8255 nucleotides (nt) with two open reading frames (ORFs). The large ORF, potentially coding for a polyprotein of 2638 amino acids (aa), is further processed into nine mature proteins at eight cleavage sites. The second ORF, PIPO (pretty interesting Potyviridae ORF) starting with a C(A)6 motif, encodes a small protein of 56 aa. The viral genome contains an additional 13 nt in the 5' untranslated region (UTR) and 6 nt in the CP gene, as well as a deletion of 13 nt at the 3' UTR in comparison to the KS isolate of CdMV. The complete viral genome and polyprotein share 76% and 85% sequence identity with the KS isolate of CdMV, indicating that the present isolate is highly divergent from the KS isolate. Sequencing and analysis of the CP sequences of 16 CdMV isolates from different regions revealed high heterogeneity among them, suggesting that they should be considered members of more than one species.

Analysis of the Tomato Aspermy Virus Complete Genomes Suggests Reassortment in Russian Isolates from Chrysanthemum

Tomato aspermy virus (TAV, genus Cucumovirus from the family Bromoviridae) is one of the most common and harmful chrysanthemum viruses, causing severe flower distortion, size reduction and color breaking. Metatranscriptome sequencing of chrysanthemum plants of the Ribonette and Golden Standard cultivars from the collection of the Nikita Botanical Garden (Yalta, Republic of Crimea) generated TAV-related reads. The complete genomes of two Russian isolates of the virus were assembled from the reads obtained. This is the first report of full-length TAV genomes from Russia. Typically of cucumoviruses, the segmented TAV genome is represented by three single-stranded positive-sense linear RNA molecules with a length of 3412 (RNA1), 3097 (RNA2) and 2219 (RNA3) nucleotides. Five open reading frames (ORF) have been identified that encode replicase (ORF1), RNA-dependent RNA polymerase (ORF2a), silencing suppressor protein (OFR2b), movement protein (OFR3a) and the virus coat protein (ORF3b). The identity of the TAV genomes from the two chrysanthemum cultivars was 99.8% for all three viral RNAs, and with other TAV isolates from Genbank – 97.5–99.7 (RNA1), 93.8–99.8 (RNA2), and 89.3–99.3% (RNA3). Phylogenetic analysis showed that RNA1 and RNA3 of the Russian isolates were assigned to heterogeneous groups of TAV isolates found on various plant species in different regions of the world. At the same time, RNA2 clearly clustered with tomato isolates SKO20ST2 from Slovenia and PV-0220 from Bulgaria and, to a lesser extent, with the Iranian isolate Ker.Mah.P from petunia and the Chinese isolate Henan from chrysanthemum. The incongruence of phylogenetic trees reconstructed from different segments of the viral genome suggests pseudo-recombination (reassortment) in the Russian TAV isolates.

Molecular characterization of a novel polerovirus from bitter gourd plants and dynamic subcellular localization of the virus-encoded proteins

Viruses within the Polerovirus genus in the family of Solemoviridae have a single-stranded positive-sense RNA genome of about 5.6–6.2 kb in length. In this study, the bitter gourd leaves showing yellowing and crumple symptoms were collected for small RNAs (sRNAs) sequencing. Analysis of the contigs de novo assembled from sRNA-sequencing data, followed by RT-PCR and cloning, determined the complete viral genome to be 5665 nucleotides. This virus isolate contains conserved ORF3a, ORF3, ORF4, and other typical features of poleroviruses. The P0 protein of this virus isolate shares less than 74.80% amino acid sequence identity with any of the previously characterized poleroviruses, indicating that it should be a novel polerovirus. We name this virus as bitter gourd yellowing crumple virus (BYCV). We further revealed the dynamic subcellular localization and protein accumulation of seven proteins encoded by BYCV in Nicotiana benthamiana plants over time, and these viral proteins displayed specific subcellular localization. Expressing the BYCV P0 protein using a potato virus X vector caused severe symptoms in N. benthamiana plants, while P0 exhibited weak RNA silencing suppression activity. These findings provide an example for investigating the dynamic subcellular localizations of viral proteins and demonstrate that P0 is a critical protein potentially playing an important role in virus infection.

Distribution and prevalence of viral genomes in Italian populations of the invasive brown marmorated stink bug Halyomorpha halys

Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species that causes significant agricultural losses, especially to orchard fruits, vegetables, herbaceous and ornamental plants. It is also a nuisance pest that seeks shelter in indoor spaces during the winter months. Harnessing the H. halys virome can result in new environmentally sustainable approaches to contain its populations and its relatated agricultural damages.In this study, RNA-Seq data were used to explore the virome associated to ten field populations collected in the Lombardy region in Northern Italy. We identified six complete viral genomes, three of which were previously unknown, belonging to the orders Reovirales, Articulavirales, Ghabrivirales, Durnavirales, and Picornavirales. The prevalence of the six viruses was evaluated by Real-time reverse transcription-quantitative PCR on eighty individuals. Halyomorpha halys ifla-like virus 2 turned out to be the most geographically widespread virus, as it was found in more than 50% of the analyzed insects and in nine out of the ten sampling locations. Moreover, in some individuals, this iflavirus was found in association with each of the other viruses in various combinations that involved up to four viruses. Further studies on such virus-virus interactions and their relationships with the insect host may open the possibility to exploit these naturally occurring viruses as specific and targeted biocontrol agents of H. halys.

A novel NADC34-like porcine reproductive and respiratory syndrome virus 2 with complex genome recombination is highly pathogenic to piglets.

The NADC34-like porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) first emerged in China in 2017 and has the potential to become the dominant PRRSV strain in China. Here, a novel PRRSV-2, SCcd2020, was isolated from diseased piglets in Sichuan province, southwest China in 2020. The complete viral genome was determined and analyzed. An ORF5-based phylogenetic analysis showed that SCcd2020 clustered with NADC34-like strains, whereas the genome sequence clustered the isolate with NADC30-like viruses and it contains a discontinuous 131-aa deletion in NSP2 when compared to NADC30 strain. Notably, recombination analyses indicated that SCcd2020 is a multiple recombinant virus from NADC30-like, NADC34-like and JXA1-like strains, which is the first description of Chinese domestic HP-PRRSV involving the recombination event of an NADC34-like strain. Importantly, an animal challenge study in 4-week-old piglets showed that SCcd2020 causes high fever and severe hemorrhagic pneumonia with pulmonary consolidation and edema, and it has a high mortality rate (60%), which indicated that SCcd2020 is a highly pathogenic PRRSV strain. The study reports the emergence of a novel highly pathogenic NADC34-like recombinant strain, and it highlights the importance of monitoring newly emerging PRRSV strains in China.

Senna multiglandulosa, a new natural host for Tobacco etch virus in Mexico.

Plants of Senna multiglandulosa (family Fabaceae), an ornamental shrub, growing adjacent to tomato and chrysanthemum greenhouses located in San Diego, Texcoco, Estado de Mexico, had leaves with putative virus symptoms, consisting of annular or irregular chlorotic spots of different sizes (Supplementary Fig. 1a). To investigate the presence of a virus, high-throughput sequencing (HTS) was performed. Total RNA was extracted from symptomatic leaves of S. multiglandulosa plants using the SV Total RNA Isolation System Kit (Promega, USA). A portion of the RNA was sent to BGI Genomics (China) for cDNA library construction and sequencing on the DNBSEQ platform (BGI Genomics). HTS yielded 14,673,469 clean paired reads (150x2), which were assembled de novo into 91,879 contigs using SPAdes v3.15 software (Prjibelski et al. 2020). The contigs ranged from 78 to 14,534 nucleotides (nts), which were subjected to BLASTx and BLASTn analyses. A single viral contig of 9,501 nts was detected (average coverage: 56,716x per nt) representing the nearly complete genome of tobacco etch virus (TEV). The highest identity was 79.26% at the nt level (92% query coverage) with TEV isolate TEV7DA (GenBank: DQ986288; length: 9,539 nts) from the USA, and 86.67% at the amino acid (aa) level considering the polyprotein, which are higher than the species demarcation threshold (<76% nt and <82% aa) for the genus Potyvirus (Inoue-Nagata et al. 2022). Additionally, the sequence obtained from S. multiglandulosa revealed 79.21-79.37% nt identities with different TEV isolates from Solanaceae plants (Capsicum annuum, MW748496; Solanum lycopersicum, OM471966.1; Nicotiana tabacum, OL311684.1). The new TEV genome was deposited in GenBank under accession number ON110203. The results obtained by HTS were confirmed by RT-PCR with the original isolated RNA using a pair of specific primers designed from the TEV sequence (TEV-NIb-F, 5'- GCGCTTAAATGCAGACTCGG-3' and TEV-NIb-R, 5'-GTGAAAGTTCAGCAGCAAGCGCA-3') that amplify a 550-bp fragment of the RNA-dependent RNA polymerase. The obtained amplicon was sequenced by the Sanger method, and was 100% identical to the sequence generated by HTS. Subsequently, N. tabacum and N. glutinosa plants were mechanically inoculated using TEV-positive S. multiglandulosa leaves as the inoculum source. Twenty days after inoculation, light chlorotic spots and necrotic lesions were observed on N. tabacum and mosaic on N. glutinosa (Supplementary Fig. 1b-c). RT-PCR analysis confirmed the presence of TEV infection in these indicator plants. To determine the incidence of S. multiglandulosa plants showing TEV-infection symptoms, a survey (n=16) was carried out on two farms in Texcoco; the survey showed a 100% incidence of symptoms. Five survey samples were randomly selected, and the presence of TEV was confirmed by RT-PCR. The discovery of Tobacco etch virus (family Potyviridae: genus Potyvirus) in tobacco was reported in Kentucky, USA in 1928 (Valleau and Johnson, 1928), one of the most common and damaging viruses for the chili crop in Mexico (Delgado, 1974). TEV causes heavy yield loss in several Solanaceae plants and infects more than 120 species in 19 families of dicotyledons (Holmes, 1946). S. obtusifolia (originally Cassia obtusifolia) was the first legume reported as a natural host of TEV in Florida, USA (Anderson, 1954). To our knowledge, this is the first report of the natural infection of S. multiglandulosa by TEV in Mexico and the first TEV genome isolated and sequenced from a legume. S. multiglandulosa is widely distributed in 16 states in Mexico, both cultivated and naturalized, however, it is not considered native to the country (Rzedowski and Calderón, 1997). The occurrence of TEV in S. multiglandulosa represents an alternative reservoir of the virus, with an important role in the epidemiology of the disease.