RNA-dependent RNA Polymerase Gene Research Articles

First Detection of Alphacoronavirus in Bats from the World's Largest Wetland, the Pantanal, Brazil.

Coronaviruses (CoV) infect a wide variety of hosts, causing epidemics in humans, birds, and mammals over the years. Bats (order Chiroptera) are one of the natural hosts of the Coronaviridae family. They represent 40% of the total number of mammal species in the Pantanal, a biodiversity hotspot in South America. Given the recent SARS-CoV-2 pandemic, we investigated the presence of CoV in bats captured in the Brazilian Pantanal. Oral and rectal swabs collected in 2021 from 419 bats were analyzed using Pancoronavirus-nested PCR targeting the RNA-dependent RNA-polymerase (RdRp) gene. Orthocoronavirinae was detected in 16.7% (70/419) of the bats; nine samples were sequenced, confirming that Carollia perspicillata (4), Phyllostomus hastatus (2), Desmodus rotundus (1), Molossus rufus (1), and Myotis cf. nigricans (1) collected in buildings formally used by humans were infected by Alphacoronavirus genera. This is the first description of Alphacoronavirus in bats from the Pantanal. As they are natural reservoirs of CoVs, constant monitoring of bats is important to comprehend the epidemiology of emerging viruses, especially in the Pantanal biome.

Infection pattern of male-killing viruses alters phenotypes in the tea tortrix moth Homona magnanima.

Male-killing is a microbe-induced reproductive manipulation in invertebrates whereby male hosts are eliminated during development. In the tea tortrix moth Homona magnanima, Osugoroshi viruses 1‒3 (OGVs), belonging to Partitiviridae induce male-killing. The infection patterns of OGVs are diverse; however, how the influence of these patterns of host phenotypes remains largely unknown. Using field-collected larvae, we established a OGV1 and OGV3 double-infection line, in addition to a triple-infection line, and examined the dsRNA segments, purified viral proteins, OGV density, and host phenotypes. PCR analysis demonstrated that the triple-infection line lost one dsRNA segment, whereas the double-infection line lost eight segments, including one RNA-dependent RNA polymerase (RdRp) gene. LC-MS analysis revealed three potential structural proteins in the OGVs. Males died at the larval stage in the triple-infection line and at the embryo-larval stage in the double-infection line of OGV1 and OGV3; the RNA load of female parents did not contribute to the developmental stage at which males died. These findings indicate that the pattern of viral infection, rather than viral RNA load transmitted from female parent, controls the stage of development at which male-killing occurs. Furthermore, the duration of the larval stage of the double-infection line was found to be significantly longer than that of the triple-infection line. The shorter duration of the larval stage of the triple-infection line could be advantageous over the double-infection line in maximizing transmission efficiency.

Identification of Two Novel Recombinant Types of Potato Virus Y from Solanum tuberosum Plants in Southern Region of Russia.

Potato virus Y (PVY, genus Potyvirus, family Potyviridae) is one of the most devastating and economically important potato pathogens. Members of the Potyviridae family demonstrate high recombination rates. In nature, 5 major parental variants of PVY were identified with at least 35 recombinants. In this study we report two novel PVY recombinants discovered in the sprouts of potato tubers produced in field conditions in Astrakhan region of Russia in 2021 using high-throughput sequencing and de novo genome assembly. These recombinants, which were named Ast-A-I and Ast-A-II, have previously unknown arrangements of genome sections derived from PVYO and PVYN with a novel recombination junction at the position 7850 nt of the PVY genome, in the middle part of the RNA-dependent RNA polymerase (NIb) gene, with PVYO-type sequence at the 5'-end of this junction and PVYN-type at the 3'-end. Other recombinant junctions in the novel PVY variants were previously found in PVYNTNa and PVYNTNb. This includes those at the positions 2390 and 9200 (PVYN at the 5'-end and PVYO at the 3'-end) which were present in both novel recombinants, and at the position 500 (PVYO at the 5'-end and PVYN at the 3'-end) which present in Ast-A-II. The presence of PVY variants with novel recombinant point is verified by Sanger sequencing. Phylogenetic analysis of genomic RNA showed that the sequences of these recombinants form a separate branch which do not cluster with previously described PVY strains.

Evolutionary Relationships of Unclassified Coronaviruses in Canadian Bat Species.

Bats are recognized as natural reservoirs for an array of diverse viruses, particularly coronaviruses, which have been linked to major human diseases like SARS-CoV and MERS-CoV. These viruses are believed to have originated in bats, highlighting their role in virus ecology and evolution. Our study focuses on the molecular characterization of bat-derived coronaviruses (CoVs) in Canada. Tissue samples from 500 bat specimens collected in Canada were analyzed using pan-coronavirus RT-PCR assays to detect the presence of CoVs from four genera: Alpha-CoVs, Beta-CoV, Gamma-CoV, and Delta-CoV. Phylogenetic analysis was performed targeting the RNA-dependent RNA polymerase (RdRP) gene. Our results showed an overall 1.4% CoV positivity rate in our bat sample size. Phylogenetic analysis based on the ~600 bp sequences led to the identification of an unclassified subgenus of Alpha-CoV, provisionally named Eptacovirus. The findings contribute to a better understanding of the diversity and evolution of CoVs found in the bat species of Canada. The current study underscores the significance of bats in the epidemiology of CoVs and enhances the knowledge of their genetic diversity and potential impact on global public health.

First report of citrus yellow vein clearing virus (CYVCV) on citrus in Italy.

In March 2024, symptoms of vein clearing (Fig. S1) were observed on leaves of six lemon (Citrus limon (L.) Burm. f.) trees grown in a private garden in Palma Campania (Campania region, Italy). Symptoms strongly resembled those of yellow vein clearing disease, caused by Potexvirus citriflavivenae (citrus yellow vein clearing virus, CYVCV), genus Potexvirus, sub-genus Mandarivirus (Loconsole et al., 2012; Cui et al., 2018). CYVCV was reported in Pakistan, India, China, Iran, Turkey, Korea and USA (Abrahamian et al., 2024; Catara et al., 1993; EPPO, 2024; Sun and Yokomi, 2024; Jin et al., 2024). Since 2022, considering the potential impact of this emerging virus, CYVCV has been included in the EPPO alert list and it has not been reported in any European Union (EU) Member State where the virus is not regulated. To ascertain possible infection with CYVCV, leaf samples were collected and tested by reverse transcription polymerase chain reaction (RT-PCR). Total nucleic acids (TNAs) from leaves of the symptomatic lemon and non-symptomatic lemon and sweet orange (Citrus sinensis (L.) Osbeck) trees grown in the same garden, were extracted using the silica-capture extraction method (Foissac et al. 2001). TNAs were tested by RT-PCR using CYVCV specific primers (Table S1) designed by Chen et al., (2014) targeting the coat protein gene. All symptomatic trees and an asymptomatic sweet orange from the same garden tested positive generating an amplicon of the expected size (612 bp). Direct Sanger sequencing of amplicons obtained from two symptomatic lemons and one non-symptomatic sweet orange (An PP842725-PP842727) followed by BLAST search showed 97.9-98.08% sequence identity (query coverage 100%) with CYVCV isolates previously reported from a mandarin (An OQ418501) and a lemon (An OQ418493) in California. In contrast, no amplicons were detected in samples from non-symptomatic lemon trees. Virus identity was confirmed by RT-PCR with different primers (Table S1) targeting the RNA-dependent RNA polymerase gene of CYVCV. Amplicons of the expected size (832 bp) were obtained only from samples previously tested positive to the virus and their sequences determined by Sanger sequence (An PQ284953-PQ284955) showed 97.98-98.11% identity (query coverage 100%) with CYVCV sequence previously reported from a mandarin (An OR251443) in New Delhi. Since CYVCV is transmitted by insects (Önelge et al., 2011a, 2011b, Zhang et al., 2018, 2019), TNAs (Foissac et al., 2001) from Aphis aurantii infesting symptomatic lemons, and of A. spiraecola and Dialeurodes citri infesting symptomatic lemon and non-symptomatic sweet orange trees were assayed by RT-PCR with primer pairs designed by Chen et al., (2014). Three samples of 15 specimens from three different trees were tested for each insect. In contrast to D. citri, which tested negative, an amplicon of 612 bp was detected from A. aurantii and A. spiraecola and Direct sequencing of amplicons (An PP842721-PP842724) confirmed the nature of the virus and showed that insect-derived viral sequences shared nucleotide identity of 99.3-100% with those from the infected trees. Altogether, these data support that CYVCV is present in Southern Italy. Likely, it has been introduced illegally through infected citrus propagation material. Given the large A. aurantii and A. spiraecola populations in the areas where CYVCV has been detected, a fast spread of the virus to other areas of Campania region can be expected. Even if the impact of CYVCV on citrus production in Italy remains to be assessed, prompt and extensive surveys may be helpful to establish the actual distribution of the virus. This would allow for a better evaluation of appropriate measures to be adopted to achieve sustainable citrus production. At the best of our knowledge, this is the first report of CYVCV in Italy and in the EU territory.

Molecular characterization and comparison of tomato zonate spot virus isolated in Japan and China.

The complete genome sequence of Orthotospovirus tomatozonae (tomato zonate spot virus, TZSV) isolated in Japan was determined and compared with that of Chinese isolates. The lengths of the S, M, and L segments of the RNA genomes of the Japanese isolate (TZSV-TZ1-3) were 3194, 4675, and 8916 nucleotides, respectively, which were similar to the Chinese isolates. Moreover, the eight motifs on the RNA-dependent RNA polymerase (RdRp) gene were conserved in both TZSV-TZ1-3 and Chinese TZSV isolates (TZSV-Bidens and TZSV-Tomato-YN). The nucleotide identity of the genes among the TZSV isolates was more than 94%, indicating low diversity among viruses. The phylogenetic analysis and the prediction of the cleavage sites in the glycoprotein showed that the TZSV-TZ1-3 isolate was closely related to TZSV-Tomato-YN isolated from China. However, there were unique frameshifts and deletions on the RdRp and glycoprotein genes of the TZSV-Tomato-YN isolate, suggesting that both isolates were genetically distinct. The findings of this study indicate that the TZSV-TZ1-3 isolate originated in China and show the sequence diversity among TZSV isolates.

Genome-Wide Identification of AGO, DCL, and RDR Genes and Their Expression Analysis in Response to Drought Stress in Peach

Small RNAs (sRNAs) control a wide range of development and physiological pathways in plants. To address the response of sRNA biogenesis to drought stress, we identified sRNA biogenesis genes, including 11 encoding argonautes (AGO), 8 encoding Dicer-like proteins (DCL), and 9 encoding RNA-dependent RNA polymerases (RDR) in the peach genome. Notably, the largest numbers of sRNA biogenesis genes are located to chromosome 1. The PAZ, PIWI, and MID domains were identified in PpAGOs, while the ribonuclease IIIa and IIIb domains were characterized in PpDCLs. The RDRP domain was recognized in PpRDRs. Orthologous similarity and collinearity analyses between Arabidopsis and peach revealed 5, 1, and 2 collinear blocks in AGOs, DCLs, and RDRs, respectively. Moreover, 41, 40, and 42 cis-acting elements were located in the promoters of PpAGOs, PpDCLs, and PpRDRs, respectively, with the majority related to drought stress response. Analysis of RNA sequencing (RNA-seq) data revealed that sRNA biogenesis genes were involved in drought stress response in different tissues. Furthermore, the expression of candidate genes was verified in two peach cultivars, Beijing 2-7 (BJ2-7) and Sinai (SN), which are tested as drought-tolerant and sensitive cultivars, respectively, based on the physiological and biochemical analyses, which revealed that the Chinese peach cultivar ‘BJ2-7’ exhibits greater drought resistance compared to the Egyptian peach cultivar ‘SN’. Interestingly, the expression of PpAGO2b, PpDCL2b, PpDCL4, and PpRDR4 genes was induced in ‘BJ2-7’ but inhibited in ‘SN’ under drought stress. Overall, this study provides insight into the roles of sRNA biogenesis genes in response to drought stress in peach.

First Report of Citrus Leaf Blotch Virus on Nagami Kumquat in Turkey.

Kumquat, known as the smallest of citrus fruits, has seen an increase in the number of commercial orchards in recent years. In May of 2022, a PCR-based survey for citrus virus diseases was conducted in commercial Nagami kumquat (Fortunella margarita (Lour.) Swingle) orchards in the Cukurova region of Turkey. During the survey, five trees with bud union disorder symptoms were found in two different kumquat orchards in the Erdemli district of Mersin province. Young leaf samples from the five trees showing bud union disorder symptoms and two symptomless trees were collected from 6-year-old kumquat. Common citrus viruses and viroids, such as citrus psorosis virus, citrus exocortis viroid, and hop stunt viroid, were not detected in PCR tests performed on all collected samples. The samples were also tested for citrus leaf blotch virus (CLBV; genus Citrivirus, family Betaflexiviridae), a virus associated with a bud union crease in kumquat (Vives et al.2001). Total RNA was isolated from collected leaf samples using the RNeasy Plant Mini Kit (Qiagen, USA). In the RT-PCR assay, two separate specific primer pairs were used to determine the presence of CLBV. The coat protein gene (CP), was amplified with primers KU-18 (5'-TTAAGATTACAGACACGAAGG- 3') and KU-19 (5' CTGTTTTTGAATTTTGCTCG-3'), and the RNA-dependent RNA polymerase gene (RdRp) was amplified with KU-27(5'-GATGCAAGCCAGGATGAATAC-3') and KU-15 (5'-CAGACACTCCAAGACCTTTCC-3') primers (Vives et al. 2002). All of symptomatic samples yielded amplicons of the expected size (456-bp RdRp and 438-bp CP). No amplicons were obtained from symptomless samples. Two PCR products were sequenced to confirm their identity by direct sequencing (GenBank accession no: OQ718432, OQ718433, PP726107, PP726108). Consensus sequences of these two genes showed 96% and 97% nt identity with CP and RdRP genes of the CLBV sequence in GenBank (MT038390, EU857539), respectively. For biological indexing, four RT-PCR-positive samples were used as inoculum sources. Four of Etrog citron (C. medica) and four of Dweet tangor (C. tangerina) seedlings were graft-inoculated for each source, and two seedlings for each cultivar were used as a negative control. Two months later, chlorotic blotching symptoms were observed on Dweet tangor leaves. Six months later, Etrog citron exhibited stem pitting symptoms. None of the negative control seedlings developed any symptoms. RT-PCR was performed to confirm the presence of CLBV in the virus-inoculated seedlings, and amplicons of the expected sizes (456-bp RdRp and 438-bp CP) were obtained. To the best of our knowledge, this is the first report of the CLBV infection in citrus in Turkey. CLBV is thought to have originated from infected grafting material in the country, although it is not known where it may have originated. Further research is needed to determine the distribution of CLBV and its potential to impact commercial citrus production in Turkey.

APPLICATION OF PCR FOR THE DETECTION OF VIRAL INFECTIONS IN THE HONEYBEE Apis mellifera IN THE NORTHERN REGIONS OF UKRAINE

Aim. This study investigated viruses of the western honeybee (Apis mellifera) from the northern regions of Ukraine using polymerase chain reaction (PCR). Methods. To identify honeybee viruses, oligonucleotide primers, which are specific to a gene fragment encoding the capsid protein and a fragment of the RNA-dependent RNA polymerase gene, were used. The reaction mixture contained 12.5 μL of master mix solution (DNA polymerase, dNTPs, and buffer), 1 μL of each primer (20 pmol/μL), 2 μL of cDNA, and water to a total volume of 25 μL. PCR products were visualized in a 2% agarose gel. Results. In the Zhytomyr region in Apis mellifera was revealed for the first time the presence of four viruses representing different taxonomic groups, in particular Israeli Acute Paralysis Virus (IAPV), Sacbrood Virus (SBV), Deformed Wing Virus (DWV) and Chronic Bee Paralysis Virus (CBPV). Three of these viruses (SBV, DWV, and CBPV) were detected in the Kyiv region, while two (SBV and DWV) were identified in the Chernihiv region. Conclusions. The obtained data indicate the widespread distribution of Apis mellifera honeybee viruses in the northern regions of Ukraine. The use of PCR to detect bee viruses will contribute to the development of effective methods for improving the health of bee colonies.

The first report of cotton leafroll dwarf virus infecting upland cotton plants in Arizona.

Cotton leafroll dwarf virus (CLRDV) represents a persistent threat to cotton production in the United States (U.S.) (Edula et al. 2023). Initially detected in Alabama (Avelar et al. 2019), CLRDV occurs in almost all the states in the U.S. cotton belt, extending from Virginia through Texas. The symptoms associated with CLRDV includes interveinal chlorosis, leaf rolling, stunting, and reduced boll sets. However, asymptomatic CLRDV-infected plants have also been reported (Edula et al. 2023). In the 2023 growing season, upland cotton plants (Gossypium hirsutum) presenting terminal splitting symptoms in the blooming stage were observed in a commercial field in Pinal County, Arizona, at 60% incidence. To evaluate if CLRDV may be associated with these symptoms, young fully expanded terminal leaves were collected from 20 symptomatic and ten asymptomatic plants. Moreover, ten samples were collected from a second asymptomatic block in the same field. Samples were shipped on dry ice to Cornell University in hermetic bags under APHIS PPQ permit 526-23-256-14384. Midribs and petioles were used to extract total nucleic acids from each sample using OPS Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics) per the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using an iScript Reverse transcriptase kit (BioRad) and used for the detection of a partial sequence of the coat protein (CP) of CLRDV using PCR assays as described previously (Mahas et al. 2022). The expected 309-bp product was obtained from only one symptomatic sample. CLRDV presence in the samples was further evaluated using CLRDV-specific primers targeting the RNA-dependent RNA polymerase (RdRp) gene. Primers CLRDV-Pol_innerF1 (5'- ACCCTCCAAGGAACAGAG -3') / R1 (5'- CGAATAATCTGATYGGGTCAC -3') and CLRDV-Pol_outerF1 (5'- AACGCGCCCAGTCCGCACAAATACC-3') / R1 (5'-ACCGGGTTTACTGGGGATTGCACGC-3'), designed based on virus isolates available in GenBank as of September 2022, were used to implement a single-tube nested RT-PCR as detailed by Dey et al. (2012) and to index the presence of CLRDV in all the samples. Two additional symptomatic samples and three asymptomatic samples (two from field one and one from field two) were positive for the virus. Direct Sanger sequencing of the one CP and two RdRp amplicons from symptomatic and asymptomatic samples (PP482918-20) demonstrated they shared >99% nucleotide identity to an isolate from South Carolina (OQ300129). To further evaluate the presence of CLRDV in Arizona, we performed double antibody sandwich (DAS)-ELISA on midrib and petiole samples using camelid single-chain antibodies against the CP as the capture antibody (Filed Patent 18/436,287) and the commercially available Anti-PLRV conjugate (Agdia, Elkhart, IN) as the secondary antibody. DAS-ELISA detected CLRDV in eight symptomatic and three asymptomatic samples, with the positive and negative controls testing positive and negative, respectively. Considering all the diagnostic approaches, ten symptomatic and five asymptomatic samples were positive for CLRDV (Table S1). To the best of our knowledge, this is the first report of CLRDV in Arizona. Future studies are required to evaluate the possible incidence and impact on the crop yield in the state and develop a reliable diagnostic assay to detect the virus in all infected samples. Since our study did not associate CLRDV with the terminal abortion or splitting symptoms, ongoing efforts are underway to elucidate if other viruses may be associated with the symptoms.

MiR-192 and miR-731 synergically inhibit RGNNV infection by targeting ULK1-mediated autophagy in sea perch (Lateolabrax japonicus)

MicroRNAs (miRNAs) are crucial regulators of gene expression and are closely linked to viral infections. Nervous necrosis virus (NNV) poses a significant threat to global fish aquaculture. This study investigates the roles of miR-192 and miR-731 in controlling NNV infection and associated autophagy in sea perch (Lateolabrax japonicus). Our findings reveal that both miRNAs are upregulated in infected brain tissues and cells of sea perch, leading to reduced NNV replication. miR-192 and miR-731 synergistically reduce NNV replication by downregulating the RNA-dependent RNA polymerase and capsid protein genes, and disrupting autophagy by altering LC3 distribution and autophagy-related protein expressions. Additionally, L. japonicus unc-51 like autophagy activating kinase 1 protein (LjULK1) was identified as the target of miR-192 and miR-731. LjULK1 is integrally associated to the ULK-VAPs-Atg13 autophagic signaling, enhancing NNV-induced autophagy, and facilitating viral infection. Modulating LjULK1 expression counteracts the inhibitory effects of miR-192 and miR-731 on NNV, suggesting these miRNAs act as negative regulators of NNV infection by targeting LjULK1-mediated autophagy. Our findings reveal a novel miRNAs-regulated antiviral mechanism against NNV, offering insights into potential strategy to prevent NNV infection in fish.

Genetic analysis of tomato brown rugose fruit virus reveals evolutionary adaptation and codon usage bias patterns

Tomato brown rugose fruit virus (ToBRFV) poses a significant threat to tomato production worldwide, prompting extensive research into its genetic diversity, evolutionary dynamics, and adaptive strategies. In this study, we conducted a comprehensive analysis of ToBRFV at the codon level, focusing on codon usage bias, selection pressures, and evolutionary patterns across multiple genes. Our analysis revealed distinct patterns of codon usage bias and selection pressures within the ToBRFV genome, with varying levels of genetic diversity and evolutionary constraints among different genes. We observed a transition/transversion bias of 2.07 across the entire ToBRFV genome, with the movement protein (MP) gene exhibiting the highest transition/transversion bias and SNP density, suggesting potential evolutionary pressures or a higher mutation rate in this gene. Furthermore, our study identified episodic positive selection primarily in the MP gene, highlighting specific codons subject to adaptive changes in response to host immune pressures or environmental factors. Comparative analysis of codon usage bias in the coat protein (CP) and RNA-dependent RNA polymerase (RdRp) genes revealed gene-specific patterns reflecting functional constraints and adaptation to the host's translational machinery. Our findings provide valuable insights into the molecular mechanisms driving ToBRFV evolution and adaptation, with implications for understanding viral pathogenesis, host-virus interactions, and the development of control strategies. Future research directions include further elucidating the functional significance of codon usage biases, exploring the role of episodic positive selection in viral adaptation, and leveraging these insights to inform the development of effective antiviral strategies and crop protection measures.

Homology Modeling and Expression of Recombinant NS5-RdRp Based on the Indonesian Local Strain of Dengue Virus for Anti-Dengue Drug Development

This study aims to isolate the RNA-dependent RNA polymerase (RdRp) gene of the dengue virus (DENV) isolates from Indonesia, determine the serotype of the local DENV strain through homology modeling, and express the recombinant RdRp protein in Escherichia coli BL21 (DE3), as the target for anti-dengue drug development. We utilize reverse transcription- polymerase chain reaction (RT-PCR) and nested PCR for serotyping the DENV isolates, obtained from Hasan Sadikin Hospital, Indonesia. Then, followed by the extraction and purification of the RdRp amplicon for gene sequencing. The resulting RdRp gene sequences are translated into amino acid sequences, which are then used for protein modeling and the construction of a recombinant plasmid for RdRp protein expression. As the result, the RdRp gene, which belongs to the non-structural protein 5 (NS5) of DENV, was successfully isolated and sequenced from DENV RNA samples. Based on serotyping results and homology modeling, the serotype of the isolate DENV was identified as serotype-3. The isolated NS5-RdRp gene from the local strain was then inserted into the pET28a(+) plasmid and transformed into E. coli BL21(DE3) as the host cell. Recombinant NS5-RdRp was predominantly found in the insoluble fraction with a molecular weight of 72 kDa. Future studies on the development of an anti-dengue drug model that confirms the inhibition of the binding domain in the recombinant RdRp protein structure will be required to provide more insight and expand the development of a universal anti-dengue strategy in Indonesia.

Diverse RNA viruses discovered in multiple seagrass species.

Seagrasses are marine angiosperms that form highly productive and diverse ecosystems. These ecosystems, however, are declining worldwide. Plant-associated microbes affect critical functions like nutrient uptake and pathogen resistance, which has led to an interest in the seagrass microbiome. However, despite their significant role in plant ecology, viruses have only recently garnered attention in seagrass species. In this study, we produced original data and mined publicly available transcriptomes to advance our understanding of RNA viral diversity in Zostera marina, Zostera muelleri, Zostera japonica, and Cymodocea nodosa. In Z. marina, we present evidence for additional Zostera marina amalgavirus 1 and 2 genotypes, and a complete genome for an alphaendornavirus previously evidenced by an RNA-dependent RNA polymerase gene fragment. In Z. muelleri, we present evidence for a second complete alphaendornavirus and near complete furovirus. Both are novel, and, to the best of our knowledge, this marks the first report of a furovirus infection naturally occurring outside of cereal grasses. In Z. japonica, we discovered genome fragments that belong to a novel strain of cucumber mosaic virus, a prolific pathogen that depends largely on aphid vectoring for host-to-host transmission. Lastly, in C. nodosa, we discovered two contigs that belong to a novel virus in the family Betaflexiviridae. These findings expand our knowledge of viral diversity in seagrasses and provide insight into seagrass viral ecology.

Deciphering the evolutionary landscape of severe fever with thrombocytopenia syndrome virus across East Asia.

Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a significant public health challenge in East Asia, necessitating a deeper understanding of its evolutionary dynamics to effectively manage its spread and pathogenicity. This study provides a comprehensive analysis of the genetic diversity, recombination patterns, and selection pressures across the SFTSV genome, utilizing an extensive dataset of 2041 sequences from various hosts and regions up to November 2023. Employing maximum likelihood and Bayesian evolutionary analysis by sampling trees (BEAST), we elucidated the phylogenetic relationships among nine distinct SFTSV genotypes (A, B1, B2, B3, B4, C, D, E, and F), revealing intricate patterns of viral evolution and genotype distribution across China, South Korea, and Japan. Furthermore, our analysis identified 34 potential reassortments, underscoring a dynamic genetic interplay among SFTSV strains. Genetic recombination was observed most frequently in the large segment and least in the small segment, with notable recombination hotspots characterized by stem-loop hairpin structures, indicative of a structural propensity for genetic recombination. Additionally, selection pressure analysis on critical viral genes indicated a predominant trend of negative selection, with specific sites within the RNA-dependent RNA polymerase and glycoprotein genes showing positive selection. These sites suggest evolutionary adaptations to host immune responses and environmental pressures. This study sheds light on the intricate evolutionary mechanisms shaping SFTSV, offering insights into its adaptive strategies and potential implications for vaccine development and therapeutic interventions.

Molecular Characterization of a Novel Rubodvirus Infecting Raspberries.

A novel negative-sense single-stranded RNA virus showing genetic similarity to viruses of the genus Rubodvirus has been found in raspberry plants in the Czech Republic and has tentatively been named raspberry rubodvirus 1 (RaRV1). Phylogenetic analysis confirmed its clustering within the group, albeit distantly related to other members. A screening of 679 plant and 168 arthropod samples from the Czech Republic and Norway revealed RaRV1 in 10 raspberry shrubs, one batch of Aphis idaei, and one individual of Orius minutus. Furthermore, a distinct isolate of this virus was found, sharing 95% amino acid identity in both the full nucleoprotein and partial sequence of the RNA-dependent RNA polymerase gene sequences, meeting the species demarcation criteria. This discovery marks the first reported instance of a rubodvirus infecting raspberry plants. Although transmission experiments under experimental conditions were unsuccessful, positive detection of the virus in some insects suggests their potential role as vectors for the virus.

Circulation of Non-Middle East Respiratory Syndrome (MERS) Coronaviruses in Imported Camels in Saudi Arabia.

Background Coronaviruses (CoVs) pose significant health risks to humans, with recent outbreaks like severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscoring their zoonotic potential. Dromedary camels (Camelus dromedarius) have been implicated as intermediate hosts for MERS-CoV, prompting heightened surveillance efforts. This study aims to identify non-MERS-CoV CoVs in imported camels at the Jeddah seaport, Saudi Arabia, using molecular techniques. Methods Camel nasal swabs (n = 337) were collected from imported dromedary camels arriving at the Jeddah Islamic seaport from Sudan and Djibouti. Samples were tested for CoVs using real-time real-time reverse transcription polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase gene. Positive samples were confirmed by conventional RT-PCR and Sanger sequencing. Selected samples underwent RNA sequencing to identify viral genomes. The study underscores the importance of molecular surveillance in camels to mitigate zoonotic risks. Results Out of 337 camel samples tested, 28 (8.30%) were positive for CoVs, predominantly from camels imported from Djibouti, compared to Sudan (13.39% vs. 5.78%). Sequence analysis confirmed the presence of non-MERS CoVs, including camel alpha-coronavirus and human CoV-229E-related strains. These findings highlight potential viral diversity and transmission risks in imported camel populations. Conclusion This study identifies diverse CoVs circulating in imported dromedary camels at the Jeddah Islamic seaport, Saudi Arabia, underscoring their potential role in zoonotic transmission. Enhanced surveillance and collaborative efforts are essential to mitigate public health risks associated with novel coronavirus strains from camel populations.

Genetic detection and analysis of porcine norovirus in pigs farmed in north Vietnam

Norovirus (NoV) causing gastroenteritis symptoms, which has been reported in several hosts, including humans, pigs, and rats. This study was conducted to identify porcine viral infection and to characterize NoV strains from pigs in some provinces in north Vietnam. Totally, 102 fecal samples from diarrheal pigs on farms in six cities and provinces in northern Vietnam during July 2022 to March 2023 were collected. Polymerase chain reaction was used to identify the viral genome. Positive samples were used for nucleotide sequencing of the partial RNA-dependent RNA polymerase gene sequence. Five (4.9 %) positive stool samples were detected from animals farmed in five different farms, with one positive animal identified in each farm. Genetic analysis indicated that nucleotide identity was in the range 97.77–99.62 % among the 5 NoVs in this study. Phylogenetic analysis pointed out that the five NoVs were Genotype II.19 viruses. Genetically, these strains were closely related to porcine NoV strains that were reported in China in 2009.

RNA virus diversity and prevalence in field and laboratory populations of melon fly throughout its distribution

Insects have a rich diversity of RNA viruses that can either cause acute infections or persist in host populations without visible symptoms. The melon fly, Zeugodacus cucurbitae (Tephritidae) causes substantial economic losses through infestation of diverse cucurbit and other crops. Of Indomalayan origin, it is now established in many tropical regions of the world. The virome diversity of Z. cucurbitae is largely unknown across large parts of its distribution, including the Indian subcontinent. We have analysed three transcriptomes each of one field–collected and one laboratory-reared Z. cucurbitae population from Bangalore (India) and discovered genomes of ten putative RNA viruses: two sigmaviruses, one chimbavirus, one cripavirus, one noda-like virus, one nora virus, one orbivirus, one partiti-like virus, one sobemovirus and one toti-like virus. Analysis of the only available host genome of a Hawaiian Z. cucurbitae population did not detect host genome integration of the detected viruses. While all ten viruses were found in the Bangalore field population only seven were detected in the laboratory population, indicating that these seven may cause persistent covert infections. Using virus-specific RNA-dependent RNA polymerase gene primers, we detected nine of the RNA viruses with an overall low variant diversity in some but not all individual flies from four out of five Indian regions. We then screened 39 transcriptomes of Z. cucurbitae laboratory populations from eastern Asia (Guangdong, Hainan, Taiwan) and the Pacific region (Hawaii), and detected seven of the ten virus genomes. We found additional genomes of a picorna-like virus and a negev-like virus. Hawaii as the only tested population from the fly’s invasive range only had one virus. Our study provides evidence of new and high RNA virus diversity in Indian populations within the original range of Z. cucurbitae, as well as the presence of persistent covert infections in laboratory populations. It builds the basis for future research of tephritid-associated RNA viruses, including their host effects, epidemiology and application potential in biological control.

Genome-wide identification and expression analyses of the Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in onion (Allium cepa L.)

ABSTRACT RNA silencing is a highly conserved gene expression regulatory mechanism participating in plant development and stress responses. Dicer-like protein (DCL), Argonaute protein (AGO) and RNA-dependent RNA polymerase (RDR) are directly involved in RNA silencing. So far, the characterisation and expression analysis of RDR, AGO and DCL proteins have not been performed in onion. A genome-wide analysis identified eight AcAGO, four AcDCL and four AcRDR putative genes in onion. Their cis-acting elements were investigated. The transcript levels of genes in different onion tissues and response to abiotic stress were studied. The results demonstrated that AcAGOs, AcDCLs and AcRDRs showed various tissue expression patterns. AcAGO2 and AcAGO4b were highly expressed in onion bulbs. AcDCLs and AcRDRs showed the highest transcript levels in inflorescence. AcAGO1, AcAGO5 and AcDCL1 were constitutively expressed in leaf and bulb during onion bulb expansion. AcRDR1 and AcRDR6 were upregulated during bulb expansion. AcAGOs were upregulated with salinity treatment, whereas AcDCLs and AcRDRs showed different expression patterns in abiotic stress response. Our results provided a basis for further clarification of RNA silencing-mediated environment stimulation response and development in onion.