RNA-dependent RNA Polymerase Gene Research Articles

Complete genome of Aedes japonicus narnavirus from wild caught mosquitoes collected in Ohio, USA.

Narnaviruses infect several genera of mosquitoes including Culex and Aedes . The narnavirus genome is a positive, single stranded RNA encoding an RNA-dependent RNA polymerase gene. The partial genome of a narnavirus identified in wild Aedes japonicus mosquitoes collected in Wooster, Ohio, USA was obtained using metagenomic analyses. Rapid amplification of 5'-cDNA ends (RACE) and Sanger sequencing were used to obtain the remaining genomic sequence of this strain. The complete genome is composed of 3153 nucleotides and has 98.4% and 99.1% nucleotide sequence identity with Aedes japonicus narnavirus genomes identified in Netherlands and Japan.

A visual detection strategy for SARS-CoV-2 based on dual targets-triggering DNA walker

SARS-CoV-2, a highly transmissible and mutagenic virus, made huge threats to global public health. The detection strategies, which are free from testing site requirements, and the reagents and instruments are portable, are vital for early screening and play a significant role in curbing the spread. This work proposed a silver-coated glass slide (SCGS)/DNA walker based on a dual targets-triggering mechanism, enzyme-catalyzed amplification, and smartphone data analysis, which build a portable visual detection strategy for the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. By this method, the detection was reflected by the ultraviolet absorbance changes and visible color changes to the naked eye which was analyzed by Red-Green-Blue (RGB) data analysis via smartphone within 30 min, simplifying the detection process and shortening the detection time. Meanwhile, the dual targets-triggering mechanism and dual signal amplification strategy ensured detection specificity and sensitivity. Further, the practicability was verified by the detection of the real sample which provided this method an application potential in SARS-CoV-2 rapid detection.

In-vitro synthesis of RNA fragments specifying envelope protein gene and RdRp gene of SARS-CoV-2 as positive standards for molecular diagnosis tests

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has rapidly spread through the entire world and has become the worst pandemic from December 2019 until now. The establishment of positive standards for molecular diagnostic testing for SARS-CoV-2 plays a critical role in development and assessment of diagnostic assays associated with the shortage of positive specimens and viral culture fluids. This study aims to establish a novel assay for in-vitro RNA fragment synthesis based on amplicons of self-priming PCR targeting envelope protein gene and RNA-dependent RNA polymerase (RdRp) gene of SARS-CoV-2. The cDNA library of the targeted genes of SARS-CoV-2 was generated by using long-primers named sMn1 forward/reverse primer (E gene) and sMn2 forward/reverse primer (RdRp gene) for self-priming PCR assays. The synthesised amplicons that overlap the target sequence of the World Health Organization (WHO) assay were cloned into a pGEM-T easy vector, then transformed into E. coli competent cells by conventional methods. The recombinant plasmids were used as materials for in-vitro RNA transcription. Concentrations of the in-vitro transcribed RNA were 200-800 ng/μl with A260/A280 ratios of 2.0-2.2. Gel electrophoresis showed a single band of each RNA molecule with sizes of 216 and 214 bases for sMn1- E and sMn2- RdRp gene, respectively. Furthermore, we effectively evaluated the in-vitro transcribed RNA by a one-step, real-time RT-PCR assay according to the standard WHO protocol. The stability of in-vitro RNA over a 6-month storage period was then investigated. In conclusion, our assay for in-vitro synthesis of RNA fragments transcribed from self-priming amplicons were successfully established and thus these positive standards were useful for molecular diagnosing of SARS-CoV-2.

Evaluation of reverse transcriptase-polymerase spiral reaction assay for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2

Background and aimExisting real-time reverse transcriptase PCR (RT-qPCR) has certain limitations for the point-of-care detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) since it requires sophisticated instruments, reagents and skilled laboratory personnel. In this study, we evaluated an assay termed the reverse transcriptase-polymerase spiral reaction (RT-PSR) for rapid and visual detection of SARS-CoV-2. MethodsThe RT-PSR assay was optimized using RdRp gene and evaluated for the detection of SARS-CoV-2. The time of 60min and a temperature of 63°C was optimized for targeting the RNA-dependent RNA polymerase gene of SARS-CoV-2. The sensitivity of the assay was evaluated by diluting the in-vitro transcribed RNA, which amplifies as low as ten copies. ResultsThe specific primers designed for this assay showed 100% specificity and did not react when tested with other lung infection-causing viruses and bacteria. The optimized assay was validated with 190 clinical samples in two phases, using automated RTPCR based TrueNat test, and the results were comparable. ConclusionsThe RT-PSR assay can be considered for rapid and sensitive detection of SARS-CoV-2, particularly in resource-limited settings. To our knowledge, there is as yet no RT-PSR-based kit developed for SARS-CoV-2.

Development and Validation of ScriptTaq COVID PCR: An In-House Multiplex rRT-PCR for Low-Cost Detection.

The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.

Phlebovirus diversity in ticks from livestock in arid ecologies in Kenya

Phleboviruses are emerging pathogens of public health importance. However, their association with ticks is poorly described, particularly in Africa. Here, adult ticks infesting cattle, goats and sheep were collected in two dryland pastoralist ecosystems of Kenya (Baringo and Kajiado counties) and were screened for infection with phleboviruses. Ticks mainly belonged to the species Rhipicephalus appendiculatus, Hyalomma impeltatum, and Hyalomma rufipes. A fragment of the RNA-dependent RNA polymerase (RdRp) gene was identified in thirty of 671 tick pools, of which twenty-nine were from livestock sampled in Baringo county. Phylogenetic analyses revealed that twenty-five sequences were falling in three clades within the group of tick-associated phleboviruses. The sequences of the three clades showed nucleotide distances 8%, 19% and 22%, respectively, to previously known viruses suggesting that these sequence fragments may belong to three distinct viruses. Viruses of the group of tick-associated phleboviruses have been found in several countries and continents but so far have not been associated with disease in humans or animals. In addition, five sequences were found to group with the sandfly-associated phleboviruses Bogoria virus, Perkerra virus and Ntepes virus recently detected in the same region. Further studies are needed to investigate the transmission and maintenance cycles of these viruses, as well as to assess their potential to infect vertebrates.

An Unwanted Association: The Threat to Papaya Crops by a Novel Potexvirus in Northwest Argentina.

An emerging virus isolated from papaya (Carica papaya) crops in northwestern (NW) Argentina was sequenced and characterized using next-generation sequencing. The resulting genome is 6667-nt long and encodes five open reading frames in an arrangement typical of other potexviruses. This virus appears to be a novel member within the genus Potexvirus. Blast analysis of RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes showed the highest amino acid sequence identity (67% and 71%, respectively) with pitaya virus X. Based on nucleotide sequence similarity and phylogenetic analysis, the name papaya virus X is proposed for this newly characterized potexvirus that was mechanically transmitted to papaya plants causing chlorotic patches and severe mosaic symptoms. Papaya virus X (PapVX) was found only in the NW region of Argentina. This prevalence could be associated with a recent emergence or adaptation of this virus to papaya in NW Argentina.

Genetic Reassortment between Endemic and Introduced Macrobrachium rosenbergii Nodaviruses in the Murray-Darling Basin, Australia.

Macrobrachium rosenbergii nodavirus (MrNV)—the aetiological agent of white tail disease—is a major limiting factor of crustacean aquaculture as it causes up to 100% mortality in M. rosenbergii larvae and juveniles. Despite the importance of MrNV, there have been few studies on the phylogenetic diversity and geographic range of this virus in Australian waterways. Here, we detected MrNV genomes in common carp (Cyprinus carpio) metatranscriptomes sampled at five freshwater sites across the Murray-Darling Basin (MDB), Australia. We identified genetic divergence of the RNA-dependent RNA polymerase gene between MrNV sequences identified in the northern and southern rivers of the MDB. Northern viruses exhibited strong phylogenetic clustering with MrNV from China, whereas the southern viruses were more closely related to MrNV from Australia. However, all five viruses were closely related in the capsid protein, indicative of genetic reassortment of the RNA1 and RNA2 segments between Australian and introduced MrNV. In addition, we identified Macrobrachium australiense in two of the five MrNV-positive libraries, suggesting that these species may be important reservoir hosts in the MDB. Overall, this study reports the first occurrence of MrNV outside of the Queensland region in Australia and provides evidence for genetic reassortment between endemic and introduced MrNV.

RNA Viral Communities Are Structured by Host Plant Phylogeny in Oak and Conifer Leaves

Wild plants can suffer devastating diseases, experience asymptomatic persistent infections, and serve as reservoirs for viruses of agricultural crops; however, we have a limited understanding of the natural plant virosphere. To access representatives of locally and globally distinct wild plants and investigate their viral diversity, we extracted and sequenced double-stranded RNA from leaves from 16 healthy oak and conifer trees in the University of California–Davis Arboretum (Davis, CA, U.S.A.). From de novo assemblies, we recovered 389 RNA-dependent RNA polymerase gene sequences from 384 putative viral species, and identified 580 putative viral contigs via virus prediction software followed by manual confirmation of virus annotation. Based on similarity to known viruses, most recovered viruses were predicted to infect plants or fungi, with the highest diversity and abundance observed in the Totiviridae and Mitoviridae families. Phyllosphere viral community composition differed significantly by host plant phylogeny, suggesting the potential for host-specific viromes. The phyllosphere viral community of one oak tree differed substantially from other oak viral communities and contained a greater proportion of putative mycoviral sequences, potentially due to the tree's more advanced senescence at the time of sampling. These results suggest that oak and conifer trees harbor a vast diversity of viruses with as-yet-unknown roles in plant health and phyllosphere microbial ecology. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Development of an Indirect Fluorescent Antibody (IFA) Assay for the Detection of Leishmania RNA Virus 2 (LRV2) in Leishmania Parasites.

Detection of Leishmania RNA virus (LRV) in Old World Leishmania species and their possible role in the disease prognosis requires sensitive and specific methods, preferably independent of the viral genome. We aimed to develop an indirect immunofluorescence antibody (IFA) assay to detect LRV in the Old World Leishmania parasites. Clinical samples were collected from 86 cutaneous leishmaniasis (CL) patients in different endemic areas of CL in Iran, during 2017-2019. For antibody preparation, the viruses were obtained from sediment of an LRV-infected L. major culture-using freeze and thaw cycles followed by gradient cesium chloride centrifugation. The purified viruses were used to immunize a male 3-4 months rabbit. Various dilutions of the LRV-immunized rabbit's serum and a conjugated antibody were deployed to detect LRV in 48 isolates by IFA assay. LRV virus was detected in four of the 48 CL cases using IFA method. Amplification of a partial fragment of RNA-dependent RNA polymerase (RdRp) gene from the isolates confirmed the IFA results. In phylogeny, the generated RdRp sequences from four isolates were grouped with the other Old World LRVs, but separate from L. aethiopica LRVs, which appeared as a highly supported distinct clade. Further optimization of this approach to detect the LRV directly in lesion scrapings can make it a more reliable tool for field studies and disclosing the virus's possible role in disseminating and unusual clinical features.

Co-circulation of coronavirus and avian influenza virus in wild birds in Shanghai (2020-2021).

Co‐infection of SARS‐CoV‐2 and influenza viruses has been reported worldwide in humans. Wild birds are natural reservoir hosts for coronaviruses (CoVs) and avian influenza viruses (AIVs). It is unknown whether co‐infection with these two types of viruses occurs in wild birds. In this study, the prevalence of co‐infection with CoV and AIV in wild birds in Shanghai, China during 2020–2021 was investigated by detecting these viruses in cloacal, tracheal, and faecal samples. Results showed that the overall rate of samples positive for both CoV and AIV was 3.3% (82/2510; 95% confidence interval [CI]: 2.6%–4.0%), and that was mainly from Anseriformes. In CoV‐positive samples, 38.9% (82/211; 95% CI: 32.5%–45.6%) of them had both CoVs and AIVs, whereas only 26.9% (82/305; 95% CI: 22.2%–32.1%) of AIV‐positive samples had both CoVs and AIVs. These results suggest that CoV infection in wild birds renders them more susceptible to AIV infection. Phylogenetic analysis based on partial RNA‐dependent RNA polymerase (RdRp) gene sequences of CoVs revealed that gamma‐CoVs mainly cluster with duck CoVs and that delta‐CoVs are more diversified and cluster with those of various wild birds. Continual surveillance is necessity to monitor the transmission and evolution of co‐infection of these two types of viruses in their natural hosts.

Detection of Alpha- and Betacoronaviruses in Frugivorous and Insectivorous Bats in Nigeria

The rise of bat-associated zoonotic viruses necessitates a close monitoring of their natural hosts. Since the detection of severe acute respiratory syndrome coronavirus (SARS-CoV), it is evident that bats are vital reservoirs of coronaviruses (CoVs). In this study, we investigated the presence of CoVs in multiple bat species in Nigeria to identify viruses in bats at high-risk human contact interfaces. Four hundred and nine bats comprising four bat species close to human habitats were individually sampled from five states in Nigeria between 2019 and 2021. Coronavirus detection was done using broadly reactive consensus PCR primers targeting the RNA-dependent RNA polymerase (RdRp) gene of CoVs. Coronavirus RNA was detected in 39 samples (9.5%, CI 95%: [7.0, 12.8]), of which 29 were successfully sequenced. The identified CoVs in Nigerian bats were from the unclassified African alphacoronavirus lineage and betacoronavirus lineage D (Nobecovirus), with one sample from Hipposideros ruber coinfected with alphacoronavirus and betacoronavirus. Different bat species roosting in similar or other places had CoVs from the same genetic lineage. The phylogenetic and evolutionary dynamics data indicated a high CoV diversity in Nigeria, while host switching may have contributed to CoV evolution. Robust sentinel surveillance is recommended to enhance our knowledge of emerging and re-emerging coronaviruses.

Incidence and genetic diversity of grapevine leafroll-associated virus 3 (GLRaV-3) isolates in Turkey

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most important virus species within the grapevine leafroll complex. It causes significant losses to growers and vineries because of its effect on grape and wine quality. A survey was conducted in the major grape-growing provinces of Turkey in 2018 to investigate distribution and the genetic diversity of GLRaV-3 in local and foreign grapevine cultivars. The genetic diversity of GLRaV-3 isolates based on partial heat-shock protein 70 homologue (Hsp70 h), partial coat protein (CP) and partial RNA-dependent RNA polymerase (RdRp) genes were determined by RT-PCR and sequencing. The infection rate of GLRaV-3 in 141 tested grapevine samples was 13.47% by DAS-ELISA and 28.36% by RT-PCR analysis. Totally, 17 amplicons from Hsp70 h, 22 isolates from RdRp and 16 isolates from CP genes were sequenced in both directions. The sequence analysis of three genes revealed that the Turkish GLRaV-3 isolates shared 91–100% nucleotide identities with the sequences of GLRaV-3 isolated deposited in the GenBank from other parts of the world without any correlation between the distribution and geographical origin.

Remdesivir-induced emergence of SARS-CoV2 variants in patients with prolonged infection

We here investigate the impact of antiviral treatments such as remdesivir on intra-host genomic diversity and emergence of SARS-CoV2 variants in patients with a prolonged course of infection.Sequencing and variant analysis performed in 112 longitudinal respiratory samples from 14 SARS-CoV2-infected patients with severe disease progression show that major frequency variants do not generally arise during prolonged infection. However, remdesivir treatment can increase intra-host genomic diversity and result in the emergence of novel major variant species harboring fixed mutations. This is particularly evident in a patient with B-cell depletion who rapidly developed mutations in the RNA-dependent RNA polymerase gene following remdesivir treatment.Remdesivir treatment-associated emergence of novel variants is of great interest in light of current treatment guidelines for hospitalized patients suffering from severe SARS-CoV2 disease, as well as the potential use of remdesivir to preventively treat non-hospitalized patients at high risk for severe disease progression.

Phylogenetic characteristics of Non-SARS human coronavirus in southern Taiwan, 2012–2013

This study characterizes the phylogenetic relatedness of non-SARS human coronaviruses (HCoVs) in southern Taiwan by sequencing the nucleocapsid (N), spike (S), and RNA-dependent RNA polymerase (RdRp) genes directly from ten HCoV PCR-positive respiratory samples collected during 2012–2013. In the N, S1, and RdRp phylogeny, HCoV-OC43 in one and three samples was clustered with genotypes F and G, respectively, and HCoV-OC43 in sample YC101/TWN/2013 represented a recombination event between genotypes F and G. Amino acid substitutions in the S1 protein of HCoV-OC43 were also identified. In the N phylogeny, HCoV-HKU1 in one and two samples clustered with genotypes A and B, respectively, and HCoV-229E in two samples was clustered with genogroup 6. The genotypes and genogroup detected here were in line with the prevalent phylogenetic lineages reported outside of Taiwan during the contemporary period. In summary, three species of non-SARS HCoVs with different genotypes cocirculated in the community, with genetic evolution observed in HCoV-OC43.

Clinical epidemiological applicability of real-time polymerase chain reaction for COVID-19.

ObjectivesReal-time polymerase chain reaction is currently used as a confirmatory test for coronavirus disease 2019 (COVID-19). The test results are interpreted as positive, negative, or inconclusive, and are used only for a qualitative classification of patients. However, the test results can be quantitated using threshold count (Ct) values to determine the amount of virus present in the sample. Therefore, this study investigated the diagnostic usefulness of Ct results through various quantitative analyzes, along with an analysis of clinical and epidemiological characteristics.Methods Clinical and epidemiological data from 4,642 COVID-19 patients in April 2021 were analyzed, including the Ct values of the RNA-dependent RNA polymerase (RdRp), envelope (E), and nucleocapsid (N) genes. Clinical and epidemiological data (sex, age, underlying diseases, and early symptoms) were collected through a structured questionnaire. A correlation analysis was used to examine the relationships between variables.Results All 3 genes showed statistically significant relationships with symptoms and severity levels. The Ct values of the RdRp gene decreased as the severity of the patients increased. Moreover, statistical significance was observed for the presence of underlying diseases and dyspnea.Conclusion Ct values were found to be related to patients’ clinical and epidemiological characteristics. In particular, since these factors are closely related to symptoms and severity, Ct values can be used as primary data for predicting patients’ disease prognosis despite the limitations of this method. Conducting follow-up studies to validate this approach might enable using the data from this study to establish policies for preventing COVID-19 infection and spread.

Detection of SARS-CoV-2 VOC-Omicron using commercial sample-to-answer real-time RT-PCR platforms and melting curve-based SNP assays

ObjectivesThe World Health Organization (WHO) had designated the SARS-CoV-2 lineage B.1.1.529 as the new Variant of Concern Omicron (VOC-Omicron) on 26th November 20211. Real-time reverse transcription polymerase chain reaction (RT-PCR), single nucleotide polymorphisms (SNP) and whole genome sequencing (WGS) tests were widely employed to detect SARS-CoV-2 and its variant. Yet, the SARS-CoV-2 Omicron detection performance of commercial real-time RT-PCR platforms and SARS-CoV-2 spike SNP assays remain to be elucidated.MethodsIn the first part of this study, we evaluated the VOC-Omicron detection performance of three commercial RT-PCR sample-to-answer platforms i.e. Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems. The detection performances were compared to one commercial conventional real-time RT-PCR assay (TIB MOLBIOL LightMix Modular SARS and Wuhan CoV E-gene) and one in-house real-time RT-PCR assay targeting RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2 in the WHO COVID-19 Reference Laboratory at Public Health Laboratory Services Branch, Centre for Health Protection, Department of Health, The Government of the Hong Kong Special Administrative Region. In the second part of this study, we evaluated the SNP detection performance of four TIB MOLBIOL melting curve-based assays (1. Spike S371L/S373P, 2. Spike E484A, 3. Spike E484K and 4. Spike N501Y) in clinical samples obtained from hospitalized COVID-19 patients in Hong Kong. The SNP results were compared to whole genome sequences generated by Illumina platform.ResultsThe VOC-Omicron detection limits of three commercial sample-to-answer assays were tested to be ≤ 2.35 Log10 dC/ml. The detection performances of the sample-to-answer platforms were comparable to the two tested conventional real-time RT-PCR assays. The test sensitivities of TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P assay and the Spike E484A assays were 100% and 96.6% respectively and the test specificities of both assays were 100%. An aberrant melting peak at Tm 42-44°C was observed when the specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay. Notably, the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike N501Y assay failed to detect the spike N501Y mutation of Omicron variant in the tested specimens.ConclusionsThe SARS-CoV-2 detection sensitivity of three commercial platforms, Roche cobas® 6800/8800, Roche cobas® Liat®, and Cepheid GeneXpert® systems were shown not to be impacted by the large number of mutations of VOC-Omicron. Also, the signature mutations i.e. Spike S371L/Spike S373P and Spike E484A in VOC-Omicron were correctly identified by the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike S371L/S373P and VirSNiP SARS-CoV-2 Spike E484A assays. Unexpected findings including a shifted melting peak or absence of amplification curve/melting peak were observed when specimens with Omicron variant were tested with the TIB MOLBIOL VirSNiP SARS-CoV-2 Spike E484K assay and Spike N501Y assay, suggesting a potential alert for Omicron variant, prior confirmation by whole genome sequencing.

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43.

Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus’s replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different human coronaviruses (HCoV). We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments.

Control of spring viremia of carp in common carp using RNA interference

In this study, the control of Carp sprivivirus, commonly known as spring viremia of carp virus (SVCV), replication in Epithelioma papulosum cyprini (EPC) cells and for the first time in common carp was investigated by using the RNA interference (RNAi) technology targeting the glycoprotein (G), matrix protein (M), and RNA-dependent RNA polymerase (L) genes. Small-interfering RNA sequences (siRNAs) were selected to target three different regions on both the SVCV-G (G-102, G-1070, and G-1456) and SVCV-M gene (M-88, M-271, and M-414). Designed specific siRNAs were investigated individually or in combinations in EPC cells. In vitro results showed that the most effective siRNAs were G-102 and M-271 that significantly reduced the virus titer in EPC cells by 1.6 logs and 1.5 logs respectively. Various combinations of the G-102 and M-271 of this study and siRNA-L-602 (targets the L-gene) from our previous study were investigated in vitro. Results showed that the most effective combination in reducing the virus replication in EPC cells was the siRNA-M-271 + L-602 combination as indicated by the significant reduction in virus titer (2.9 logs). In-vivo investigation of the siRNA-M-271 + L-602 combination by single or two successive doses in common carp challenged by IP injection of SVCV showed a significant reduction in mortality rate by 77.78% and 88.89% respectively. Results were confirmed by measuring the downregulation of SVCV-M and -L gene expression in vivo.Results of this study proved that SVCV can be controlled in common carp through targeting the SVCV-M and -L genes by using the RNAi technology that results in a significant in-vivo reduction of the SVCV replication.

SARS-CoV-2 RNA Detection on Environmental Surfaces in a University Setting of Central Italy.

The transmission of SARS-CoV-2 occurs through direct contact (person to person) and indirect contact by means of objects and surfaces contaminated by secretions from individuals with COVID-19 or asymptomatic carriers. In this study, we evaluated the presence of SARS-CoV-2 RNA on surfaces made of different materials located in university environments frequented by students and staff involved in academy activity during the fourth pandemic wave (December 2021). A total of 189 environmental samples were collected from classrooms, the library, computer room, gym and common areas and subjected to real-time PCR assay to evaluate the presence of SARS-CoV-2 RNA by amplification of the RNA-dependent RNA polymerase (RdRp) gene. All samples gave a valid result for Internal Process Control and nine (4.8%) tested very low positive for SARS-CoV-2 RNA amplification with a median Ct value of 39.44 [IQR: 37.31–42.66] (≤1 copy of viral genome). Our results show that, despite the prevention measures implemented, the presence of infected subjects cannot be excluded, as evidenced by the recovery of SARS-CoV-2 RNA from surfaces. The monitoring of environmental SARS-CoV-2 RNA could support public health prevention strategies in the academic and school world.