Complete Viral Genome Research Articles

Identification of Novel Reassortant Shuni Virus Strain in Clinical Cases of Israeli Ruminants, 2020-2021.

The Shuni virus (SHUV) causes an endemic viral infection in Israel and South Africa. It belongs to the Simbu serogroup within the order Bunyavirales, family Peribunyaviridae, genus Orthobunyavirus. Recently, it has been identified in aborted cases of domestic ruminants, young cattle and horses manifesting neural signs and acute death, symptomatic cows, and in carcasses of wild animals. Moreover, SHUV was isolated and identified in humans. In this study, we describe clinical cases of SHUV infection in Israeli domestic ruminants in 2020–2021, which represented clinical manifestations of simbuviral infection including abortions, a neural lethal case in a fattening calf, and an acute symptomatic case in a beef cow. In all cases, SHUV was confirmed by complete or partial viral genome sequencing. There is a significant difference of M and L segments of the novel strains compared with those of all known SHUV strains, while the S segments have more than 99% nucleotide (nt) identity with Israeli and African “Israeli-like” strains previously circulated in 2014–2019. This indicates a reassortment origin of the strain. At the same time, M and S segment nt sequences showed about 98–99% nt identity with some South African strains collected in 2016–2018. Nevertheless, the viral origin and the geographical place of the reassortment stayed unknown.

Whole-genome analysis of human papillomavirus 67 isolated from Japanese women with cervical lesions

BackgroundHuman papillomavirus (HPV) type 67 is phylogenetically classified into Alphapapillomavirus species 9 (alpha-9) together with other carcinogenic types (HPV16, 31, 33, 35, 52 and 58), but is the only alpha-9 type defined as possibly carcinogenic. This study aimed to determine whole-genome sequences of HPV67 isolated from Japanese women with cervical cancer or cervical intraepithelial neoplasia (CIN) to better understand the genetic basis of the oncogenic potential of HPV67.MethodsTotal cellular DNA isolated from cervical exfoliated cells that were single positive for HPV67 (invasive cervical cancer, n = 2; CIN3, n = 6; CIN 2, n = 1; CIN1, n = 2; the normal cervix, n = 1) was subjected to PCR to amplify HPV67 DNA, followed by next generation sequencing to determine the complete viral genome sequences. Variant lineages/sublineages were assigned through viral whole-genome phylogenetic analysis. The transcriptional activity of the virus early promoter was assessed by luciferase reporter assays in cervical cancer cell lines.ResultsPhylogenetic analyses of HPV67 genomes from Japan (n = 12) revealed that 11 belonged to lineage A (sublineage A1, n = 9; sublineage A2, n = 2) and one belonged to lineage B. All cancer cases contained sublineage A1 variants, and one of these contained an in-frame deletion in the E2 gene. The long control region of the HPV67 genome did not induce transcription from the virus early promoter in HeLa cells, but showed weak transcriptional activity in CaSki cells.ConclusionsThe single detection of HPV67 in cervical cancer and precancer specimens strongly suggests the carcinogenic potential of this rare genotype. The phylogenetic analysis indicates a predominance of lineage A variants among HPV67 infections in Japan. Since only 23 complete genome sequences of HPV67 had been obtained until now, the newly determined genome sequences in this study are expected to contribute to further functional and evolutionary studies on the genetic diversity of HPV67.

First report of Tomato leaf curl Karnataka virus associated with leaf curl disease of Zinnia elegans in India

Tomato leaf curl Karnataka virus (ToLCKV) is a species of begomovirus which has a circular, ssDNA monopartite genome and is transmitted by the whitefly (Bemisia tabaci). Zinnia elegans is a popular ornamental bedding plant which is grown worldwide for its beautiful flowers. A few begomovirus species have been reported to infect Z. elegans, including Ageratum yellow vein China virus in Vietnam (Li et al., 2013), and Ageratum enation virus and Zinnia leaf curl virus in India (Kumar et al., 2010; Pandey & Tiwari, 2012). In March 2019 symptoms of excessive leaf curling with mild mosaic were observed on Z. elegans plants growing in a nursery in Bhopal, central India (Fig. 1). To investigate the aetiology of the disease total DNA was extracted from leaf samples from two diseased and one asymptomatic Z. elegans plant using a plant genomic DNA purification kit (Promega, USA), following the manufacturer's instructions. A begomovirus was detected from both diseased samples (but not the asymptomatic sample) by PCR using begomovirus-specific primers: CPIT-I/CPIT-T (AM180920/AM180921) (Singh, 2005), PALIv 1978/ PARIc 496 and PALIv 722/ PALIc 1960 (Rojas et al., 1993). The expected amplicons (~800 bp, 1.2 Kb and 1.2 Kb, respectively) were purified using a PCR Clean-up System (Promega, USA) and Sanger sequenced in both directions by Bioinnovations Connecting Technologies to Life (Mumbai, India). The consensus sequence from both samples was identical and the complete viral DNA-A genome of 2761 nucleotides was deposited in GenBank (Accession No. MK965196). Sequence analysis was done by BLASTn and showed that the DNA-A genome had the highest identity (96-97%) with ToLCKV (KX219744, HM007094, LN878123, U38239); Sunflower leaf curl virus (JX678965) and 94–95% with Tomato leaf curl virus (JX524172, KX831454). Phylogenetic analysis using neighbour-joining in MEGA v.7.0 showed a close relationship with isolates of ToLCKV and relationships with other begomoviruses (Fig. 2). The disease was successfully transmitted by B. tabaci from naturally infected Z. elegans to healthy seedlings of Nicotiana tabacum, Solanum lycopersicum and Z. elegans (each 3/3), symptoms being observed 30 days post inoculation. The test plants all tested positive by PCR using the begomovirus-specific primer pairs above. Positive amplicons were sequenced and the nucleotide sequences were identical to each other and the original isolate (MK965196). The present study demonstrates that Z. elegans is a host of ToLCKV. ToLCKV is a serious threat to the production of several economically important crops in India and has been reported at an incidence of >80% on soybean, sunflower and tomato in India (Malathi et al., 2017). Recently, ToLCKV and satellite molecules have been found associated with enation leaf curl disease of another ornamental plant, Pseuderanthemum reticulatum, in India (Ashwathappa et al., 2021). The whitefly transmission study provides additional evidence that ToLCKV causes the observed disease in Z. elegans and it is possible that ornamental plants may act as a reservoir of the virus. To the best of our knowledge, this is the first report of the association of ToLCKV with leaf curl disease of Z. elegans. The authors are thankful to the Vice Chancellor of Barkatullah University, Bhopal, India, for use of the facilities and the UGC, New Delhi, India for financial support under the UGC-BSR Research Start-Up grant.

A genetically novel avian Hepatitis E virus in China.

Hepatitis E virus (HEV) infection has a global distribution with diverse hosts, including mammals and avians. In this study, an avian Hepatitis E virus (aHEV) strain with a high mortality rate of about 30%, designated as SDXT20, was obtained from the liver of 30-week-old Hubbard chickens with severe hepatosplenomegaly in 2020 in Eastern China and HEV was proved to be the only pathogen by next-generation sequencing. Its complete genome, which encodes three open reading frames (ORFs), is 6649 nt in length. ORF1-3 encodes three proteins with lengths of 1532 aa, 606 aa, and 82 aa, respectively, and ORF2 and ORF3 overlap with each other. BLAST-based similarity analysis of the complete viral genome demonstrated that SDXT20 had merely 80.5-92.2% similarity with avian Avihepevirus magniiecur strains and 50.4%-54.8% lower similarity with Paslahepevirus balayani, Rocahepevirus ratti, and Chirohepevirus eptesici species. Further genetic evolution analysis of the complete genome and ORF2 revealed that the isolate was genetically distinct from known aHEVs, and it belonged to a novel genetically distinct aHEV. This study provides data for further analysis of the multi-host and cross-host genetic evolution of HEVs.

Genetic characteristics of archival noroviruses detected from the 1970s to the 1990s.

The genetic characterization of archival specimens is important for evaluating the evolutionary processes of noroviruses. Complete viral genome sequences, GVIII.1[GII.P28] and GIX.1[GII.P15], were determined from two archival specimens collected in Tokyo, Japan, in 1986 and 1995. In addition, complete VP1 and partial RdRp sequences of four samples collected between 1975 and 1983 were determined. Two viruses were classified as GI.5[P5] and GI.9[P9]; however, the viruses from the other two samples could not be assigned to any known genotypes using norovirus typing tools and phylogenetic analysis, suggesting that they might be untypable genotypes. Further evolutionary analysis of these viruses is warranted.

Elucidating the sweet potato virome in Greece with the aid of high‐throughput sequencing technology

AbstractIn 2018, typical symptoms of sweet potato virus disease (SPVD) such as purple ringspots, distortion and crinkling of the leaves appeared on a sweet potato crop located in southern Greece. High‐throughput sequencing (HTS) analysis revealed the presence of seven viruses, sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato virus C (SPVC), sweet potato virus G (SPVG), sweet potato virus 2 (SPV2), sweet potato leaf curl virus (SPLCV) and sweet potato pakakuy virus (SPPV). In silico analysis of the HTS data in combination with Sanger sequencing succeeded in obtaining the complete genome sequences of all seven viruses. Nucleotide sequence comparisons of complete viral genomes showed high similarity of all detected potyviruses and SPLCV with homologous sequences retrieved from GenΒank, whereas a new variant of SPCSV RNA1 was characterized and successfully transmitted onto Nicotiana benthamiana plants with the whitefly species Bemisia tabaci MED and MEAM1. Moreover, the Greek strain of SPPV showed higher similarity with SPPV strain B, even though it possesses a variable RT‐RNase H domain. Further phylogenetic analysis clustered Greek sweet potato virus isolates into various phylogenetic groups, whereas few new clades were formed in the cases of SPFMV, SPVG and SPCSV RNA1. Small‐scale surveys on three different sweet potato crops exhibiting typical, mild or no symptoms also indicated the close association of SPCSV with SPVD. Taken together, this study characterizes for the first time the virome of sweet potato crops in Greece and provides a glimpse into the evolutionary relationships of the identified viruses.

Comparative genomic analysis of five freshwater cyanophages and reference-guided metagenomic data mining

BackgroundAs important producers using photosynthesis on Earth, cyanobacteria contribute to the oxygenation of atmosphere and the primary production of biosphere. However, due to the eutrophication of urban waterbodies and global warming, uncontrollable growth of cyanobacteria usually leads to the seasonal outbreak of cyanobacterial blooms. Cyanophages, a group of viruses that specifically infect and lyse cyanobacteria, are considered as potential environment-friendly agents to control the harmful blooms. Compared to the marine counterparts, only a few freshwater cyanophages have been isolated and genome sequenced to date, largely limiting their characterizations and applications.ResultsHere, we isolated five freshwater cyanophages varying in tail morphology, termed Pam1~Pam5, all of which infect the cyanobacterium Pseudanabaena mucicola Chao 1806 that was isolated from the bloom-suffering Lake Chaohu in Anhui, China. The whole-genome sequencing showed that cyanophages Pam1~Pam5 all contain a dsDNA genome, varying in size from 36 to 142 Kb. Phylogenetic analyses suggested that Pam1~Pam5 possess different DNA packaging mechanisms and are evolutionarily distinct from each other. Notably, Pam1 and Pam5 have lysogeny-associated gene clusters, whereas Pam2 possesses 9 punctuated DNA segments identical to the CRISPR spacers in the host genome. Metagenomic data-based calculation of the relative abundance of Pam1~Pam5 at the Nanfei estuary towards the Lake Chaohu revealed that the short-tailed Pam1 and Pam5 account for the majority of the five cyanophages. Moreover, comparative analyses of the reference genomes of Pam1~Pam5 and previously reported cyanophages enabled us to identify three circular and seven linear contigs of virtual freshwater cyanophages from the metagenomic data of the Lake Chaohu.ConclusionsWe propose a high-throughput strategy to systematically identify cyanophages based on the currently available metagenomic data and the very limited reference genomes of experimentally isolated cyanophages. This strategy could be applied to mine the complete or partial genomes of unculturable bacteriophages and viruses. Transformation of the synthesized whole genomes of these virtual phages/viruses to proper hosts will enable the rescue of bona fide viral particles and eventually enrich the library of microorganisms that exist on Earth.4WYBiJEHr_CXoYqKn7ZoJMVideo abstract

Few Amino Acid Mutations in H6 Influenza A Virus From South American Lineage Increase Viral Replication Efficiency in Poultry.

In chickens, infections due to influenza A virus (IAV) can be mild to severe and lethal. The study of IAV infections in poultry has been mostly limited to strains from the North American and Eurasian lineages, whereas limited information exists on similar studies with strains from the South American lineage (SAm). To better evaluate the risk of introduction of a prototypical SAm IAV strain into poultry, chickens were infected with a wild-type SAm origin strain (WT557/H6N2). The resulting virus progeny was serially passaged in chickens 20 times, and the immunopathological effects of the last passage virus, 20Ch557/H6N2, in chickens were compared to those of the parental strain. A comparison of complete viral genome sequences indicated that the 20Ch557/H6N2 strain contained 13 amino acid differences compared to the wild-type strain. Five of these mutations are in functionally relevant regions of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, despite higher and more prolonged virus shedding in chickens inoculated with the 20Ch557/H6N2 strain compared to those that received the WT557/H6N2 strain, transmission to naïve chickens was not observed for either group. Analyses by flow cytometry of mononuclear cells and lymphocyte subpopulations from the lamina propria and intraepithelial lymphocytic cells (IELs) from the ileum revealed a significant increase in the percentages of CD3+TCRγδ+ IELs in chickens inoculated with the 20Ch557/H6N2 strain compared to those inoculated with the WT557/H6N2 strain.

Fenugreek plants showing the severe leaf curl disease are associated with tomato leaf curl Kerala virus, DNA-B molecule of tomato leaf curl New Delhi virus and a novel betasatellite

Fenugreek plants showing typical leaf curl disease symptoms were collected from Aurangabad, Maharashtra State, India. Begomovirus association with the five diseased in fenugreek was confirmed through PCR using begomovirus specific primers which resulted in expected amplicon of 1.2 kb product of virus genome. Analysis of the partial genome sequences obtained revealed that they have more than 95% nucleotide identity among themselves and showing similarity with begomoviruses reported earlier. Further, complete viral genome segments (DNA-A and DNA-B molecule) were amplified by RCA and DNA satellites by PCR were cloned and sequenced for one representative sample (F-TK). No PCR amplification resulted for alphasatellite specific primers in the infected fenugreek samples. Sequence analysis showed the nucleotide (nt) identity of 91.6—96.3 per cent for DNA-A and 90.2 to 91.9 per cent for DNA-B with several isolates of tomato leaf curl Kerala virus (ToLCKeV) and tomato leaf curl New Delhi virus (ToLCNDV) infecting tomato, respectively. Betasatellite showed maximum nt identity of 80.9 per cent with ToLCB infecting papaya will be considered as novel betasatellite as per ICTV classification criteria (less than 91% nt identity for complete DNAβ), for which we proposed the name, fenugreek leaf curl betasatellite. Recombination analysis of sequences of DNA-A, DNA-B and betasatellite of begomovirus associated with the fenugreek using RDP4 software indicated that they have are originated through recombination of begomoviruses infecting other crops. To the best of our knowledge this is the first report DNA-A of ToLCKeV, DNA-B of ToLCNDV and novel betasatellite associated with severe leaf curl disease of fenugreek from India.

Diversity of novel archaeal viruses infecting methanogens discovered through coupling of stable isotope probing and metagenomics.

Diversity of viruses infecting non-extremophilic archaea has been grossly understudied. This is particularly the case for viruses infecting methanogenic archaea, key players in the global carbon biogeochemical cycle. Only a dozen of methanogenic archaeal viruses have been isolated so far. In the present study, we implemented an original coupling between stable isotope probing and complementary shotgun metagenomic analyses to identify viruses of methanogens involved in the bioconversion of formate, which was used as the sole carbon source in batch anaerobic digestion microcosms. Under our experimental conditions, the microcosms were dominated by methanogens belonging to the order Methanobacteriales (Methanobacterium and Methanobrevibacter genera). Metagenomic analyses yielded several previously uncharacterized viral genomes, including a complete genome of a head-tailed virus (class Caudoviricetes, proposed family Speroviridae, Methanobacterium host) and several near-complete genomes of spindle-shaped viruses. The two groups of viruses are predicted to infect methanogens of the Methanobacterium and Methanosarcina genera and represent two new virus families. The metagenomics results are in good agreement with the electron microscopy observations, which revealed the dominance of head-tailed virus-like particles and the presence of spindle-shaped particles. The present study significantly expands the knowledge on the viral diversity of viruses of methanogens.

Complete sequence and genome characterization of a new potexvirus isolated from Chaenostoma cordatum.

Leaves from the ornamental plant Chaenostoma cordatum (Thunb.) Benth. expressing virus-like symptoms were collected for pathogen testing. A virus with features consistent with those of members of the genus Potexvirus was identified by high-throughput sequencing. The genome sequence was confirmed and completed using RT-PCR, cloning, rapid amplification of cDNA ends kits, and Sanger sequencing, revealing a complete viral genome of 6,071 nucleotides, excluding the poly-A tail. Phylogenetic analysis of the RNA-dependent RNA polymerase sequence from the viral genome indicated that its closest relative is Plantago asiatica mosaic virus. Further analysis of the nucleotide and amino acid sequences revealed that it had diverged enough from other potexviruses to be considered a member of a new species.

Untangling an AGS Outbreak Caused by the Recombinant GII.12[P16] Norovirus With Nanopore Sequencing.

For a rapidly spreading virus such as NoV (norovirus), pathogen identification, genotype classification, and transmission tracing are urgent for epidemic control. Here, we applied the Nanopore metatranscriptomic sequencing to determine the causative pathogen of a community AGS (Acute gastroenteritis) outbreak. The results were also confirmed by RT-PCR. The NGS (Next Generation Sequencing) library was constructed within 8 hours and sequence analyses were carried out in real-time. NoV positive reads were detected in 13 of 17 collected samples, including two water samples from sewage treatment tank and cistern. A nearly complete viral genome and other genome fragments could be generated from metatranscriptomic sequencing of 13 samples. The NoV sequences from water samples and cases are identical suggesting the potential source of the outbreak. The sequencing results also indicated the outbreak was likely caused by an emerging recombinant GII.12[P16] virus, which was only identified in the United States and Canada in 2017–2018. This is the first report of this emerging variant in mainland China, following the large outbreaks caused by the recombinant GII.17[P17] and GII.2[P16] in 2014 and 2016, respectively. Closely monitoring of the prevalence of this recombinant strain is required. Our data also highlighted the importance of real-time sequencing in emerging pathogens’ surveillance.

Complete Genome Sequence of a Novel Monopartite Mastrevirus, Soybean Geminivirus B, Isolated from Soybean (Glycine max (L.) Merrill).

Soybean is one of the most important crops in Korea. To identify the viruses infecting soybean, we conducted RNA sequencing with samples displaying symptoms of viral disease. A contig displaying sequence similarity to the known Geminivirus was identified. A polymerase chain reaction (PCR) using two different pairs of back-to-back primers and rolling circle amplification (RCA) confirmed the complete genome of a novel virus named soybean geminivirus B (SGVB), consisting of a circular monopartite DNA genome measuring 2616 nucleotides (nt) in length. SGVB contains four open reading frames (ORFs) and three intergenic regions (IRs). IR1 includes a nonanucleotide origin of replication in the stem-loop structure. Phylogenetic and BLAST analyses demonstrated that SGVB could be a novel virus belonging to the genus Mastrevirus in the family Geminiviridae. We generated infectious clones for SGVB by adding a copy of the IR1 region of SGVB, comparing the V-ori in addition to the full-length genome of SGVB. Using the infectious clones, we observed chlorosis and leaf curling with a latent infection in the inoculated Nicotiana benthamiana plants, while none of the inoculated soybean plants showed any visible symptoms of disease. This study provides the complete genome sequence and infectious clones of a novel Mastrevirus referred to as SGVB from soybean in Korea.

Genome stability of the vaccine strain VACΔ6.

Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, f ive virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and f ive intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certif ied for vaccine production in accordance with the “Guidelines for Clinical Trials of Medicinal Products”. PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells.

Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology.

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution.

Molecular Characterization of a Novel Polerovirus Infecting Soybean in China.

Poleroviruses are positive-sense, single-stranded viruses. In this study, we describe the identification of a novel polerovirus isolated from soybean displaying curled leaves. The complete viral genome sequence was identified using high-throughput sequencing and confirmed using rapid amplification of cDNA ends (RACE), RT-PCR and Sanger sequencing. Its genome organization is typical of the members of genus Polerovirus, containing seven putative open reading frames (ORFs). The full genome is composed of single-stranded RNA of 5822 nucleotides in length, with the highest nucleotide sequence identity (79.07% with 63% coverage) for cowpea polerovirus 2 (CPPV2). Amino acid sequence identities of the protein products between the virus and its relatives are below the threshold determined by the International Committee of Taxonomy of Viruses (ICTV) for species demarcation, and this strongly supports this virus’ status as a novel species, for which the name soybean chlorotic leafroll virus (SbCLRV) is proposed. Recombination analysis identified a recombination event in the ORF5 of the 3’ portion in the genome. Phylogenetic analyses of the genome and encoded protein sequences revealed that the new virus is closely related to phasey bean mild yellows virus, CPPV2 and siratro latent polerovirus. Subsequently, we demonstrated the infectivity of SbCLRV in Nicotiana benthamiana via infectious cDNA clone generation and agroinoculation.

Uncovering 1058 Novel Human Enteric DNA Viruses Through Deep Long-Read Third-Generation Sequencing and Their Clinical Impact

Lack of viral reference genomes poses a challenge to virome study. We investigated human gut virome and its clinical implication by ultra-deep metagenomic sequencing. We extracted sufficient viral DNA from human feces for ultra-deep PacBio sequencing (>10 μg) and Illumina sequencing (>1 μg). Upon de novo assembly and 6 stages of strict filtering, viral genomes were generated and validated in 3 cohorts of 2819 published fecal metagenomes. Diagnostic performance of assembled viruses for colorectal cancer were tested in a training cohort and 2 independent validation cohorts. Virus mapping ratio, evolutionary history, and virus status (lytic or temperate) were also examined. The mean amount of extracted viral DNA increased by 14-fold compared with previous protocols. We obtained PacBio long reads and Illumina short reads with 290-fold higher depth than previous studies. We assembled and validated 1178 contigs as complete viral genomes, of which 1058 were newly identified. Thirteen viral genomes (398-839 kb) that are longer than the largest bacteriophage found in humans (393 kb) were discovered. Phylogenetic tree was constructed based on Hidden Markov Models alignment scores of 4 conserved viral proteins. Incorporating our assembled genomes into the National Center for Biotechnology Information database improved the mapping ratio of published metagenomes ≤18 times. Lytic viruses (75.9% ± 12.2% of total) were predominantly present in our sample. A biomarker panel of 14 novel viruses could discriminate patients with colorectal cancer from controls with an area under the receiver operating characteristics curve of 0.87 in the training cohort, which was validated with areas under the receiver operating characteristics curve of 0.85 and 0.73 in 2 independent cohorts. We uncovered 1058 novel human gut viruses. These findings can contribute to clinical diagnosis, current viral reference genome, and future virome investigation.

Recombination and darwinian selection as drivers of genetic diversity and evolution of sweet potato leaf curl viruses in Tanzania

Using High-throughput Sequencing (HTS) and Sanger sequencing techniques, we detected sweet potato leaf curl viruses (SPLCVs; Genus Begomovirus) and determined their genetic diversities, recombination, and evolutionary selection pressure imposed on their genes. Eleven complete (2767–2824 nucleotides) and fifty partial (V1 and C1 proteins) sequences of SPLCVs were generated, and one complete genome of sweet potato pakakuyu virus was also generated. The detected SPLCVs were genetically diverse, with complete nucleotide sequence identities ranging from 78.7 to 100%. These sequences were closely related (>89% identical) to the sweet potato leaf curl virus, the sweet potato Sao Paulo virus, and the sweet potato leaf curl Uganda virus. The diversity of SPLCVs indicated the occurrence of their strains and variants in Tanzania. Intra- and interspecies recombination events were detected in four and two complete and partial sequences of SPLCVs, respectively. The genes of SPLCVs, except the C4 gene, are evolving under purifying selection pressure. In the C4 gene, numerous codon sites are evolving under positive selection pressure. High genetic diversity and diversifying selection imply an increased chance of SPLCVs to interact and adapt to diverse sweet potato genotypes and their wild relatives in Tanzania.

Characteristics of 24 SARS-CoV-2-Sequenced Reinfection Cases in a Tertiary Hospital in Spain.

BackgroundSince the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the main concern is whether reinfections are possible, and which are the associated risk factors. This study aims to describe the clinical and molecular characteristics of 24 sequence-confirmed reinfection SARS-CoV-2 cases over 1 year in Barcelona (Catalonia, Spain).MethodsPatients with > 45 days between two positive PCR tests regardless of symptoms and negative tests between episodes were initially considered as suspected reinfection cases from November 2020 to May 2021. Whole-genome sequencing (WGS) was performed to confirm genetic differences between consensus sequences and for phylogenetic studies based on PANGOLIN nomenclature. Reinfections were confirmed by the number of mutations, change in lineage, or epidemiological criteria.ResultsFrom 39 reported suspected reinfection cases, complete viral genomes could be sequenced from both episodes of 24 patients, all were confirmed as true reinfections. With a median age of 44 years (interquartile range [IQR] 32–65), 66% were women and 58% were healthcare workers (HCWs). The median days between episodes were 122 (IQR 72–199), occurring one-third within 3 months. Reinfection episodes were frequently asymptomatic and less severe than primary infections. The absence of seroconversion was associated with symptomatic reinfections. Only one case was reinfected with a variant of concern (VOC).ConclusionSevere acute respiratory syndrome coronavirus 2 reinfections can occur in a shorter time than previously reported and are mainly found in immunocompetent patients. Surveillance through WGS is useful to identify viral mutations associated with immune evasion.

The Genetic Characterization of a Novel Natural Recombinant Pseudorabies Virus in China.

We sequenced the complete genome of the pseudorabies virus (PRV) FJ epidemic strain, and we studied the characteristics and the differences compared with the classical Chinese strain and that of other countries. Third-generation sequencing and second-generation sequencing technology were used to construct, sequence, and annotate an efficient, accurate PRV library. The complete FJ genome was 143,703 bp, the G+C content was 73.67%, and it encoded a total of 70 genes. The genetic evolution of the complete genome and some key gene sequences of the FJ strain and PRV reference strains were analyzed by the maximum likelihood (ML) method of MEGA 7.0 software. According to the ML tree based on the full-length genome sequences, PRV FJ strain was assigned to the branch of genotype II, and it showed a close evolutionary relationship with PRV epidemic variants isolated in China after 2011. The gB, gC, gD, gH, gL, gM, gN, TK, gI, and PK genes of the FJ strain were assigned to the same branch with other Chinese epidemic mutants; its gG gene was assigned to the same branch with the classic Chinese Fa and Ea strains; and its gE gene was assigned to a relatively independent branch. Potential recombination events were predicted by the RDP4 software, which showed that the predicted recombination sites were between 1694 and 1936 bp, 101,113 and 102,660 bp, and 107,964 and 111,481 bp in the non-coding region. This result broke the previously reported general rule that pseudorabies virus recombination events occur in the gene coding region. The major backbone strain of the recombination event was HLJ8 and the minor backbone strain was Ea. Our results allowed us to track and to grasp the recent molecular epidemiological changes of PRV. They also provide background materials for the development of new PRV vaccines, and they lay a foundation for further study of PRV.