Integrated Use of Molecular Techniques to Detect and Genetically Characterise DNA Viruses in Italian Wolves (Canis lupus italicus)

In this study, internal organs (tongue, intestine, and spleen) of 23 free-ranging Italian wolves (Canis lupus italicus) found dead between 2017 and 2019 were tested for Carnivore protoparvovirus 1, Canine adenovirus (CAdV), and Canine circovirus (CanineCV) using real-time PCR assays. Genetic characterisation of the identified viruses was carried out by amplification, sequencing, and analysis of the complete viral genome or informative viral genes. All the wolves tested positive for at least one of the DNA viruses screened, and 11/23 were coinfected. Carnivore protoparvoviruses were the most frequently detected viruses (21/23), followed by CanineCV (11/23) and CAdV (4/23). From the analysis of the partial VP2 gene of 13 carnivore protoparvoviruses, 12 were canine parvovirus type 2b, closely related to the strains detected in dogs and wild carnivores from Italy, and one was a feline panleukopenia-like virus. Of the four CAdV identified, two were CAdV-1 and two were CAdV-2. The complete genome of seven CanineCVs was sequenced and related to the CanineCV identified in dogs, wolves, and foxes worldwide. Close correlations emerged between the viruses identified in wolves and those circulating in domestic dogs. Further studies are needed to investigate if these pathogens may be potentially cross-transmitted between the two species.

Identification of two distinct begomoviruses infecting Malvastrum coromandelianum

Malvastrum coromandelianum is a common weed plant frequently found around agricultural fields. Three virus isolates (Y249, Y278 and Y281) were obtained from M. coromandelianum with yellow vein symptoms in Honghe and Baoshan, Yunnan Province, China. Specific 500 bp products were amplified from total DNA extracts using universal primers for members of the genus Begomovirus. The complete viral genome sequences of both Y278 and Y281 were determined to be 2743 nucleotides, and that of Y249 was determined to be 2740 nucleotides. Sequence alignments and phylogenetic analyses support the proposal of creating new species in the genus Begomovirus, for which the name malvastrum yellow vein Baoshan virus (MaYVBsV) is proposed for Y278 and Y281, and malvastrum yellow vein Honghe virus (MaYVHhV) is proposed for Y249.

Divergent RNA Viruses In Macrophomina phaseolina exhibit potential as virocontrol agents

Macrophomina phaseolina is an important necrotrophic phytopathogenic fungus and cause extensive damage in many oilseed crops. Twelve M. phaseolina isolates with diverse biological phenotypes were selected for a high-throughput sequencing-based metatranscriptomic and bioinformatics analysis to identify viruses infecting M. phaseolina. The analysis identified 40 partial or nearly complete viral genome segments, 31 of which were novel viruses. Among these viral sequences, 43% of the viral genomes were double-stranded RNA (dsRNA), 47% were positive single-stranded RNA (ssRNA+), and the remaining 10% were negative sense-stranded RNA (ssRNA-). The 40 viruses showed affinity to 13 distinct viral lineages, including Bunyavirales (four viruses), Totiviridae (three viruses), Chrysoviridae (five viruses), Partitiviridae (four viruses), Hypoviridae (one virus), Endornaviridae (two viruses), Tombusviridae (three viruses), Narnaviridae (one virus), Potyviridae (one virus), Bromoviridae (one virus), Virgaviridae (six viruses), “Fusagraviridae” (five viruses), and Ourmiavirus (four viruses). Two viruses are closely related to two families, Potyviridae and Bromoviridae, which previously contained no mycovirus species. Moreover, nine novel viruses associated with M. phaseolina were identified in the family Totiviridae, Endornaviridae, and Partitiviridae. Coinfection with multiple viruses is prevalent in M. phaseolina, with each isolate harboring different numbers of viruses, ranging from three to eighteen. Furthermore, the effects of the viruses on the fungal host were analyzed according to the biological characteristics of each isolate. The results suggested that Macrophomina phaseolina hypovirus 2, Macrophomina phaseolina fusagravirus virus 1-5 (MpFV1-5), Macrophomina phaseolina endornavirus 1-2 (MpEV1-2), Macrophomina phaseolina ourmia-like virus 1-3 (MpOLV1-3), Macrophomina phaseolina mitovirus 4 (MpMV4), and Macrophomina phaseolina mycobunyavirus 1-4 (MpMBV1-4) were only detected in hypovirulent isolates. Those viruses associated with hypovirulence might be used as biological control agents as an environmentally friendly alternative to chemical fungicides. These findings considerably expand our understanding of mycoviruses in M. phaseolina and unvailed the presence of a huge difference among viruses in isolates from different hosts in distant geographical regions. Together, the present study provides new knowledge about viral evolution and fungus-virus coevolution.

A gapless unambiguous RNA metagenome-assembled genome sequence of a unique SARS-CoV-2 variant encoding spike S813I and ORF1a A859V substitutions

The novel severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is causing an unprecedented pandemic, threatening global health, daily life, and economy. Genomic surveillance continues to be a critical effort towards tracking the virus and containing its spread, and more genomes from diverse geographical areas and different time points are needed to provide an appropriate representation of the virus evolution. We here report the successful assembly of one single gapless, unambiguous contiguous sequence representing the complete viral genome from a nasopharyngeal swab of an infected healthcare worker in Cairo, Egypt. The sequence has all typical features of SARS-CoV-2 genomes, with no protein-disrupting mutations; however, three mutations are worth highlighting and future tracking: a synonymous mutation causing a rare spike S-813-I variation) and two less frequent ones leading to an A41V variation in NSP3, encoded by ORF1a (ORF1a A895V), and a Q677H variation in the spike protein. Both affected proteins, S and NSP3, are relevant to vaccine and drug development. While the genome, named CU_S3, belongs to the prevalent global genotype, marked by the D614G spike variation, the combined variations in the spike proteins and ORF1a have not been observed in any of the 197,000 genomes reported to date. Future studies will assess the biological, pathogenic, and epidemiologic implications of this set of genetic variations.

A gapless, unambiguous metagenome-assembled genome variant of SARS-CoV-2 encoding a rare spike S813I substitution

The novel severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) is causing an unprecedented pandemic, threatening global health, lifestyle, and economy. Genomic surveillance continues to be a critical effort towards tracking and containing the virus. We here report the successful assembly of one single gapless, unambiguous contiguous sequence representing the complete viral genome from a nasopharyngeal swab of an infected healthcare worker in Cairo, Egypt. The genome has all typical features of SARS-CoV-2 genomes, with no protein-disrupting mutations; however, three mutations are worth highlighting and future tracking: a synonymous mutation causing a rare spike S-813-I variation) and two less frequent ones leading to an A41V variation in NSP3, encoded by ORF1a, and a Q677H variation in the spike protein. Both affected proteins, S and NSP3, are relevant to vaccine and drug development.

Coding-Complete Genome Sequence of a Recombinant Human Norovirus Strain Identified as Subtype GII.p12_GII.3

A human norovirus (HuNoV) strain was obtained from a patient with acute gastroenteritis, and its complete coding sequence was determined. The coding-complete viral genome, with three open reading frames, was 7,565 bp long, with a GC content of 49.9%. The genotype of the HuNoV strain obtained in this study was identified as GII.p12_GII.3.

Complete Coding Sequence of a Chikungunya Virus Strain Imported into Slovenia from Thailand in Late 2018

A case of chikungunya virus infection was imported from Thailand into Slovenia in late 2018. The infection was diagnosed using real-time reverse transcription-PCR, the virus was isolated in cell culture, and the whole genome was sequenced. Phylogenetic analysis of the nearly complete viral genome indicated that the virus belongs to the Indian Ocean lineage but does not possess the A226V mutation in the envelope protein E1.

Lettuce Chlorosis Virus Disease: A New Threat to Cannabis Production

In a survey conducted in Cannabis sativa L. (cannabis) authorized farms in Israel, plants showed disease symptoms characteristic of nutrition deprivation. Interveinal chlorosis, brittleness, and occasional necrosis were observed in older leaves. Next generation sequencing analysis of RNA extracted from symptomatic leaves revealed the presence of lettuce chlorosis virus (LCV), a crinivirus that belongs to the Closteroviridae family. The complete viral genome sequence was obtained using RT-PCR and Rapid Amplification of cDNA Ends (RACE) PCR followed by Sanger sequencing. The two LCV RNA genome segments shared 85–99% nucleotide sequence identity with LCV isolates from GenBank database. The whitefly Bemisia tabaci Middle Eastern Asia Minor1 (MEAM1) biotype transmitted the disease from symptomatic cannabis plants to un-infected ‘healthy’ cannabis, Lactuca sativa, and Catharanthus roseus plants. Shoots from symptomatic cannabis plants, used for plant propagation, constituted a primary inoculum of the disease. To the best of our knowledge, this is the first report of cannabis plant disease caused by LCV.

Discovery of a Highly Divergent Coronavirus in the Asian House Shrew from China Illuminates the Origin of the Alphacoronaviruses

ABSTRACT Although shrews are one of the largest groups of mammals, little is known about their role in the evolution and transmission of viral pathogens, including coronaviruses (CoVs). We captured 266 Asian house shrews (Suncus murinus) in Jiangxi and Zhejiang Provinces, China, during 2013 to 2015. CoV RNA was detected in 24 Asian house shrews, with an overall prevalence of 9.02%. Complete viral genome sequences were successfully recovered from the RNA-positive samples. The newly discovered shrew CoV fell into four lineages reflecting their geographic origins, indicative of largely allopatric evolution. Notably, these viruses were most closely related to alphacoronaviruses but sufficiently divergent that they should be considered a novel member of the genus Alphacoronavirus, which we denote Wénchéng shrew virus (WESV). Phylogenetic analysis revealed that WESV was a highly divergent member of the alphacoronaviruses and, more dramatically, that the S gene of WESV fell in a cluster that was genetically distinct from that of known coronaviruses. The divergent position of WESV suggests that coronaviruses have a long association with Asian house shrews. In addition, the genome of WESV contains a distinct NS7 gene that exhibits no sequence similarity to genes of any known viruses. Together, these data suggest that shrews are natural reservoirs for coronaviruses and may have played an important and long-term role in CoV evolution. IMPORTANCE The subfamily Coronavirinae contains several notorious human and animal pathogens, including severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and porcine epidemic diarrhea virus. Because of their genetic diversity and phylogenetic relationships, it has been proposed that the alphacoronaviruses likely have their ultimate ancestry in the viruses residing in bats. Here, we describe a novel alphacoronavirus (Wénchéng shrew virus [WESV]) that was sampled from Asian house shrews in China. Notably, WESV is a highly divergent member of the alphacoronaviruses and possesses an S gene that is genetically distinct from those of all known coronaviruses. In addition, the genome of WESV contains a distinct NS7 gene that exhibits no sequence similarity to those of any known viruses. Together, these data suggest that shrews are important and longstanding hosts for coronaviruses that merit additional research and surveillance.