Virus-specific RNA Research Articles

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.

Isolation, Identification, and Pathogenicity of a Goose Astrovirus Genotype 1 Strain in Goslings in China.

Goose astrovirus genotype 1 (GAstV-1) has emerged in goose farms in some provinces of China in recent years and is considered to be one of the pathogens of gout in goslings in China. However, few studies have been conducted on the dynamic distribution, tissue tropism, and pathogenesis of GAstV-1 in goslings. In 2022, an epidemiological investigation of goose astrovirus (GAstV) in goslings was conducted in seven provinces of China. During the investigation, a GAstV-1 designated as GAstV-JSXZ was identified in the kidney of an 8-day-old gosling and was successfully isolated from a goose embryo. The full genome sequence of GAstV-JSXZ was determined using the next-generation sequencing technique. The complete genome of GAstV-JSXZ was 7299-nt-long. Interestingly, the phylogenetic analysis revealed that Chinese GAstV-1 has formed two distinct subgroups based on the ORF 2 genomes, designated GAstV-1 1a and GAstV-1 1b. The GAstV-JSXZ shared the highest identity with GAstV-1 1a strain FLX and TZ03 in nucleotides (ORF1a: 98.3-98.4%; ORF1b: 92.3-99.1%; ORF2: 95.8-98.8%) and amino acid sequences (ORF1a: 99.4-99.5%; ORF1b: 98.2-98.8%; ORF2: 97.0-99.4%). To evaluate the pathogenicity of GAstV-1, 1-day-old goslings were inoculated with the virus by oral and subcutaneous injection routes, respectively. The results revealed that the virus causes extensive pathological organ damage, especially in the kidney, liver, and thymus. Virus-specific genomic RNA could be detected in the cloacal swabs and tissues of infected goslings throughout the experiment. The viral copy numbers examined in the kidney and intestine were the highest, followed by the liver and spleen. These results are likely to provide a new understanding of the pathogenicity of GAstV-1 in geese.

Role of Plant Virus Movement Proteins in Suppression of Host RNAi Defense.

One of the systems of plant defense against viral infection is RNA silencing, or RNA interference (RNAi), in which small RNAs derived from viral genomic RNAs and/or mRNAs serve as guides to target an Argonaute nuclease (AGO) to virus-specific RNAs. Complementary base pairing between the small interfering RNA incorporated into the AGO-based protein complex and viral RNA results in the target cleavage or translational repression. As a counter-defensive strategy, viruses have evolved to acquire viral silencing suppressors (VSRs) to inhibit the host plant RNAi pathway. Plant virus VSR proteins use multiple mechanisms to inhibit silencing. VSRs are often multifunctional proteins that perform additional functions in the virus infection cycle, particularly, cell-to-cell movement, genome encapsidation, or replication. This paper summarizes the available data on the proteins with dual VSR/movement protein activity used by plant viruses of nine orders to override the protective silencing response and reviews the different molecular mechanisms employed by these proteins to suppress RNAi.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48-100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48-100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

1095. Retrospective Review of SARS-CoV-2 Infection in Immunocompromised Children at a Single Pediatric Institution

Abstract Background Immunocompromised individuals are considered higher risk for severe COVID-19 disease. Much of this data was collected from adults with limited pediatric reporting, although a recent study showed no worse outcome in immunocompromised children. In this study, we characterize our experience of immunocompromised children who were hospitalized with acute SARS-CoV-2 infection and aimed to identify a sub-group of patients who had worse outcomes. Methods We reviewed charts of all immunocompromised children hospitalized with SARS-CoV-2 RT-PCR positive results at our hospital from March 2020-April 2022. Disease severity was characterized according to the NIH COVID-19 guidelines. Clinical characteristics and outcomes were assessed. Individuals were grouped into solid organ transplant recipients, solid tumors, liquid tumors, and stem cell transplant recipients. Proportions were calculated per group in addition to the exact binomial confidence intervals (95%). Results We identified 39 pediatric aged patients ranging from 7 months to 20 years of age with a median age of 10. 35.9% (14/39) of individuals were solid organ transplant recipients, 25.6% (10/39) had solid tumors, 33% (13/39) had liquid tumors, and 5% (2/39) were stem cell transplant recipients. 17.9% were asymptomatic, 66.7% had mild disease, and 15.4% had severe/critical COVID-19 disease. The median time of positive SARS CoV-2 RT-PCR was 32 days. Six children had prolonged SARS-CoV-2 RT-PCR positive testing between 29-97 days with a mean of 49 days. Viral strand specific RNA testing for replicating virus was also persistently positive. Six experienced severe/critical disease resulting in death of three. Most of the patients were treated with predominantly T cell depleting agents (∼74%). However, four patients who had prolonged active critical infection were treated with B cell depleting agents or had known B cell dysfunction. Conclusion The majority of the immunocompromised children in our cohort had mild COVID-19 disease. A subset of individuals experienced ongoing prolonged RT-PCR positivity and testing indicating persistent viral replication, with three demonstrating an inability to control the virus and resulting in death. Worse outcomes may be related to their immune dysfunction and use of B cell depleting agents. Disclosures All Authors: No reported disclosures.

Phylogenetic And Recombination Analysis Of Yellow Mosaic Disease In Soybean Plant

Numerous viruses have been discovered in plants, both domestic and wild, in recent years. The biodiversity of plant viruses has been studied using a variety of methods, including virus-specific DNA or RNA, followed by sequencing and bioinformatics analyses. All the analysis reveals more knowledge about plant viruses. Phylogenetic analyses are increasingly being used in epidemiology, to understand the pattern of DNA/RNA virus infection in soybean plants, while the Recombination detection program is also used to analyse nucleotide sequence data and to identify the evidence of genetic recombination between the viruses infecting soybean.This study have found evidence that recombination is an important impelling cause in the evolution and divergence ofmany plant viruses, including begomoviruses, potyviruses and other viruses.

Rapid molecular assays for the detection of the four dengue viruses in infected mosquitoes.

The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48-100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48-100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.

Drug-Membrane Interactions: Effects of Virus-Specific RNA-Dependent RNA Polymerase Inhibitors Remdesivir and Favipiravir on the Structure of Lipid Bilayers.

The two RNA-dependent RNA polymerase inhibitors remdesivir and favipiravir were originally developed and approved as broad-spectrum antiviral drugs for the treatment of harmful viral infections such as Ebola and influenza. With the outbreak of the global SARS-CoV-2 pandemic, the two drugs were repurposed for the treatment of COVID-19 patients. Clinical studies suggested that the efficacy of the drugs is enhanced in the case of an early or even prophylactic application. Because the contact between drug molecules and the plasma membrane is essential for a successful permeation process of the substances and therefore for their intracellular efficiency, drug-induced effects on the membrane structure are likely and have already been shown for other substances. We investigated the impact of remdesivir and favipiravir on lipid bilayers in model and cell membranes via several biophysical approaches. The measurements revealed that the embedding of remdesivir molecules in the lipid bilayer results in a disturbance of the membrane structure of the tested phospholipid vesicles. Nevertheless, in a cell-based assay, the presence of remdesivir induced only weak hemolysis of the treated erythrocytes. In contrast, no experimental indication for an effect on the structure and integrity of the membrane was detected in the case of favipiravir. Regarding potential prophylactic or accompanying use of the drugs in the therapy of COVID-19, the physiologically relevant impacts associated with the drug-induced structural modifications of the membrane might be important to understand side effects and/or low effectivities.

Novel PCR Test to Differentiate Between Infections with SARS-CoV-2, Influenza A and B

PurposeInfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a worldwide spreading disease with acute respiratory distress syndrome as one of the major complications. In the early disease stage, COVID-19 cannot be distinguished from influenza based on the clinical symptoms. During viraemia, direct pathogen detection by reverse transcription polymerase chain reaction (RT-PCR) is the diagnostic gold standard. This study evaluated a novel real-time RT-PCR test for fast detection and differentiation of RNA from SARS-CoV-2 and influenza virus types A and B.Methods & MaterialsThe assays´ diagnostic performance was compared to CE-IVD/FDA-EUA-marked reference PCR tests. RNA was extracted from patient samples collected as nasopharyngeal or oropharyngeal swabs. Virus-specific RNA was amplified after reverse transcription using the EURORealTime SARS-CoV-2/Influenza A/B PCR test (EUROIMMUN) allowing simultaneous detection of two target sequences in the SARS-CoV-2 ORF1ab and N genes as well as one target sequence each for influenza virus A and B. Assays were carried out on the CFX96 cycler (Bio-Rad) and evaluated with the EURORealTime Analysis Software (EUROIMMUN). The 95% limit of detection (LoD) was determined by Probit analysis using a dilution series of quantified target RNA. To exclude cross-reactivity and interference, the assay was run against human genomic DNA/RNA, nucleic acids from different viral, bacterial and fungal pathogens, and potentially interfering substances.ResultsCompared to the reference PCR tests, the EURORealTime SARS-CoV-2/Influenza A/B showed positive agreements of 97.8%, 93.0% and 100% and negative agreements of 100%, 100% and 98.9% for SARS-CoV-2, influenza A and influenza B, respectively. The 95% LoD values were calculated to be 0.55cp/µl for SARS-CoV-2, 0.92cp/µl for influenza A H3N2, 0.67cp/µl for influenza A H1N1 and 1.21cp/µl for influenza B. No cross-reactivities with human or pathogen-specific nucleic acids or interferences were detected.ConclusionThe novel test is able to detect SARS-CoV-2, influenza A and influenza B with high sensitivity and clearly discriminate between these viruses. It is therefore optimally suited for differential diagnostics for patients presenting with symptoms compatible with COVID-19 and influenza. Combined detection of the three pathogens in one multiparameter assay helps to save time and resources in the diagnostic workup.

Small RNA Response to Infection of the Insect-Specific Lammi Virus and Hanko Virus in an Aedes albopictus Cell Line

RNA interference (RNAi)-mediated antiviral immunity is believed to be the primary defense against viral infection in mosquitoes. The production of virus-specific small RNA has been demonstrated in mosquitoes and mosquito-derived cell lines for viruses in all of the major arbovirus families. However, many if not all mosquitoes are infected with a group of viruses known as insect-specific viruses (ISVs), and little is known about the mosquito immune response to this group of viruses. Therefore, in this study, we sequenced small RNA from an Aedes albopictus-derived cell line infected with either Lammi virus (LamV) or Hanko virus (HakV). These viruses belong to two distinct phylogenetic groups of insect-specific flaviviruses (ISFVs). The results revealed that both viruses elicited a strong virus-derived small interfering RNA (vsiRNA) response that increased over time and that targeted the whole viral genome, with a few predominant hotspots observed. Furthermore, only the LamV-infected cells produced virus-derived Piwi-like RNAs (vpiRNAs); however, they were mainly derived from the antisense genome and did not show the typical ping-pong signatures. HakV, which is more distantly related to the dual-host flaviviruses than LamV, may lack certain unknown sequence elements or structures required for vpiRNA production. Our findings increase the understanding of mosquito innate immunity and ISFVs’ effects on their host.

Characterization of an outbreak of cardiomyopathy syndrome (CMS) in young Atlantic salmon, Salmo salar L.

Cardiomyopathy syndrome (CMS) is the most common viral cardiac disease in Norwegian Atlantic salmon farming and typically affects large, market size fish. Only six months after seawater transfer, Atlantic salmon were diagnosed with CMS at a fish farm in the south-western part of Norway. Due to the unexpected young age and the remarkable large amounts of virus-specific RNA (Ct <10), the fish group was monitored with five additional samplings until slaughtered almost 10months later. At three weeks after the first CMS diagnosis (weeks post-diagnosis, wpd) and at slaughter (39 wpd), more comprehensive samplings were performed of the study cage, with specific focus on three different cardiac compartments. The clinical, autopsy and histopathological findings at first diagnosis and at all succeeding samplings were similar to previous descriptions of typical CMS. A slightly elevated mortality was observed in the cage with diseased fish at the time of the first CMS diagnosis and continued throughout the study. The prevalence and load of PMCV-specific RNA in the fish remained high until slaughtering, with similar amounts in all sampled cardiac compartments. No fish from the other five cages at the site were diagnosed with CMS, until fish sampled from the last cage at the site were diagnosed 10weeks after slaughtering of the study cage (49 wpd). Sequence analysis of the PMCV on the site showed that the outbreak virus was similar to PMCV variants previously sequenced from Norwegian field outbreaks. In conclusion, CMS in young Atlantic salmon had clinical signs and histopathological cardiac lesions typical for the disease, and diseased fish could be found in the study cage until slaughtering.

Role and Virology of RSV and RSV RNA Polymerase

Respiratory syncytial virus (RSV) is a highly contagious, respiratory virus that commonly infects children under the age of five. RSV infections can present as cold-like symptoms which include a runny nose, coughing, and fever among other common symptoms; however, RSV can cause more severe infections such as bronchiolitis and pneumonia in young children and older, immunosuppressed patients. Despite RSV being a leading cause for respiratory illness in small children, there is limited research on its molecular biology, and especially its RNA polymerase. That being said, there has been slow progress in the development of a vaccine, partly due to the history of failed trials in the past. What is known is that the viral infection of RSV is initiated through the delivery of a virus-specific RNA synthesis machine into the host cell. This machine is responsible for both genome replication and gene transcription, thus including the RNA polymerase as a component. The RSV RNA polymerase consists of two units, a core large (L) protein, and a modulatory phosphoprotein (P). The L protein is 250 kDa with three functional domains: the RNA dependent RNA polymerization (RdRp), the capping (Cap), and the cap methyltransferase (MT) domains. As for the P protein, it has two domains that are understood to be the Oligomerization domain (POD) and the C-terminal domain (PPTD). These two domains are known to interact with the RdRp and Cap domains of the L protein which is essential in stabilizing the structure of the overall protein. The L protein serves as the core of the RSV structure and is responsible for the catalyzation of the RNA polymerization in replication and transcription, the cap addition, and cap methylation of nascent viral mRNAs. When synthesizing RNA, RSV RNA uses the nucleoprotein coated genomic RNA (N:RNA) as the template. In this process, the L protein requires the tetrameric P protein to displace the nucleoproteins. The synthesis of the mRNA caps first begins when a polyribonucleotide transferase forms the mRNA caps. This is a notable distinction from eukaryotes and other viral families which use guanylyltransferase instead. The caps are then methylated by the RNA polymerase at the 2’-O position, followed by the N-7 position, a pattern opposite from the order followed in mammalian mRNA. The refined mechanisms of these processes have yet to be described, but it is speculated that they share similarities with the cap addition and methylation assays of vesicular stomatitis virus (VSV), whose assays have been reported. The Walton High School MAPS team worked in partnership with the MSOE Center for Biomolecular Modeling to design a 3D model of RSV RNA Polymerase using a recently published cryo-electron microscopy structure to further investigate and examine the structure-function relationship of RSV RNA polymerase.

Effects of Virus Specific Short-Interfering RNAs on Enterovirus D68 Induced Lung Injury

Enterovirus D68 (EV-D68) is an emerging respiratory pathogen. We previously described siRNAs targeting the EV-D68 RNA dependent RNA polymerase, which were able to suppress EV-D68 replication and cytopathic effect in vitro. We sought to assess the effects of intranasal application of these siRNAs in vivo, utilizing a previously established animal model of EV-D68 infection.

Pathogenicity of novel goose-origin astrovirus causing gout in goslings

BackgroundA novel goose-origin astrovirus (GoAstV) has broken out across China in recent years, causing gout in goslings with a mortality rate of around 50%. However, our understanding of the dynamic distribution, tissue tropism and pathogenesis of GoAstV is incomplete. In order to assess its pathogenicity, one-day-old goslings were inoculated separately with GoAstV via oral and subcutaneous injection routes.ResultsClinical symptoms, gross and microscopic lesions, blood biochemical parameters and viral loads were detected and recorded for 20 days after infection. Typical gout was observed in experimental goslings. GoAstV can be replicated in tissues and cause pathological damage, especially in the kidney, liver, heart and spleen. Virus-specific genomic RNA was detected in blood, cloacal swabs and all representative tissues, and virus shedding was detected up to 20 days after inoculation, suggesting that GoAstV has a wide tissue tropism and spread systematically after inoculation. The viral copy numbers examined in kidney were the highest, followed by spleen and liver.ConclusionThis experiment determined the accurate value of viral loads and biochemical indicators of GoAstV-induced goslings. These findings increase our understanding of the pathogenicity of GoAstV in goslings and provide more reference for future research.

BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle.

Latent infection with herpesviruses constitutively activates inflammasomes, while lytic replication suppresses their activation through distinct mechanisms. However, how Epstein-Barr virus (EBV) lytic replication inhibits the activation of inflammasomes remains unknown. Here, we reveal that the EBV immediate-early protein BRLF1 inhibits inflammasome activation, and BRLF1 deficiency significantly increases the activation of inflammasomes and pyroptosis during early lytic lifecycle. BRLF1 interacts with RNA polymerase III subunits to suppress immunostimulatory small RNA transcription, RIG-I inflammasome activation, and antiviral responses. Consequently, BRLF1-deficient EBV primary infection induces robust T-cell and NK cell activation and killing through IL-1β and IL-18. A BRLF1-derived peptide that inhibits inflammasome activation is sufficient to suppress T-cell and NK cell responses during BRLF1-deficient EBV primary infection in lymphocytes. These results reveal a novel mechanism involved in the evasion of inflammasome activation and antiviral responses during EBV early lytic infection and provide a promising approach for the manipulation of inflammasomes against infection of oncogenic herpesviruses.

Variplex\u2122 test system fails to reliably detect SARS-CoV-2 directly from respiratory samples without RNA extraction

Diagnosis of COVID is performed by PCR methods, but their capacity is limited by the requirement of high-level facilities and instruments. The loop-mediated isothermal amplification (LAMP) method has been utilized for the detection of isolated virus-specific RNA. Preliminary data suggest the possibility of isothermal amplification directly from respiratory samples without RNA extraction. All patients admitted to our hospital were screened for SARS-CoV-2 by routine. Respiratory samples were tested by variplex system based on LAMP method directly without RNA extraction and by PCR. Primary endpoint was the false-negative rate of variplex test compared with PCR as gold standard. In 109 patients variplex test and PCR assay were performed simultaneously. Median age was 80 years and male/female ratio was 40/60%. The prevalence of PCR-confirmed COVID diagnosis was 43.1%. Variplex test was positive in 13.8%. False-negative rate of variplex test compared with PCR was 83.0%. The potential of LAMP technology using isolated RNA has been demonstrated impressively by others, and excellent sensitivity and specificity of detecting SARS-CoV-2 has been reported. However, without RNA extraction, the variplex test system failed to reliably detect SARS-CoV-2 directly in respiratory samples.

The Correlation of Neutralizing Antibody Responses with SARS-CoV-2 Virus Clearance and Lung Disease Prognosis: Implication from a Family Cluster Study

Background: A novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing pneumonia-associated respiratory disease pandemic across the world. Until now, the viral prevalence patterns, radiological characters, and antibody responses at initial stage of SARS-CoV-2 infection have been reported. The prolonged viral RNA shedding and coexistence with viral specific IgG antibody in discharged patients have also been reported recently. To date, however, correlations among viral specific RNA shedding, radiological pattern shift, and serum neutralizing antibody response covering the whole acute- and convalescent- phase in patients infected with SARS-CoV-2 are unknown. Focusing on a family cluster, present study is aim to give a bird view on the correlation of neutralizing antibody responses with viral clearance and lung disease prognosis from acute to convalescent phases. Methods: We conducted a surveillance study on a familial cluster of COVID-19 patients from Jan 17 to Apr 27 (99 days), 2020, in Guangzhou, Guangdong province, China. Epidemiological study was performed to clarify the virus transmission inside a family. NPAs, stool, and serum samples were collected and conducted RT-PCR to test the presence of SARS-CoV-2. Serum was used to perform neutralizing antibody tests to determine dynamic levels of neutralizing antibody. Radiological images as well as high-resolution CT scans for each patients were continually recorded to monitor outcomes of lung recovery. Findings: In this family cluster, three members (P2, P3, P4) in Guangzhou were infected by SARS-CoV-2 due to a close contact with P1 (index), who came back Guangzhou from Wuhan on Jan, 17, 2020, while another two members (H1 and H2) were unsusceptible and negative to SARS-CoV-2 test. The patients (P1, P2, P3) presented varying degrees of clinical symptoms on admission including recurrent fever, cough, and dyspnea, while P4 was asymptomatic. CT images showed ground-glass opacities in lungs of P1-3. The median length from illness onset to discharge for four-confirmed cases was 21.3 (4.7) days (range from 15 to 26). However, all patients returned positive for SARS-CoV-2 in follow-up investigation. The mean time for RNA shedding was 36.5 (3.2) days in P1, P2, and P4. The neutralizing antibody of P1, P2, and P4 was detectable during illness day 16 to 22. Then, the level was apparently ascent to a relative high titer and remained stable during 34-40, 31-41, and 35 days of illness onset for P1, P2, and P4, respectively, while declined gradually with time. The last positive time for SARS-CoV-2 test for P1, P2, and P4 was 40, 35, and 34 days of illness onset, respectively. Follow-up chest CT showed good prognosis in lung disease of P1 and P2, and CT scan was normal for P4. P3 was an exceptional case in this family cluster, who experienced the longest RNA shedding time (up to 68 days) as well as lung disease. In particular, radiological worsen was observed at day 34 of illness onset, high-resolution CT image revealed a glass-ground opacity in middle segment of right lung since day 45 to 99 of illness onset. The neutralizing antibody level in P3 was low during the entire study and significantly lower than that in other patients. Interpretation: The efficiency of SARS-CoV-2 virus clearance and lung disease prognosis were closely related to the levels of neutralizing antibody in COVID-19 patients. Viral clearance was observed along with high level of neutralizing antibody in P1, P2, and P4. However, the level of neutralizing antibody in P3 was not high enough to clear the virus completely, leading a prolong RNA shedding and lung disease. We proposed that high level of neutralizing antibody was essential for viral clearance and lung recovery, and virus was thoroughly eliminated during the period when neutralizing antibody reached high level in serum. Funding Statement: This work was supported by grants from the Guangzhou Science and Technology Program (201904010012), Guangdong Provincial Science and Technology Program (2018B020207006), Guangdong Provincial Novel Coronavirus Scientific and Technological Project (2020111107001). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was approved by the Institutional Review Board of the Guangdong Second Provincial General Hospital. All patients involved in this study signed the clinical research informed consent.

Evidence for Gastrointestinal Infection of SARS-CoV-2

No abstract available Keywords: ACE2; Gastrointestinal Infection; Oral-Fecal Transmission; SARS-CoV-2.

Transgenic Wheat Harboring an RNAi Element Confers Dual Resistance Against Synergistically Interacting Wheat Streak Mosaic Virus and Triticum Mosaic Virus.

Wheat streak mosaic virus (WSMV) and triticum mosaic virus (TriMV) are economically important viruses of wheat (Triticum aestivum L.), causing significant yield losses in the Great Plains region of the United States. These two viruses are transmitted by wheat curl mites, which often leads to mixed infections with synergistic interaction in grower fields that exacerbates yield losses. Development of dual-resistant wheat lines would provide effective control of these two viruses. In this study, a genetic resistance strategy employing an RNA interference (RNAi) approach was implemented by assembling a hairpin element composed of a 202-bp (404-bp in total) stem sequence of the NIb (replicase) gene from each of WSMV and TriMV in tandem and of an intron sequence in the loop. The derived RNAi element was cloned into a binary vector and was used to transform spring wheat genotype CB037. Phenotyping of T1 lineages across eight independent transgenic events for resistance revealed that i) two of the transgenic events provided resistance to WSMV and TriMV, ii) four events provided resistance to either WSMV or TriMV, and iii) no resistance was found in two other events. T2 populations derived from the two events classified as dual-resistant were subsequently monitored for stability of the resistance phenotype through the T4 generation. The resistance phenotype in these events was temperature-dependent, with a complete dual resistance at temperatures ≥25°C and an increasingly susceptible response at temperatures below 25°C. Northern blot hybridization of total RNA from transgenic wheat revealed that virus-specific small RNAs (vsRNAs) accumulated progressively with an increase in temperature, with no detectable levels of vsRNA accumulation at 20°C. Thus, the resistance phenotype of wheat harboring an RNAi element was correlated with accumulation of vsRNAs, and the generation of vsRNAs can be used as a molecular marker for the prediction of resistant phenotypes of transgenic plants at a specific temperature.

Lack of nsP2-specific nuclear functions attenuates chikungunya virus replication both in vitro and in vivo

Chikungunya virus (CHIKV) is an important arthritogenic human pathogen that is already circulating in both hemispheres. In the present study, we substituted VLoop, located on the surface of nsP2, by other amino acid sequences. These modifications had deleterious effects on viral nuclear functions and made CHIKV incapable of interfering with the induction of type I interferon and the antiviral response in both mouse and human cells. Importantly, the identified mutations have no significant effects on the synthesis of virus-specific RNAs and viral structural proteins. The designed mutants induced a few orders of magnitude lower viremia but remained highly immunogenic in mice. Thus, the proposed modifications of nsP2 can additionally improve the safety of the attenuated strain CHIKV 181/25. Furthermore, defined mutations in the macro domain of another nonstructural protein, nsP3, additionally reduce cytopathogenicity of nsP2 mutants in human cells, and can be potentially applied for CHIKV attenuation.