RdRp Region Research Articles

Molecular Evolutionary Analyses of the RNA-Dependent RNA Polymerase (RdRp) Region and VP1 Gene in Sapovirus GI.1 and GI.2

Human sapovirus (HuSaV) is a significant cause of gastroenteritis. This study aims to analyze the evolutionary dynamics of the RNA-dependent RNA polymerase (RdRp) and capsid (VP1) genes of the HuSaV GI.1 and GI.2 genotypes between 1976 and 2020. Using bioinformatics tools such as the Bayesian phylogenetics software BEAST 2 package (v.2.7.6), we constructed time-scale evolutionary trees based on the gene sequences. Most of the recent common ancestors (MRCAs) of the RdRp region and VP1 gene in the present HuSaV GI.1 diverged around 1930 and 1933, respectively. The trees of the HuSaV GI.1 RdRp region and VP1 gene were divided into two clusters. Further, the MRCAs of the RdRp region and VP1 gene in HuSaV GI.2 diverged in 1960 and 1943, respectively. The evolutionary rates were higher for VP1 gene in HuSaV GI.1 than that in HuSaV GI.2, furthermore, were higher in GI.1 Cluster B than GI.1 Cluster A. In addition, a steep increase was observed in the time-scaled genome population size of the HuSaV GI.1 Cluster B. These results indicate that the HuSaV GI.1 Cluster B may be evolving more actively than other genotypes. The conformational B-cell epitopes were predicted with a higher probability in RdRp for GI.1 and in VP1 for GI.2, respectively. These results suggest that the RdRp region and VP1 gene in HuSaV GI.1 and GI.2 evolved uniquely. These findings suggest unique evolutionary patterns in the RdRp region and VP1 gene of HuSaV GI.1 and GI.2, emphasizing the need for a ‘One Health’ approach to better understand and combat this pathogen.

Discovery of novel 'sugarcane totivirus 1' from Saccharum officinarum by high-throughput sequencing.

Sugarcane is cultivated globally and affected by more than 125 pathogens, which lead to various plant diseases. In recent years, high-throughput sequencing (HTS)-based genome analyses have been broadly adopted for the discovery of both characterized and un-characterized viruses from plant samples. In this study, the HTS data of sugarcane pooled sample retrieved from sequence read archive (SRA) were de novo re-assembled using CLC Genomic Workbench. The genomic sequence of a novel dsRNA totivirus, 5,384 nucleotides (nt) long, excluding the 5' untranslated region (UTR) and 3' UTR, was discovered and named sugarcane totivirus 1 (STV 1). The genome contains two open reading frames (ORFs): a putative coat protein (CP) encoding 866 amino acids (aa) and RNA-dependent RNA polymerase (RdRp) encoding 824 aa. Phylogenetic studies based on the genomic sequences (nt), and the aa sequences of CP as well as RdRp regions revealed that STV 1 is closely related to other members of the genus Totivirus. Pairwise sequence identity of CP and RdRp aa sequences showed 30.0-51.5% and 26.3-47.2% similarity, respectively with other members of the family Totiviridae. The HTS results were further validated and confirmed through OneStep RT-PCR assay and Sanger sequencing. A novel totivirus (STV 1) in the genus Totivirus, family Totiviridae has been identified. This is the first report of dsRNA totivirus STV 1 associated with sugarcane from India.

Molecular Characterization and Phylogenetic Analysis of Honeybee (Apis mellifera) Mite-Borne Pathogen DWV-A and DWV-B Isolated from Lithuania.

Deformed wing virus (DWV) is known as one of the main viruses that affect honeybees' health all around the world. The virus has two widespread genotypes, DWV-A and DWV-B (VDV-1), transmitted mainly by V. destructor mites. In this study, we collected honeycombs with covered broods from 73 apiaries in eight Lithuanian regions and initially investigated the prevalence of V. destructor mites. Mites were collected from May to the end of July in 2021 from 124 hives. The prevalence of V. destructor infestations in beehives reached 30% and 63% in investigated apiaries. The presence of DWV-A and DWV-B pathogens in mites and broods was examined by RT-qPCR targeting the CRPV-capsid region. The molecular characterization of the virus in mite samples was based on sequence analysis of the RNA-dependent RNA polymerase (RdRp) region. In addition, leader polypeptide (LP), structural protein (Vp3), Helicase, and RdRp genes were used for phylogenetic characterization of dual infection. The prevalences of DWV-B in mites and broods were 56.5% and 31.5%, respectively, while DWV-A was detected in 12.9% of mite samples and 24.7% of brood samples. Some of the examined mite samples harboured dual virus infections. Our findings showed that bee colonies from the same apiary were not always infected by the same viruses. Some bee colonies were virus-free, while others were highly infected. Phylogenetic analysis of 21 sequences demonstrated the presence of highly variable DWV-B and DWV-A genotypes in Lithuania and possible recombinant variants of the virus. This study represents the first molecular characterization of mite-borne pathogens hosted by honeybees (Apis mellifera) in Lithuania.

Quantitative detection of cassava common mosaic virus for health certification of cassava (Manihot esculenta Crantz) germplasm using qPCR analysis

Cassava (Manihot esculenta Crantz) is a crop of global economic and food safety importance, used for human consumption and in various industrial applications. The genebank of the Genetic Resources Program of the Alliance of Bioversity International and CIAT currently holds the world's largest cassava collection, with 5965 in vitro accessions from 28 countries. Managing this extensive collection involves indexing quarantine pathogens as a phytosanitary certification requirement for safely distributing cassava germplasm. The study therefore aimed to optimize a quantitative diagnostic protocol to detect cassava common mosaic virus (CsCMV) using quantitative PCR (qPCR) as a better alternative to other molecular techniques. This was done through designing primers and a probe in the RdRP region of CsCMV, and optimizing the qPCR conditions of the diagnostic protocol using primer concentration assays, and reaction amplification conditions such as volume and reaction time. We also evaluated the qPCR protocol by comparing the results of 140 cassava accession evaluations using three diagnostic methodologies (DAS-ELISA, end-point PCR, and qPCR) for CsCMV. Our protocol established that qPCR technique analysis is ten-times more sensitive in detecting CsCMV compared to end-point PCR, showing a maximum detection level of 77.97 copies/μL of plasmid, with 76 min of reaction time. The comparison allowed us to verify the level of CsCMV detection through the techniques evaluated, concluding that qPCR was more sensitive and allowed the quantification of viral concentration. The optimized qPCR protocol will be used to accelerate diagnostic screening of cassava germplasm for the presence or absence of CsCMV to ensure safe movement and distribution of disease-free germplasm.

Genomic variation in pepper vein yellows viruses in Australia, including a new putative variant, PeVYV-10.

Since the first identification and full sequence of the polerovirus pepper vein yellows virus in Australia in 2016, virus surveys of crops and weeds have sporadically identified PeVYV in different hosts and locations. Genomic comparisons of 14 PeVYV-like isolates using RT-PCR products spanning the 3' end of the RdRp region (ORF 2), the intergenic region, ORF 3a, ORF 4, and ORF 3 (1388 nt) showed that four of the PeVYV isolates might be a new variant or PeVYV-like virus. From six PeVYV-positive plants, eight PeVYV-like sequences were obtained by high-throughput sequencing, as two hosts, 5352 and 5634, contained two slightly different PeVYV-like isolates. Three of the PeVYV-like isolates were most closely related to PeVYV-6 and PeVYV-5, and two isolates were closely related to PeVYV-9 and PeVYV-2. The other three isolates shared only 69-74% nucleotide sequence identity across the whole genome with any of the other PeVYVs, despite sharing 73-98%, 87-91%, and 84-87% amino acid sequence identity in ORF 3a, ORF 3, and the RdRp (ORF 2), respectively, suggesting that this virus is a new PeVYV-like virus, which we have tentatively called PeVYV-10. This is also the first report of a PeVYV-like virus infecting garlic.

Norovirus Epidemiology and Genotype Circulation during the COVID-19 Pandemic in Brazil, 2019-2022.

Norovirus stands out as a leading cause of acute gastroenteritis (AGE) worldwide, affecting all age groups. In the present study, we investigated fecal samples from medically attended AGE patients received from nine Brazilian states, from 2019 to 2022, including the COVID-19 pandemic period. Norovirus GI and GII were detected and quantified using RT-qPCR, and norovirus-positive samples underwent genotyping through sequencing the ORF1/2 junction region. During the four-year period, norovirus prevalence was 37.2%, varying from 20.1% in 2020 to 55.4% in 2021. GII genotypes dominated, being detected in 92.9% of samples. GII-infected patients had significantly higher viral concentrations compared to GI-infected patients (median of 3.8 × 107 GC/g and 6.7 × 105 GC/g, respectively); and patients aged >12-24 months showed a higher median viral load (8 × 107 GC/g) compared to other age groups. Norovirus sequencing revealed 20 genotypes by phylogenetic analysis of RdRp and VP1 partial regions. GII.4 Sydney[P16] was the dominant genotype (57.3%), especially in 2019 and 2021, followed by GII.2[P16] (14.8%) and GII.6[P7] (6.3%). The intergenogroup recombinant genotype, GIX.1[GII.P15], was detected in five samples. Our study is the first to explore norovirus epidemiology and genotype distribution in Brazil during COVID-19, and contributes to understanding the epidemiological dynamics of norovirus and highlighting the importance of continuing to follow norovirus surveillance programs in Brazil.

Molecular epidemiology and genetic diversity of norovirus among hospitalized patients with acute gastroenteritis in Shandong, China, 2016-2018.

Norovirus (NoV) infection is a leading cause of acute gastroenteritis (AGE) for people of all ages. Here, we reported the molecular epidemiology and genetic diversity of NoVs among hospitalized patients with AGE between 2016 and 2018 in Shandong Province, China. Two thousand sixty-nine AGE patients from sentinel hospitals were enrolled. The stool samples were collected and tested for NoVs by real-time RT-PCR. The RNA-dependent RNA polymerase (RdRp) and capsid gene of 163 strains were amplified and sequenced for genotyping. Phylogenetic analyses and genomic characterization were conducted with the VP1 and RdRp region of the full genome sequences. Four hundred seventy two(21.76%) samples were NoV-positive. The positive rate in 2016 was higher than those of 2017 and 2018. We observed diverse NoV genotypes. GII.2[P16] emerged in January 2017 and became the dominant genotype between May and June 2017. Phylogenetic analyses showed that our GII.2[P16] genomes clustered in the SC1 in VP1 region, while they belonged to the Emerging GⅡ.P16 (2015-2017) clade in RdRp region. Our GⅡ.4 strains displayed two amino acid mutations, positions R297H and D372N, in epitope A of the VP1 region. Our study highlighted that NoV is an important pathogen of viral AGE in Shandong and, therefore, it is necessary to strengthen its surveillance.

Evidence for the natural infection of cucurbit chlorotic yellows virus (CCYV) in lettuce plants from India.

In the years 2021 and 2022, lettuce plants showing blistering, chlorosis, mosaic, rosetting/ excess proliferation, and stunting symptoms were subjected to leaf-dip transmission electron microscopy, RT-PCR followed by sequence analysis and bio-assay to unfold the identity ofassociated virus(es). The association of longfilamentous virions (~ 850nm in length) as seen through leaf-dip transmission electron microscopy suggested the possible infection by a potyvirus or crinivirus, either singly or in combination. RT-PCR assays using generic primers targeting the RdRp region of criniviruses and the NIb region of potyviruses revealed the association of both a crinivirus as well as a potyvirus. The gel-purified RT-PCR products derived from the RdRp region of criniviruses upon cloning, sequencing, and NCBI BLAST analysis indicated the associated crinivirus as cucurbit chlorotic yellows virus (CCYV). Further, RT-PCR assays using specific primers targeting CP and CP minor genes of CCYV followed by cloning and sequencing confirmed its association with the diseased lettuce plants. Besides, the bioassay based on whitefly-mediated virus transmission followed by RT-PCR confirmed the infectivity of CCYV from diseased to healthy lettuce plants. The results of this study confirmed the natural infection of CCYV in lettuce host for the first time in the world indicating its distribution across the crop families.

Phylogenetic Relationship of Cymbidium Mosaic Virus from the Native Orchids of South Kalimantan, Indonesia

Information on viral genetics, including their phylogenetic relationship, is valuable in controlling viral infection and screening for the development of virus-resistant cultivars in the future. The objectives of this study were to detect and characterize the Cymbidium mosaic virus (CymMV) from the native orchids of South Kalimantan, Indonesia, by the RT-PCR method. Also, to determine their phylogenetic relationship based on a partial genome of RdRp by the ML and PCA methods. Following RT-PCR analysis, one of 10 samples of native orchids used was positively infected by CymMV. In early detection, the RdRp region of CymMV has approximately 530 bp in size. After being sequenced and aligned with other isolates, this region has 121 polymorphic or mutation sites, a GC content of 45.21%, a transition/transversion bias value of 3.52, and nucleotide diversity (0.0415). The phylogenetic analysis revealed that CymMV from South Kalimantan, Indonesia, has closest related to similar isolates from Korea Type 2 (AF016914.1), Niigata, Japan (AB197937.1), Hawaii (EF125180.1), and Taiwan M2 (EU314803.1), with the coefficient divergence of 0.025. But, it has very distantly related to Hawaii 18-1 (EF125178.1) with a coefficient of 0.142. The results provide urgent information in supporting the native orchid's conservation and breeding efforts, locally and globally, including mitigating or controlling the viral infection and screening for the development of virus-free or resistant cultivars in the future.

Metagenomic Analysis of Environmental Samples from Wildlife Rescue Station at Poyang Lake, China

ObjectiveTo improve the understanding of the virome and bacterial microbiome in the wildlife rescue station of Poyang Lake, China. MethodsTen smear samples were collected in March 2019 Metagenomic sequencing was performed to delineate bacterial and viral diversity. Taxonomic analysis was performed using the Kraken2 and Bracken methods. A maximum-likelihood tree was constructed based on the RNA-dependent RNA polymerase (RdRp) region of picornavirus. ResultsWe identified 363 bacterial and 6 viral families. A significant difference in microbial and viral abundance was found between samples S01–S09 and S10. In S01–S09, members of Flavobacteriia and Gammaproteobacteria were the most prevalent, while in S10, the most prevalent bacteria class was Actinomycetia. Among S01–S09, members of Myoviridae and Herelleviridae were the most prevalent, while the dominant virus family of S10 was Picornaviridae. The full genome of the pigeon mesivirus-like virus (NC-BM-233) was recovered from S10 and contained an open reading frame of 8,124 nt. It showed the best hit to the pigeon mesivirus 2 polyprotein, with 84.10% amino acid identity. Phylogenetic analysis showed that RdRp clustered into Megrivirus B. ConclusionThis study provides an initial assessment of the bacteria and viruses in the cage-smeared samples, broadens our knowledge of viral and bacterial diversity, and is a way to discover potential pathogens in wild birds

Molecular Evolutionary Analyses of the RNA-Dependent RNA Polymerase (RdRp) Region and VP1 Gene in Human Norovirus Genotypes GII.P6-GII.6 and GII.P7-GII.6.

To understand the evolution of GII.P6-GII.6 and GII.P7-GII.6 strains, the prevalent human norovirus genotypes, we analysed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than for the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains.

Comparative Analysis of the Effect of Different Nucleic Acid Extraction Methods on SARS-CoV-2 Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction Results

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of Coronavirus diseases-2019 (COVID-19) disease. Rapid and accurate detection of the virus is vital to prevent transmission and effectively manage the pandemic. The gold standard diagnostic method for this agent is the real-time reverse transcription polymerase chain reaction (qrRT-PCR) test conducted on respiratory tract samples and one of the most critical steps affecting the sensitivity of this test is the nucleic acid extraction stage. However, restrictive factors such as reagent supply and storage conditions limit the testing capacity. Therefore, innovative and cost-effective alternatives are needed to speed up testing and minimize pre-processing steps. The aim of this study was to evaluate the impact and applicability of different methods to enhance the efficiency of the nucleic acid extraction stage in the SARS-CoV-2 qrRT-PCR test. As an alternative to the routinely used viral nucleic acid extraction buffer (vNAT), the modified vNAT method (MvNAT), which includes centrifugation, the R1-R2 kit and the heat treatment (HT) method, was applied to 118 respiratory tract samples. Samples determined with threshold cycle values of (Cq) of ≤ 35 (n= 10), > 35 (n= 42), indeterminate (n= 56) in routine results and negative controls (n= 10) were included in the study. The RNA quantities obtained after extraction for each method were measured and recorded using a spectrophotometric measurement device. All samples were processed using the SARS-CoV-2 qrRT-PCR kit targeting the RdRp region. The results were statistically analyzed using unpaired and paired t-tests and results with a p-value of < 0.05 were considered statistically significant. Excluding negative control samples, while the standard method yielded a Cq value of 48.1% (mean Cq value (Cqmean)= 39.5 ± 6.9) for a total of 108 samples, the MvNAT method produced a Cq value of 11.1% (Cqmean= 38.4 ± 5.2), the R1-R2 kit yielded 14.8% (Cqmean= 35.9 ± 7.1) and HT method resulted in 25% (Cqmean= 31.4 ± 6.3). When the variability in target gene Cq values was analyzed in all samples compared to the standard method, the HT method significantly provided lower Cq values (n= 16; p= 0.007; paired t-test) while the MvNAT method and R1-R2 kit yielded higher Cq values (n= 6; p= 0.025, n= 11; p= 0.004; paired t-test). Sensitivity rates were MvNAT= 31.6%, R1-R2= 57.9%, HT= 84.2%, with 100% specificity for all three methods. The HT method demonstrated a positive extraction efficiency because it is fast, easy and not dependent on reagents. Although this method provided lower Cq values than the standard method, especially in samples with a high viral load, it should be considered that it also has the potential to yield false-negative results in samples with Cq> 35. With this study, it was concluded that the extraction phase of the SARS-CoV-2 qrRT-PCR test can be carried out using various methods that do not require kits or reagents, such as the HT method. However, it is believed that multicenter studies involving a larger number of samples are necessary to standardize the test and assess the possibility of false negatives.

Coronaviruses Are Abundant and Genetically Diverse in West and Central African Bats, including Viruses Closely Related to Human Coronaviruses.

Bats are at the origin of human coronaviruses, either directly or via an intermediate host. We tested swabs from 4597 bats (897 from the Democratic Republic of Congo (DRC), 2191 from Cameroon and 1509 from Guinea) with a broadly reactive PCR in the RdRp region. Coronaviruses were detected in 903 (19.6%) bats and in all species, with more than 25 individuals tested. The highest prevalence was observed in Eidolon helvum (239/733; 39.9%) and Rhinolophus sp. (306/899; 34.1%), followed by Hipposideros sp. (61/291; 20.9%). Frugivorous bats were predominantly infected with beta coronaviruses from the Nobecovirus subgenus (93.8%), in which at least 6 species/genus-specific subclades were observed. In contrast, insectivorous bats were infected with beta-coronaviruses from different subgenera (Nobecovirus (8.5%), Hibecovirus (32.8%), Merbecovirus (0.5%) and Sarbecovirus (57.6%)) and with a high diversity of alpha-coronaviruses. Overall, our study shows a high prevalence and genetic diversity of coronaviruses in bats and illustrates that Rhinolophus bats in Africa are infected at high levels with the Sarbecovirus subgenus, to which SARS-CoV-2 belongs. It is important to characterize in more detail the different coronavirus lineages from bats for their potential to infect human cells, their evolution and to study frequency and modes of contact between humans and bats in Africa.

Identification of grapevine virus G and grapevine virus H in Sardinia, Italy

Grapevine virus G (GVG) and grapevine virus H (GVH) (genus Vitivirus) are recently discovered viruses. Analysis of 38 samples from Sardinian grapevine cultivars for the presence of GVG and GVH was carried out using RT-PCR. All samples were also tested for grapevine Pinot gris virus (GPGV) using RT-PCR, and for grapevine leafroll virus -1, -2 and -3, grapevine virus A (GVA) and B (GVB), arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV) and grapevine fleck virus (GFkV) using multiplex RT-PCR. GVG was confirmed in four vines, and GVH was detected in only one sample. In phylogenetic analyses of the coat protein (CP) region, the Sardinian GVG isolates clustered separately from isolates from Croatia and New Zealand. The Sardinian GVH isolate clustered with most sequences from other countries, but with greater affinity to isolates from California (USA) for the CP region, whereas it clustered with isolates from Croatia in the RNA-dependent RNA polymerase (RdRp) region. In addition to GVG and GVH, many samples were coinfected with GVA, viruses from the leafroll complex, and GPGV. This is the first record of GVG and GVH occurring in Italy.

Improvement of nested reverse transcription-polymerase chain reaction (RT-PCR) with high specificity and sensitivity detection of sapovirus in food matrix

Sapovirus (SaV) is a causative agent of human gastroenteritis in both community outbreaks and sporadic cases worldwide. Shellfish accumulate a variety of pathogens during filter feeding. In particular, the contamination of shellfish by SaV has caused several outbreaks. As reported previously, nested RT-PCR (nRT-PCR) has been widely used in clinical samples, but has not proven suitable for food samples, such as oysters. This study aimed to identify a primer set for the detection of SaV with high specificity and sensitivity in food samples. To accomplish this, primers were improved in RNA-dependent RNA polymerase (RdRp) regions of SaV whole genome sequences. The sensitivity of the improved nRT-PCR was 100–1000 times higher than that of previous nRT-PCR and > 10 times higher than that of the previous real-time RT-PCR assay. Notably, cross-reaction with other viruses or food matrices was not observed by the specificity test. This study improved the reliable primer set to detect SaV in various food matrices with high sensitivity.

Evaluation of False Negative Among SARS-COV-2 Patients with Negative Real-time PCR Result Using Nested-RT PCR

Background: Fast and precise detection of SARS-CoV-2 RNA in clinical samples and subsequent quarantine are two critical factors in preventing virus transmission and distribution through the community. The false-negative result is a major problem in the SARS-CoV-2 detection because of the kind of sample (swab sample), sampling error, and sensitivity of PCR test, which can be reduced by a much more sensitive test such as nested PCR. Objectives: This study aimed to evaluate the false-negative rate among samples that were negative by a real-time PCR test using RT-nested PCR. Methods: One hundred eighty-four negative samples were included in the study, and nucleic acid was extracted using a commercial kit based on a silica filter column and then subjected to RT-nested PCR using three sets of primers targeting Orf1ab, N, and RdRp regions. Results: Among 184 negative swab samples for SARS-CoV-2, 27 (14.6%) cases were positive for the Orf1ab gene using RT-nested PCR. The samples were tested using N and RdRp primer sets. Also, seven (3.8%) cases were positive for the N gene, and four (2.1%) cases were positive for the RdRp gene. Conclusions: The results indicated that RT-nested PCR could be more sensitive than real-time PCR and reduce the false-negative rate.

Identification and genetic characterization of a minor norovirus genotype, GIX.1[GII.P15], from China

BackgroundHuman noroviruses, single-stranded RNA viruses in the family Caliciviridae, are a leading cause of nonbacterial acute gastroenteritis in people of all ages worldwide. Despite three decades of genomic sequencing and epidemiological norovirus studies, full-length genome analyses of the non-epidemic or minor norovirus genotypes are rare and genomic regions other than ORF2 and 3′-end of ORF1 have been largely understudied, which hampers a better understanding of the evolutionary mechanisms of emergence of new strains. In this study, we detected a rare norovirus genotype, GIX.1[GII.P15], in a vomit sample of a 60 year old woman with acute gastroenteritis using Raji cells and sequenced the complete genome.ResultsUsing electron microscopy, a morphology of spherical and lace-like appearance of norovirus virus particles with a diameter of approximately 30 nm were observed. Phylogenetic analysis of VP1 and the RdRp region indicated that the KMN1 strain could be genotyped as GIX.1[GII.P15]. In addition, the VP1 region of KMN1 strain had 94.15% ± 3.54% percent nucleotide identity (PNI) compared to 26 genomic sequences available in GenBank, indicating a higher degree similarity between KMN1 and other GIX.1[GII.P15] strains. Further analysis of the full genome sequence of KMN1 strain showed that a total of 96 nucleotide substitutions (63 in ORF1, 25 in ORF2, and 8 in ORF3) were found across the genome compared with the consensus sequence of GIX.1[GII.P15] genome, and 6 substitutions caused amino acid changes (4 in ORF1, 1 in ORF2, and 1 in ORF3). However, only one nucleotide substitution results in the amino acid change (P302S) in the VP1 protein and the site was located near one of the predicted conformational B epitopes on the dimer structure.ConclusionsThe genomic information of the new GIX.1[GII.P15] strain KMN1, which was identified in Kunming, China could provide helpful insights for the study of the genetic evolution of the virus.

Complete genome analysis of a novel iflavirus from the spotted lanternfly Lycorma delicatula.

The spotted lanternfly (Lycorma delicatula) is an invasive pest that causes serious economic losses in fruit and wood production. Here, we identified a novel iflavirus named "Lycorma delicatula iflavirus 1" (LDIV1), in a spotted lanternfly. The full genome sequence of LDIV1 is 10,222 nt in length and encodes a polyprotein containing a picornavirus capsid-protein-domain-like domain, a cricket paralysis virus capsid superfamily domain, an RNA helicase domain, a peptidase C3 superfamily domain, and an RNA-dependent RNA polymerase (RdRp) domain. LDIV1 replicates in the host insect and activates small interfering RNA (siRNA)-based host antiviral immunity. Phylogenetic analysis demonstrated that LDIV1 is most closely related to an unspecified member of the order Picornavirales, with 61.7% sequence identity in the RdRp region and 57.6% sequence identity in the coat protein region, and thus meets the demarcation criteria for new species in the genus Iflavirus. To the best of our knowledge, LDIV1 is the first virus discovered in L. delicatula.

Genetic diversity of norovirus associated with outbreaks in school children with acute gastroenteritis in Changzhou, China, 2018-2019.

Norovirus is one of the major causes of outbreaks and sporadic cases of acute gastroenteritis in school children. Obtaining local genotype diversity information regarding norovirus is important for developing and evaluating prevention strategies of the transmission of this virus in school children. Clinical specimens, obtained from the routine acute gastroenteritis surveillance network from 2018 to 2019, were primarily tested using commercial real-time PCR Kit. Samples with Ct value less than 25 were selected and used for complete genome sequencing and those with Ct value between 25 and 30 were selected and used for he partial VP1 and RdRp regions sequencing. Phylogenetic trees of the viral genome were constructed by using the neighbor-joining method with bootstrap analysis of 1000 replicates in MEGA 6.0. Epidemiological surveillance of acute intestinal infections (n = 384) showed high-level detection (73.18%) of human norovirus in school endemic acute gastroenteritis events in Changzhou, with obvious epidemic characteristics in autumn and winter. Through genotyping, it was found that 93.12% of norovirus were GII, including GII.2, GII.3, GII.4, GII.6, GII.7, and GII.17. By October 2019, two norovirus genotypes, GII.4[P31] and GII.17[P17], became the preponderant epidemic strains. Phylogenetic analysis of the new GII.17[P17] complete genomes showed close relationship with Miyagi strain identified in Japan in 2015, and GII.4[P31] showed close relationship with Jinan strain indentified in China in 2017. The study highlights the emerging role of GII.4[P31] and GII.17[P17] in causing endemic acute gastroenteritis outbreaks at school children, in Changzhou, China in 2019.

The Complexity of Swine Caliciviruses. A Mini Review on Genomic Diversity, Infection Diagnostics, World Prevalence and Pathogenicity.

Caliciviruses are single stranded RNA viruses, non-enveloped structurally, that are implicated in the non-bacterial gastroenteritis in various mammal species. Particularly in swine, viral gastroenteritis represents a major problem worldwide, responsible for significant economic losses for the pig industry. Among the wide range of viruses that are the proven or suspected etiological agents of gastroenteritis, the pathogenicity of the members of Caliciviridae family is among the less well understood. In this context, the present review presents and discusses the current knowledge of two genera belonging to this family, namely the Norovirus and the Sapovirus, in relation to swine. Aspects such as pathogenicity, clinical evidence, symptoms, epidemiology and worldwide prevalence, genomic diversity, identification tools as well as interchanging hosts are not only reviewed but also critically evaluated. Generally, although often asymptomatic in pigs, the prevalence of those microbes in pig farms exhibits a worldwide substantial increasing trend. It should be mentioned, however, that the factors influencing the symptomatology of these viruses are still far from well established. Interestingly, both these viruses are also characterized by high genetic diversity. These high levels of molecular diversity in Caliciviridae family are more likely a result of recombination rather than evolutionary or selective adaptation via mutational steps. Thus, molecular markers for their detection are mostly based on conserved regions such as the RdRp region. Finally, it should be emphasized that Norovirus and the Sapovirus may also infect other domestic, farm and wild animals, including humans, and therefore their surveillance and clarification role in diseases such as diarrhea is a matter of public health importance as well.