Viral RNA Segments Research Articles

Exposure of Culicoides sonorensis to Enzootic Strains of Bluetongue Virus Demonstrates Temperature- and Virus-Specific Effects on Virogenesis.

Bluetongue virus (BTV) is a segmented RNA virus transmitted by Culicoides midges. Climatic factors, animal movement, vector species, and viral mutation and reassortment may all play a role in the occurrence of BTV outbreaks among susceptible ruminants. We used two enzootic strains of BTV (BTV-2 and BTV-10) to explore the potential for Culicoides sonorensis, a key North American vector, to be infected with these viruses, and identify the impact of temperature variations on virogenesis during infection. While BTV-10 replicated readily in C. sonorensis following an infectious blood meal, BTV-2 was less likely to result in productive infection at biologically relevant exposure levels. Moreover, when C. sonorensis were co-exposed to both viruses, we did not detect reassortment between the two viruses, despite previous in vitro findings indicating that BTV-2 and BTV-10 are able to reassort successfully. These results highlight that numerous factors, including vector species and exposure dose, may impact the in vivo replication of varying BTV strains, and underscore the complexities of BTV ecology in North America.

Characterization of the Molecular Interactions That Govern the Packaging of Viral RNA Segments into Rift Valley Fever Phlebovirus Particles.

Rift Valley fever phlebovirus (RVFV) has a single-stranded, negative-sense RNA genome, consisting of L, M, and S segments. The virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes (RNPs), composed of encapsidated genomes carrying N protein and the viral polymerase, L protein. A quantitative analysis of the profile of viral RNA segments packaged into RVFV particles showed that all three genomic RNA segments had similar packaging abilities, whereas among antigenomic RNA segments, the antigenomic S RNA, which serves as the template for the transcription of mRNA expressing the RVFV virulence factor, NSs, displayed a significantly higher packaging ability. To delineate the factor(s) governing the packaging of RVFV RNA segments, we characterized the interactions between Gn and viral RNPs in RVFV-infected cells. Coimmunoprecipitation analysis demonstrated the interaction of Gn with N protein, L protein, and viral RNAs in RVFV-infected cells. Furthermore, UV-cross-linking and immunoprecipitation analysis revealed, for the first time in bunyaviruses, the presence of a direct interaction between Gn and all the viral RNA segments in RVFV-infected cells. Notably, analysis of the ability of Gn to bind to RVFV RNA segments indicated a positive correlation with their respective packaging abilities and highlighted a binding preference of Gn for antigenomic S RNA, among the antigenomic RNA segments, suggesting the presence of a selection mechanism for antigenomic S RNA incorporation into infectious RVFV particles. Collectively, the results of our study illuminate the importance of a direct interaction between Gn and viral RNA segments in determining their efficiency of incorporation into RVFV particles. IMPORTANCE Rift Valley fever phlebovirus, a bunyavirus, is a mosquito-borne, segmented RNA virus that can cause severe disease in humans and ruminants. An essential step in RVFV life cycle is the packaging of viral RNA segments to produce infectious virus particles for dissemination to new hosts. However, there are key gaps in knowledge regarding the mechanisms that regulate viral RNA packaging efficiency in bunyaviruses. Our studies investigating the mechanism of RNA packaging in RVFV revealed the presence of a direct interaction between the viral envelope glycoprotein, Gn, and the viral RNA segments in infected cells, for the first time in bunyaviruses. Furthermore, our data strongly indicate a critical role for the direct interaction between Gn and viral RNAs in determining the efficiency of incorporation of viral RNA segments into RVFV particles. Clarifying the fundamental mechanisms of RNA packaging in RVFV would be valuable for the development of antivirals and live-attenuated vaccines.

Cell culture-based production and in vivo characterization of purely clonal defective interfering influenza virus particles

BackgroundInfections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.ResultsIn the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E−2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1r/r mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.ConclusionIn summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.

Rotavirus as an Expression Platform of Domains of the SARS-CoV-2 Spike Protein.

Among vaccines administered to children are those targeting rotavirus, a segmented double-stranded RNA virus that represents a major cause of severe gastroenteritis. To explore the feasibility of establishing a combined rotavirus-SARS-CoV-2 vaccine, we generated recombinant (r)SA11 rotaviruses with modified segment 7 RNAs that contained coding cassettes for NSP3, a translational 2A stop-restart signal, and a FLAG-tagged portion of the SARS-CoV-2 spike (S) protein: S1 fragment, N-terminal domain (NTD), receptor-binding domain (RBD), extended RBD (ExRBD), or S2 core (CR) domain. Generation of rSA11 containing the S1 coding sequence required a sequence insertion of 2.2 kbp, the largest such insertion yet introduced into the rotavirus genome. Immunoblotting showed that rSA11 viruses containing the smaller NTD, RBD, ExRBD, and CR coding sequences expressed S-protein products of expected size, with ExRBD expressed at highest levels. These rSA11 viruses were genetically stable during serial passage. In contrast, the rSA11 virus containing the full-length S coding sequence (rSA11/NSP3-fS1) failed to express its expected 80 kDa fS1 product, for unexplained reasons. Moreover, rSA11/NSP3-fS1 was genetically unstable, with variants lacking the S1 insertion appearing during serial passage. Nonetheless, these results emphasize the potential usefulness of rotavirus vaccines as expression vectors of immunogenic portions of the SARS-CoV-2 S protein, including NTD, RBD, ExRBD, and CR, that have sizes smaller than the S1 fragment.

Segment-specific kinetics of mRNA, cRNA and vRNA accumulation during influenza infection.

Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major epidemics and pandemics. The replication of IAV is complex, involving the production of three distinct RNA species; mRNA, cRNA, and vRNA for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here we report the development of a novel pipeline using total stranded RNA-Seq, which we named Influenza Virus Enumerator of RNA Transcripts (InVERT), that allows for the simultaneous quantification of all three RNA species produced by IAV. Using InVERT, we provide a full landscape of the IAV replication kinetics and found that different groups of viral genes follow different kinetics. The segments coding for RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other segments (NP, NS, HA) consistently made more mRNA than vRNA. vRNA expression levels did not correlate with cRNA expression, suggesting complex regulation of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall, InVERT is a powerful tool for quantifying IAV RNA species to elucidate key features of IAV replication.ImportanceInfluenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood. This is due in part to both technical limitations and the complexity of the virus replication, which has a segmented genome and produces three distinct RNA species for each gene segment. We developed a new approach that allows the methodical study of IAV replication kinetics, shedding light on many interesting features of IAV replication biology. This study advances our understanding of the kinetics of IAV replication and will help to facilitate future research in the field.

Genomic Analysis of the First Virological Evidence of a Human Case of Crimean-Congo Haemorrhagic Fever Virus (CCHFV) in Nigeria

Several studies in both humans and animals in Nigeria have presented evidence for the endemicity of CCHFV in Nigeria. In spite of the aforementioned, there has been no confirmed human case of CCHF infection in Nigeria. We present a study giving the first serologic and virological evidence of CCHF in a 15-year-old female, She was admitted in March 2012 into the female medical ward of the University of Maiduguri Teaching Hospital, a tertiary health facility in northeastern Nigeria with a 6 day history of fever, body pain, bloody diarrhoea, and epistaxis. She resides in a peri-urban settlement in old Maiduguri located not far from a major municipal abattoir in Maiduguri, the capital city of Borno state, Nigeria. The abattoir serves as the major animal slaughter house within Maiduguri city. Animals (Camel, Cattle, Goat and Sheep) are brought from within the Stat Borno), neighbouring states and mostly from neighbouring countries such as Cameroun, Chad, Niger, and as far as Sudan, Central Africa. The RNA extracted from this sample (N428) was further characterized by next generation sequencing (NGS) which resulted in complete S, M, and L viral RNA segment sequences. Phylogenetic analysis clustered the S-segment in the Africa 3 clade or phylogenetic group. The S-segment open-reading frame showed close homology with a previous isolate of CCHFV from Nigeria (IbAr10200), as well as isolates from Mauritania (ArD39554) and South Africa (SPU415/85 and SPU128/61/7). The M and L clustered closely with the Sudan ABI-2009 isolate and the Nigeria IbAr10200. The L, M, and S sequences were submitted to GenBank with accession numbers KX238956, KX238957 and KX238958 respectively. This genomic analysis provides the first published evidence of a human case of CCHFV in Nigeria and its phylogenetic context.

Identification of a Novel Cis-Acting Regulator of HIV-1 Genome Packaging.

Human immunodeficiency virus type 1 (HIV-1) uptakes homo-dimerized viral RNA genome into its own particle. A cis-acting viral RNA segment responsible for this event, termed packaging signal (psi), is located at the 5′-end of the viral genome. Although the psi segment exhibits nucleotide variation in nature, its effects on the psi function largely remain unknown. Here we show that a psi sequence from an HIV-1 regional variant, subtype D, has a lower packaging ability compared with that from another regional variant, HIV-1 subtype B, despite maintaining similar genome dimerization activities. A series of molecular genetic investigations narrowed down the responsible element of the selective attenuation to the two sequential nucleotides at positions 226 and 227 in the psi segment. Molecular dynamics simulations predicted that the dinucleotide substitution alters structural dynamics, fold, and hydrogen-bond networks primarily of the psi-SL2 element that contains the binding interface of viral nucleocapsid protein for the genome packaging. In contrast, such structural changes were minimal within the SL1 element involved in genome dimerization. These results suggest that the psi 226/227 dinucleotide pair functions as a cis-acting regulator to control the psi structure to selectively tune the efficiency of packaging, but not dimerization of highly variable HIV-1 genomes.

Different survival of three populations of European sea bass (Dicentrarchus labrax) following challenge with two variants of nervous necrosis virus (NNV)

Viral Nervous Necrosis (VNN, also called viral encephalo- and retinopathy (VER)), is a widespread disease of marine aquaculture caused by betanodavirus (or nervous necrosis virus - NNV), a segmented positive sense RNA virus, member of the nodaviridae family. VNN affects predominantly marine fish and cause significant losses to the Mediterranean fish farming industry, including the production of European sea bass (Dicentrarchus labrax). Of the four circulating genotypes of betanodavirus, red-spotted grouper NNV (RGNNV) and the reassortant genotype red-spotted grouper/striped jack NNV (RG/SJNNV) are most prevalent in the Mediterranean. Inheritable resistance against VNN has been detected in sea bass, and selective breeding could be a mean to limit this untreatable disease. In the current study, we compare resistance to disease among three populations from the Atlantic Ocean (AT), Eastern Mediterranean (EM) and Western Mediterranean (WM), by challenge trials using both a highly pathogenic isolate of RGNNV and a lower pathogenic reassortant isolate of RG/SJNNV. The survival of the three populations were modelled with a logistic regression, and the odds ratio (OR) of surviving was calculated. The challenge with RG/SJNNV reduced the odds of surviving three-fold (OR = 0.29 [0.07-0.87]), whereas the challenge with RGNNV reduced the odds of surviving 100-fold (OR = 0.01 [0.00-0.03]). Overall, the EM population had 3.32 (1.92–5.86) times higher odds of surviving the challenge than the AT and WM stocks. All survivors were harboring viral RNA in the brain, as demonstrated by RT-qPCR. However, viral RNA levels were in average lower in survivors from the EM population in both challenges, though only significantly lower in the challenge with RG/SJNNV (p < 0.01). The survival results combined with the RT-qPCR results indicate that the EM sea bass population has a natural resistance to disease caused by RGNNV, possibly associated with limited viral entry into and/or replication in the brain.

Late Male-Killing Viruses in Homona magnanima Identified as Osugoroshi Viruses, Novel Members of Partitiviridae.

Late male-killing, a male-specific death after hatching, is a unique phenotype found in Homona magnanima, oriental tea tortrix. The male-killing agent was suspected to be an RNA virus, but details were unknown. We herein successfully isolated and identified the putative male-killing virus as Osugoroshi viruses (OGVs). The three RNA-dependent RNA polymerase genes detected were phylogenetically related to Partitiviridae, a group of segmented double-stranded RNA viruses. Purified dsRNA from a late male-killing strain of H. magnanima revealed 24 segments, in addition to the RdRps, with consensus terminal sequences. These segments included the previously found male-killing agents MK1068 (herein OGV-related RNA16) and MK1241 (OGV-related RNA7) RNAs. Ultramicroscopic observation of purified virions, which induced late male-killing in the progeny of injected moths, showed sizes typical of Partitiviridae. Mathematical modeling showed the importance of late male-killing in facilitating horizontal transmission of OGVs in an H. magnanima population. This study is the first report on the isolation of partiti-like virus from insects, and one thought to be associated with late male-killing, although the viral genomic contents and combinations in each virus are still unknown.

A novel emaravirus comprising five RNA segments is associated with ringspot disease in oak

We report the complete nucleotide sequence of the genome of a novel virus in ringspot-diseased common oak (Quercus robur L.). The newly identified pathogen is associated with leaf symptoms such as mottle, chlorotic spots and ringspots on diseased trees. High-throughput sequencing (HTS, Illumina RNASeq) was used to explore the virome of a ringspot-diseased oak that had chlorotic ringspots of suspected viral origin on leaves for several years. Bioinformatic analysis of the HTS dataset followed by RT-PCR enabled us to determine complete sequences of four RNA genome segments of a novel virus. These sequences showed high similarity to members of the genus Emaravirus, which includes segmented negative-stranded RNA viruses of economic importance. To verify the ends of each RNA, we conducted rapid amplification of cDNA ends (RACE). We identified an additional genome segment (RNA 5) by RT-PCR using a genus-specific primer (PDAP213) to the conserved 3´ and 5´termini in order to amplify full-length genome segments. RNA 5 encodes a 21-kDa protein that is homologous to the silencing suppressor P8 of High Plains wheat mosaic virus. The five viral RNAs were consistently detected by RT-PCR in ringspot-diseased oaks in Germany, Sweden, and Norway. We conclude that the virus represents a new member of the genus Emaravirus affecting oaks in Germany and in Scandinavia, and we propose the name “common oak ringspot-associated emaravirus” (CORaV).

Structural and functional characterization of the replication and transcription activities of Hantaan virus polymerase

The Bunyavirales order is a large and worldwide distributed group of segmented negative sense single-stranded RNA viruses (sNSV) that includes more than 350 species (nine families). Arthropods and rodents are their natural reservoirs and humans are occasionally infected resulting in severe diseases. Particularly, the pathogenic prototype viruses Hantaan virus (HTNV, family Hantaviridae) and Crimean-Congo Haemorrhagic fever virus (CCHFV, family Nairoviridae) have increased their geographical expansion and as a consequence, also cases of human diseases. Thus, it is necessary to (i) understand their mechanism of infection and (ii) to develop effective drugs to counteract them. In this perspective, we are working on two critical steps of bunyavirus viral cycle : replication and transcription. These processes are carried out by the multifunctional viral polymerase (L). In order to decipher the molecular mechanisms of bunyavirus replication, we present the study of interactions between hantavirus L proteins and their genomic RNA and replication assays of the full-length Hantaan polymerase. By electrophoresis mobility shifts assays and fluoresce anisotropy we determined the viral sequences specifically binding the L proteins anddefined their length and measured their affinity. We could see differences in the interactions between bunyaviral families L proteins suggesting differences on their mechanisms of replication and the way they are regulated. On the other hand, we performed replication assays for HNTV and LACV L and we have obtained a different result patron. These studies on replication and transcription reactions have shown that Hantaan L is more active than LACV L in the presence or absence of N terminal TAG. Also, the expected replication products are different and we observed some reproducible abortive products.We observed an unexpected UTP transferase activity by the Hantaan L protein that seems related to the processing of its genomic RNA for preventing recognition by the cell innate immune system and maintaining genome integrity. TheseRNA-protein interactions studies, along with the replication assays, will provide the basis for subsequent biochemical and structural studies to understand the molecular mechanism uncovering these reactions. This will be crutial for the development of effective antiviral drugs.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Molecular Characteristics of Jujube Yellow Mottle-Associated Virus Infecting Jujube (Ziziphus jujuba Mill.) Grown at Aksu in Xinjiang of China.

Chinese jujube (Ziziphus jujuba Mill.) is a native fruit crop in China. Leaf mottle and dapple fruit disease is prevalent in cultivated jujube plants grown at Aksu in Xinjiang Uygur Autonomous Region of China. Jujube yellow mottle-associated virus (JYMaV), a tentative member in the genus Emaravirus, was recently identified from mottle-diseased jujube plants grown in Liaoning Province in China, but its incidence and genetic diversity in China is unknown. In this study, the genome sequences of three JYMaV isolates from two jujube cultivars and one jujube variant were determined by high-throughput sequencing (HTS) for small RNA and rRNA-depleted RNA coupled with RT-PCR assays. Comparison of these sequences together with sequences of the viral RNA segments derived by primer set 3C/5H-based RT-PCR revealed that genetic diversity was present in the virus populations and high sequence variation occurred at the non-translational regions of each of the viral genomic segments. Field investigation confirmed the close association of the virus with leaf mottle symptoms of jujube plants. Furthermore, this study revealed that P5 encoded in the viral RNA5 displayed a nuclear localization feature differing from the plasmodesma (PD) subcellular localization of the virus movement protein (P4), and the two proteins could interact with each other in the BiFC assays. Our study provides a snapshot of JYMaV genetic diversity in its natural hosts.

Randomly primed, strand-switching, MinION-based sequencing for the detection and characterization of cultured RNA viruses.

RNA viruses rapidly mutate, which can result in increased virulence, increased escape from vaccine protection, and false-negative detection results. Targeted detection methods have a limited ability to detect unknown viruses and often provide insufficient data to detect coinfections or identify antigenic variants. Random, deep sequencing is a method that can more fully detect and characterize RNA viruses and is often coupled with molecular techniques or culture methods for viral enrichment. We tested viral culture coupled with third-generation sequencing for the ability to detect and characterize RNA viruses. Cultures of bovine viral diarrhea virus, canine distemper virus (CDV), epizootic hemorrhagic disease virus, infectious bronchitis virus, 2 influenza A viruses, and porcine respiratory and reproductive syndrome virus were sequenced on the MinION platform using a random, reverse primer in a strand-switching reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for reference-based sequence building using a stock personal computer. This method accurately detected and identified complete coding sequence genomes with a minimum of 20× coverage depth for all 7 viruses, including a sample containing 2 viruses. Each lineage-typing region had at least 26× coverage depth for all viruses. Furthermore, analyzing the CDV sample through a pipeline devoid of CDV reference sequences modeled the ability of this protocol to detect unknown viruses. Our results show the ability of this technique to detect and characterize dsRNA, negative- and positive-sense ssRNA, and nonsegmented and segmented RNA viruses.

First expert elicitation of knowledge on drivers of emergence of influenza D in Europe.

The influenza D virus (IDV) was first identified and characterized in 2011. Considering the virus' zoonotic potential, its genome nature (segmented RNA virus), its worldwide circulation in livestock and its role in bovine respiratory disease, an increased interest is given to IDV. However, few data are available on drivers of emergence of IDV. We first listed fifty possible drivers of emergence of IDV in ruminants and swine. As recently carried out for COVID-19 in pets (Transboundary and Emerging Diseases, 2020), a scoring system was developed per driver and scientific experts (N=28) were elicited to (a) allocate a score to each driver, (b) weight the drivers' scores within each domain and (c) weight the different domains among themselves. An overall weighted score was calculated per driver, and drivers were ranked in decreasing order. Drivers with comparable likelihoods to play a role in the emergence of IDV in ruminants and swine in Europe were grouped using a regression tree analysis. Finally, the robustness of the expert elicitation was verified. Eight drivers were ranked with the highest probability to play a key role in the emergence of IDV: current species specificity of the causing agent of the disease; influence of (il)legal movements of live animals (ruminants, swine) from neighbouring/European Union member states and from third countries for the disease to (re-)emerge in a given country; detection of emergence; current knowledge of the pathogen; vaccine availability; animal density; and transport vehicles of live animals. As there is still limited scientific knowledge on the topic, expert elicitation of knowledge and multi-criteria decision analysis, in addition to clustering and sensitivity analyses, are very important to prioritize future studies, starting from the top eight drivers. The present methodology could be applied to other emerging animal diseases.

Characterization of mutations of Haemagglutinin (HA) and Neuraminidase (NA) genes of Influenza A H1N1pdm09 isolated during the 2018 outbreak in India

Background: The influenza virus is a segmented RNA virus, has higher propensity to undergo mutations, which carry major implications, both clinically and epidemiologically. Mutations in HA and NA are of particular importance. Changes in the HA region are associated with increased virulence, antigenicity and host specificity as it is involved in receptor binding and membrane fusion. Hence is a major target for vaccine development. The NA gene is involved in virus release as well as drug susceptibility. These changes can trigger the emergence of new strains, which could lead to epidemics and pandemics. Therefore, it is important to monitor and characterize the mutations closely. Thus, this study was undertaken to understand the genetic variation in Influenza H1N1 pdm09 for the outbreak in 2018. Methods and materials: This study conducted in Regional Influenza Laboratory, Puducherry on the isolates positive for Influenza A pdm09 by real time RT PCR. Conventional PCR was done using the WHO primers. The PCR positive products were then subjected to Sanger sequencing in ABI3500. The sequences were then analysed on the MEGA X software and Flusurver software for detection of the mutations in HA and NA. Results: Phylogenetic analysis was performed for both HA and NA gene along with the clade-specific reference strains. All our isolates clustered along with the clade 6B.1. In HA region, of the 28 mutations documented, the most common mutation was R240Q (100%) followed by S181T, S200P and S91R. The mutation of N173K was detected for the 1st time globally in the antigenic site. Of the 12 mutations in the HA stem region, 4 new mutations documented – K71Q, V466L, R467T and Q529R. The most common mutations in NA observed were G77R, I188T, S200G/V, F74S/L and V81A. We documented 4 new mutations that could be associated with drug resistance – W179G, R225K, V267L and R293K. Conclusion: Continuous surveillance of Influenza virus is important to monitor the changes in the HA genes for providing recommendations for vaccine strain. Further, as the treatment options for influenza is limited, it makes it all the more important to have continuous surveillance to track the antiviral resistance.

Organization of the Influenza A Virus Genomic RNA in the Viral Replication Cycle-Structure, Interactions, and Implications for the Emergence of New Strains.

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA–RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future.

Meta-Transcriptomic Identification of Divergent Amnoonviridae in Fish.

Tilapia lake virus (TiLV) has caused mass mortalities in farmed and wild tilapia with serious economic and ecological consequences. Until recently, this virus was the sole member of the Amnoonviridae, a family within the order Articulavirales comprising segmented negative-sense RNA viruses. We sought to identify additional viruses within the Amnoonviridae through total RNA sequencing (meta-transcriptomics) and data mining of published transcriptomes. Accordingly, we sampled marine fish species from both Australia and China and discovered several segments of two new viruses within the Amnoonviridae, tentatively called Flavolineata virus and Piscibus virus, respectively. In addition, by mining vertebrate transcriptome data, we identified nine additional virus transcripts matching to multiple genomic segments of TiLV in both marine and freshwater fish. These new viruses retained sequence conservation with the distantly related Orthomyxoviridae in the RdRp subunit PB1, but formed a distinct and diverse phylogenetic group. These data suggest that the Amnoonviridae have a broad host range within fish and that greater animal sampling will identify additional divergent members of the Articulavirales.

A CARDIOVASCULAR COMPLICATION OF COVID-19: DELAYED PRESENTATION OF ACUTE MYOPERICARDITIS

SESSION TITLE: Medical Student/Resident Cardiovascular Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: “SARS CoV-2” is an enveloped non segmented positive-sense RNA virus,. In December 2019, SARS CoV-2 started as a pandemic in Wuhan city of China and spread to the rest of the world. The classical clinical picture of COVID-19 is variable ranging from asymptomatic, a flu-like syndrome of mild severity, to interstitial pneumonia and a variable degree of respiratory failure with elevation of cardiac troponin I (cTnI) levels and arrhythmia in most cases. In here we present a case of delayed presentation of acute Viral Myopericarditis by COVID 19 even after the patient had recovered from infection. CASE PRESENTATION: A previously healthy 29 year old female presented with acute chest pressure of 5 hour duration, 10/10 in intensity, located at left sternal border. It was worsened with lying flat, and deep breathing and better with sitting up and bending forward. Forty five days before this presentation, she had cough, fever and fatigue and was diagnosed with COVID 19. After 14 days, patient was cleared by the state health department. Physical examination including the CVS and respiratory systems was normal. Laboratory tests showed on elevated troponin-1 level of 0.5 µg/L, which peaked to 8.09 µg/L the following day, (ESR) elevated at 35mm/hr, lactate dehydrogenase (LDH) elevated at 244U/L, C-reactive protein (CRP) elevated at 19.2mg/L. and D-dimer elevated at 561 FEUng/m. Two repeat SARS CoV-2 testing were negative. Electrocardiogram showed some non-specific T wave inversions in leads V3 with normal sinus rhythm at a rate of 87 beats per minute. Transthoracic echocardiography performed which did not demonstrate wall motion abnormalities or pericardial effusion. Chest CTA was negative for acute pulmonary embolism. The patient was diagnosed with viral myopericarditis caused by COVID 19. The patient was managed with pain medications, ibuprofen, and colchicine and was discharged in 2 days. DISCUSSION: Viral myopericarditis is the most common cause of myopericarditis. The exact mechanism COVID 19 causing cardiac injury is not clear. It could be either due to direct invasion of the cardiac myocytes or through immune mediated which is more likely in our patient. The cardiac manifestation of COVID 19 ranges from patients having asymptomatic troponin leak, acute CHF exacerbation due to dilated cardiomyopathy and arrhythmias. The treatment at this time is mainly supportive with medications such as remdesivir, favipivir, protease inhibitors under trial. CONCLUSIONS: A thorough history and physical for previous history and timing of exposure, and also help establish diagnosis and exclusion for acute coronary syndrome, to avoid delay in anti-inflammatory therapy and unnecessary testing. Post-viral acute myopericardial damage should be suspected even in the absence of residual viral genome Reference #1: Management of Acute and Recurrent Pericarditis. J Am Coll Cardiol 2020;75:76-92. Chiabrando JG, Bonaventura A, Vecchié A, et al. DISCLOSURES: No relevant relationships by Navya Akula, source=Web Response No relevant relationships by Miluska Mejia Trebejo, source=Web Response No relevant relationships by tausif syed, source=Web Response

Full-length genome sequence of segmented RNA virus from ticks was obtained using small RNA sequencing data

BackgroundIn 2014, a novel tick-borne virus of the Flaviviridae family was first reported in the Mogiana region of Brazil and named the Mogiana tick virus (MGTV). Thereafter, the Jingmen tick virus (JMTV), Kindia tick virus (KITV), and Guangxi tick virus (GXTV)—evolutionarily related to MGTV—were reported.ResultsIn the present study, we used small RNA sequencing (sRNA-seq) to detect viruses in ticks and discovered a new MGTV strain in Amblyomma testudinarium ticks collected in China’s Yunnan Province in 2016. We obtained the full-length genome sequence of this MGTV strain Yunnan2016 (GenBank: MT080097, MT080098, MT080099 and MT080100) and recommended it for its inclusion in the NCBI RefSeq database for future studies on MGTV, JMTV, KITV and GXTV. Phylogenetic analysis showed that MGTV, JMTV, KITV and GXTV are monophyletic and belong to a MGTV group. Furthermore, this MGTV group of viruses may be phylogenetically related to geographical regions that were formerly part of the supercontinents Gondwana and Laurasia.ConclusionsTo the best of our knowledge, this is the first study in which 5′ and 3′ sRNAs were used to generate full-length genome sequences of, but not limited to, RNA viruses. We also demonstrated the feasibility of using the sRNA-seq based method for the detection of viruses in pooled two and even possible one small ticks. MGTV may preserve the characteristic of ancient RNA viruses, which can be used to study the origin and evolution of RNA viruses. In addition, MGTV can be used as novel species for studies in phylogeography.