Open Reading Frame 1a Research Articles

An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific RNA silencing and is graft transmissible to commercial citrus varieties.

A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Classic human astrovirus 4, 8, MLB-3, and likely new genotype 5 sublineage in stool samples of children in Nigeria.

Human astrovirus (HAstV) is a nonenveloped RNA virus and has been implicated in acute gastroenteritis among children and elderly. However, there exists a substantial dearth of information on HAstV strains circulating in Nigeria. Viral-like particles were purified from archived 254 stool samples of children with acute flaccid paralysis between January and December 2020 from five states in Nigeria, using the NetoVIR protocol. Extracted viral RNA and DNA were subjected to a reverse transcription step and subsequent random polymerase chain reaction amplification. Library preparation and Illumina sequencing were performed. Using the virome paired-end reads pipeline, raw reads were processed into genomic contigs. Phylogenetic and pairwise identity analysis of the recovered HAstV genomes was performed. Six near-complete genome sequences of HAstV were identified and classified as HAstV4 (n = 1), HAstV5 (n = 1), HAstV8 (n = 1), and MLB-3 (n = 3). The HAstV5 belonged to a yet unclassified sublineage, which we tentatively named HAstV-5d. Phylogenetic analysis of open reading frames 1a, 1b, and 2 suggested recombination events inside the MAstV1 species. Furthermore, phylogenetic analysis implied a geographic linkage between the HAstV5 strain from this study with two strains from Cameroon across all the genomic regions. We report for the first time the circulation of HAstV genotypes 4, 8, and MLB-3 in Nigeria and present data suggestive for the existence of a new sublineage of HAstV5. To further understand the burden, diversity, and evolution of HAstV, increased research interest as well as robust HAstV surveillance in Nigeria is essential.

Gambaran Nilai Ct Value qRT-Pcr Sars Cov-2 Dengan Metode Ekstraksi Manual Dan Otomatis

Corona Virus Infection Disease (COVID)-19 is a disease caused by a new coronavirus C derivative. The SARS-CoV-2 genome has six main open reading frames (ORFs): ORF 1a and 1b, envelope protein/E genes, membrane protein/M genes, spike protein/S gene, and nucleocapsid protein/N genes. Realtime RT-PCR is a DNA amplification technique in which amplification products can be analyzed at each cycle using fluorogenic probes. The RT-PCR method is used for amplification, isolation, or identification of sequences from RNA cells or tissues. The extraction of nucleic acids in the form of DNA and RNA is the initial process for biomolecular studies. The principle in the extraction of genetic material in the form of DNA and RNA is to break down cells and genetic material in the cell from other cellular components in the form of fats, proteins, carbohydrates, and other substances. The purpose of this study is to find out the picture of the value of CT Value qRT-PCR SARS-CoV-2 using two extraction methods, namely manual and automatic. This study was descriptive using accidental sampling techniques of positive patients confirmed with COVID-19. Swab samples are carried out using manual and automatic extraction methods, then qRT-PCR examination is carried out. The results of descriptive statistical tests obtained the Mean Gene E value of manual extraction 19.1290 and automatic extraction 18.8187, as well as the Mean Gene value of ORF1ab manual extraction 19.1290 and automatic extraction 19.5177.

Targeting SARS-CoV-2 Non-Structural Proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped respiratory β coronavirus that causes coronavirus disease (COVID-19), leading to a deadly pandemic that has claimed millions of lives worldwide. Like other coronaviruses, the SARS-CoV-2 genome also codes for non-structural proteins (NSPs). These NSPs are found within open reading frame 1a (ORF1a) and open reading frame 1ab (ORF1ab) of the SARS-CoV-2 genome and encode NSP1 to NSP11 and NSP12 to NSP16, respectively. This study aimed to collect the available literature regarding NSP inhibitors. In addition, we searched the natural product database looking for similar structures. The results showed that similar structures could be tested as potential inhibitors of the NSPs.

The Roles of the 5' and 3' Untranslated Regions in Human Astrovirus Replication.

Astroviruses are small nonenveloped single-stranded RNA viruses with a positive sense genome. They are known to cause gastrointestinal disease in a broad spectrum of species. Although astroviruses are distributed worldwide, a gap in knowledge of their biology and disease pathogenesis persists. Many positive-sense single-stranded RNA viruses show conserved and functionally important structures in their 5' and 3' untranslated regions (UTRs). However, not much is known about the role of the 5' and 3' UTRs in the viral replication of HAstV-1. We analyzed the UTRs of HAstV-1 for secondary RNA structures and mutated them, resulting in partial or total UTR deletion. We used a reverse genetic system to study the production of infectious viral particles and to quantify protein expression in the 5' and 3' UTR mutants, and we established an HAstV-1 replicon system containing two reporter cassettes in open reading frames 1a and 2, respectively. Our data show that 3' UTR deletions almost completely abolished viral protein expression and that 5' UTR deletions led to a reduction in infectious virus particles in infection experiments. This indicates that the presence of the UTRs is essential for the life cycle of HAstV-1 and opens avenues for further research.

Triple Intergenotype Recombination of Human Astrovirus 5, Human Astrovirus 8, and Human Astrovirus 1 in the Open Reading Frame 1a, Open Reading Frame 1b, and Open Reading Frame 2 Regions of the Human Astrovirus Genome.

Human astrovirus (HAstV) strains exhibit high levels of genetic diversity, and many recombinant strains with different recombination patterns have been reported. The aims of the present study were to investigate the emergence of HAstV recombinant strains and to characterize the recombination patterns of the strains detected in pediatric patients admitted to the hospital with acute gastroenteritis in Chiang Mai, Thailand. A total of 92 archival HAstV strains detected in 2011 to 2020 were characterized regarding their open reading frame 1a (ORF1a) genotypes in comparison with their ORF1b genotypes to identify recombinant strains. The recombination breakpoints of the putative recombinant strains were determined by whole-genome sequencing and were analyzed by SimPlot and RDP software. Three HAstV strains (CMH-N178-12, CMH-S059-15, and CMH-S062-15) were found to be recombinant strains of three different HAstV genotypes, i.e., HAstV5, HAstV8, and HAstV1 within the ORF1a, ORF1b, and ORF2 regions, respectively. The CMH-N178-12 strain displayed recombination breakpoints at nucleotide positions 2681 and 4357 of ORF1a and ORF1b, respectively, whereas the other two recombinant strains, CMH-S059-15 and CMH-S062-15, displayed recombination breakpoints at nucleotide positions 2612 and 4357 of ORF1a and ORF1b, respectively. This is the first study to reveal nearly full-length genome sequences of HAstV recombinant strains with a novel recombination pattern of ORF1a-ORF1b-ORF2 genotypes. This finding may be useful as a guideline for identifying other recombinant HAstV strains in other geographical regions and may provide a better understanding of their genetic diversity, as well as basic knowledge regarding virus evolution. IMPORTANCE Recombination is one of the mechanisms that plays a crucial role in the genetic diversity and evolution of HAstV. We wished to investigate the emergence of HAstV recombinant strains and to analyze the whole-genome sequences of the putative HAstV recombinant strains detected in pediatric patients with acute gastroenteritis in 2011 to 2020. We reported 3 novel intergenotype recombinant strains of HAstV5-HAstV8-HAstV1 at the ORF1a-ORF1b-ORF2 regions of the HAstV genome. The hot spots of recombination occur frequently near the ORF1a-ORF1b and ORF1b-ORF2 junctions of the HAstV genome. The findings indicate that intergenotype recombination of HAstV occurs frequently in nature. The emergence of a novel recombinant strain allows the new virus to adapt and successfully escape from the host immune system, eventually emerging as the predominant genotype to infect human populations that lack herd immunity against novel recombinant strains. The virus may cause an outbreak and needs to be monitored continually.

Genetic Recombination Sites Away from the Insertion/Deletion Hotspots in SARS-Related Coronaviruses.

The genome sequences of severe acute respiratory syndrome (SARS)-related coronaviruses (sarbecoviruses) have been reported to include many long and complex insertions/deletions (indels) in specific genomic regions, including open reading frame 1a (ORF1a), S1 domain of the spike, and ORF8 genes. These indel hotspots incorporate various non-classical, long, and complex indels with uncertain developmental processes. A possible explanation for these complex and diversified indels at the hotspots is genetic recombination. To determine the possible association between recombination events and development of indel hotspots, this study investigated the genome sequences of many sarbecoviruses from different countries and hosts and compared the distributions of the indel hotspots and recombination sites by performing multiple sequence alignments and recombination analyses. The genomes of 19 SARS-related coronaviruses (15 coronaviruses that infect bats, two that infect humans, one that infects pangolins, and one that infects civets), including human-infecting SARS-CoV and SARS-CoV-2, were evaluated. Hotspots of complex indels with diverse RNA sequences around gaps were clustered in non-structural protein 2 (Nsp2) and Nsp3 of ORF1a, S1, and ORF8. Phylogenetic reconstructions revealed different structures of the inferred phylogenetic trees between genomic regions, and recombination analyses identified multiple recombination sites across ORF1ab and S genes. However, the nucleotide positions of the indel hotspots were not identical with the identified recombination sites in the recombinant viruses, suggesting the involvement of different developmental processes of indel hotspots and genetic recombination. Further research is required to elucidate the developmental mechanisms underpinning clustered complex indels and recombination events in the evolutionary history of sarbecoviruses.

In vivo efficacy of 2% povidone iodine, chlorhexidine gluconate, and herbal extract mouthwash on SARS-CoV-2 viral load in saliva: A randomized clinical trial.

One of the major techniques to reduce the transmission rate of COVID-19 would be to decrease the viral titers of SARS-CoV-2 in the saliva of infected patients and it is particularly useful in a dental setting. The present study evaluated the change in salivary viral load of COVID-19 patients using povidone iodine (PI), chlorhexidine (CHX), and an herbal extract (RightSure®) oral antiviral herbal mouthwash (HM) at clinically recommended duration and concentrations. Thirty individuals with SARS-CoV-2 were randomly allocated to three groups: (1) Group 1, PI mouthwash; (2) Group 2, CHX mouthwash; and (3) Group 3, HM mouthwash. A baseline salivary throat sample was collected from all the participants who were later instructed to rinse with their respective mouthwash for 30 s. A second salivary sample was collected 30 min after rinsing. The SARS-CoV-2 viral load was analyzed using real-time reverse-transcription polymerase chain reaction wherein the cyclic threshold (Ct) values were evaluated. Independent t-test analysis reported a statistically significant difference concerning the PI group (before-after comparison) (P < 0.05). The rest of the two study groups failed to report any significant difference in the nucleocapsid gene and open reading frame 1a gene levels. While all three types of mouthwash increased the Ct values, a statistically significant difference was observed with PI mouthwash, indicating that it might potentially reduce the spread of the SARS-CoV-2 virus, especially via aerosol but further studies with larger sample size and longer follow-up periods are required to investigate this relationship.

Identification of MHC-I-Presented Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) Peptides Reveals Immunogenic Epitopes within Several Non-Structural Proteins Recognized by CD8+ T Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most relevant porcine pathogens worldwide. Active control of the disease relies on modified live virus vaccines (MLVs), as most inactivated vaccines provide very limited protection. Neutralizing antibodies occur late in infection; therefore, CD8+ T cells are considered important correlates of protection and are a frequent focus of investigation. Our aim was to identify viral peptides naturally bound by the class I major histocompatibility complex (MHC-I) and to confirm their ability to stimulate CD8+ T cells. For this purpose, we immunoprecipitated MHC-I/peptide complexes of PRRSV (strain AUT15-33) -infected cells (SLA-I Lr-Hp 35.0/24 mod) to isolate the viral epitopes and analyzed them with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Furthermore, we employed these identified peptides to stimulate peripheral blood mononuclear cells (PBMCs) of previously PRRSV-infected pigs and measured the PRRSV-specific CD8+ T-cell response with an intracellular cytokine staining (ICS). Our data revealed that PRRSV non-structural proteins (NSPs), encoded in open reading frame 1a and 1b (ORF1), present the major source of MHC-I-presented peptides. Additionally, we show that our identified epitopes are able to trigger IFNγ responses in vitro. These findings are a basis for understanding the proteasomal degradation of PRRSV proteins, the cellular ability to display them via MHC-I, and their potential to restimulate CD8+ T cells.

The COVID-19 viral 3a protein forms a potassium channel that can be inhibited by antiarrhythmic drugs

Funding AcknowledgementsType of funding sources: Public Institution(s). Main funding source(s): Deutsche Gesellschaft für Kardiologie /German Cardiac Society Joachim Herz Stiftung /Joachim Herz FoundationBackground/IntroductionSupraventricular and ventricular arrhythmias can often be observed in patients with COVID-19 infection. Both, the clinically observed increase in cardiac biomarkers as well as histological studies indicate virus replication within cardiomyocytes. The 3a open reading frame of the viral genome encodes for a transmembrane protein that is transported to the cell membrane where it can serve as a potassium channel.PurposeThe aim of this study was to investigate whether COVID-19 infected induced pluripotent stem cell (iPSC)-derived cardiomyocytes also express the 3a protein and whether the potassium currents that are conducted through the 3a protein can be inhibited by clinically used antiarrhythmic drugs.Methods and ResultsiPSC-derived cardiomyocytes were infected with COVID-19 and subsequently subjected to immunoblotting, where expression of the 3a protein could be observed. Plasmid DNA, encoding the COVID-19 3a protein, was generated by gene synthesis and used for in vitro transcription of cRNA. 3–5 days after intracytoplasmic injection of the 3a protein cRNA into Xenopus laevis oocytes, potassium currents could be measured by two-electrode voltage clamp recordings. While class I and class IV antiarrhythmic drugs showed only minor effects on the potassium currents of the 3a protein, a robust inhibition by several beta-blockers and by class III antiarrhythmic drugs could be observed. The strongest effects were found with dofetilide (58.1 % inhibition at 100 µM) and amiodarone (50.1 % inhibition at 100 µM, IC50 level 4.7 µM). An in silico docking analysis, based on the recently revealed crystal structure of the 3a protein, identified the amino acid residues K61 and D142 as part of the binding site of amiodarone. After deactivation of these amino acid residues by site-directed mutagenesis, the inhibition by amiodarone was significantly attenuated.ConclusionThe COVID-19 viral 3a protein is expressed in COVID-19-infected iPS-derived cardiomyocytes and forms a potassium channel that can be inhibited by antiarrhythmic drugs.

Genomic Epidemiology of Imported Cases of COVID-19 in Guangdong Province, China, October 2020 – May 2021

ObjectiveThe pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been engendering enormous hazards to the world. We obtained the complete genome sequences of SARSCoV-2 from imported cases admitted to the Guangzhou Eighth People's Hospital, which was appointed by the Guangdong provincial government to treat coronavirus disease 2019 (COVID-19). The SARS-CoV-2 diversity was analyzed, and the mutation characteristics, time, and regional trend of variant emergence were evaluated. MethodsIn total, 177 throat swab samples were obtained from COVID-19 patients (from October 2020 to May 2021). High-throughput sequencing technology was used to detect the viral sequences of patients infected with SARS-CoV-2. Phylogenetic and molecular evolutionary analyses were used to evaluate the mutation characteristics and the time and regional trends of variants. ResultsWe observed that the imported cases mainly occurred after January 2021, peaking in May 2021, with the highest proportion observed from cases originating from the United States. The main lineages were found in Europe, Africa, and North America, and B.1.1.7 and B.1.351 were the two major sublineages. Sublineage B.1.618 was the Asian lineage (Indian) found in this study, and B.1.1.228 was not included in the lineage list of the Pangolin web. A reasonably high homology was observed among all samples. The total frequency of mutations showed that the open reading frame 1a (ORF1a) protein had the highest mutation density at the nucleotide level, and the D614G mutation in the spike protein was the commonest at the amino acid level. Most importantly, we identified some amino acid mutations in positions S, ORF7b, and ORF9b, and they have neither been reported on the Global Initiative of Sharing All Influenza Data nor published in PubMed among all missense mutations. ConclusionThese results suggested the diversity of lineages and sublineages and the high homology at the amino acid level among imported cases infected with SARS-CoV-2 in Guangdong Province, China.

The status and analysis of common mutations found in the SARS-CoV-2 whole genome sequences from Bangladesh

Rapid emergence of covid-19 variants by continuous mutation made the world experience continuous waves of infections and as a result, a huge number of death-toll recorded so far. It is, therefore, very important to investigate the diversity and nature of the mutations in the SARS-CoV-2 genomes. In this study, the common mutations occurred in the whole genome sequences of SARS-CoV-2 variants of Bangladesh in a certain timeline were analyzed to better understand its status. Hence, a total of 78 complete genome sequences available in the NCBI database were obtained, aligned and further analyzed. Scattered Single Nucleotide Polymorphisms (SNPs) were identified throughout the genome of variants and common SNPs such as: 241:C>T in the 5′UTR of Open Reading Frame 1A (ORF1A), 3037: C>T in Non-structural Protein 3 (NSP3), 14,408: C>T in ORF6 and 23,402: A>G, 23,403: A>G in Spike Protein (S) were observed, but all of them were synonymous mutations. About 97% of the studied genomes showed a block of tri-nucleotide alteration (GGG>AAC), the most common non-synonymous mutation in the 28,881–28,883 location of the genome. This block results in two amino acid changes (203–204: RG>KR) in the SR rich motif of the nucleocapsid (N) protein of SARS-CoV-2, introducing a lysine in between serine and arginine. The N protein structure of the mutant was predicted through protein modeling. However, no observable difference was found between the mutant and the reference (Wuhan) protein. Further, the protein stability changes upon mutations were analyzed using the I-Mutant2.0 tool. The alteration of the arginine to lysine at the amino acid position 203, showed reduction of entropy, suggesting a possible impact on the overall stability of the N protein. The estimation of the non-synonymous to synonymous substitution ratio (dN/dS) were analyzed for the common mutations and the results showed that the overall mean distance among the N-protein variants were statistically significant, supporting the non-synonymous nature of the mutations. The phylogenetic analysis of the selected 78 genomes, compared with the most common genomic variants of this virus across the globe showed a distinct cluster for the analyzed Bangladeshi sequences. Further studies are warranted for conferring any plausible association of these mutations with the clinical manifestation.

Detection of the ORF1 Gene Is an Indicator of the Possible Isolation of Severe Acute Respiratory Syndrome Coronavirus 2.

In the ongoing coronavirus diseases 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), real-time RT-PCR based diagnostic assays have been used for the detection of infection, but the positive signal of real-time RT-PCR does not necessarily indicate the infectivity of the patient. Due to the unique replication system of the coronavirus, primer/probe sets targeted nucleocapsid (N) and spike (S) protein detect the abundantly synthesized subgenomic RNAs as well as the virus genome, possibly making the assay unsuitable for estimation of the infectivity of the specimen, although it has an advantage for the diagnostic tests. In this study, the primer/probe set targeting the open reading frame 1a (ORF1a) gene was developed to specifically detect viral genomic RNA. Then the relation between the ORF1a signal and infectivity of the clinical specimens was validated by virus isolation using VeroE6 cells, which constitutively express transmembrane protease, serine 2, (VeroE6/TMPRSS2). The analytical sensitivity of developed ORF1a set was similar to that of previously developed N and S sets. Nevertheless, in the assay of the clinical specimen, detection rate of the ORF1a gene was lower than that of the N and S genes. These data indicated that clinical specimens contain a significant amount of subgenomic RNAs. However, as expected, the isolation-succeeded specimen always showed an RT-PCR-positive signal for the ORF1a gene, suggesting ORF1a detection in combination with N and S sets could be a more rational indicator for the possible infectivity of the clinical specimens.

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus.

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

Profiling CD8+ Tcell epitopes of COVID-19 convalescents reveals reduced cellular immune responses to SARS-CoV-2 variants.

Cellular immunity is important in determining the disease severity of COVID-19 patients. However, current understanding of SARS-CoV-2 epitopes mediating cellular immunity is limited. Here we apply T-Scan, a recently developed method, to identify CD8+ T cell epitopes from COVID-19 patients of four major HLA-A alleles. Several identified epitopes are conserved across human coronaviruses, which might mediate pre-existing cellular immunity to SARS-CoV-2. In addition, we identify and validate four epitopes that were mutated in the newly circulating variants, including the Delta variant. The mutations significantly reduce T cell responses to the epitope peptides in convalescent and vaccinated samples. We further determine the crystal structure of HLA-A∗02:01/HLA-A∗24:02 in complex with the epitope KIA_S/NYN_S, respectively, which reveals the importance of K417 and L452 of the spike protein for binding to HLA. Our data suggest that evading cellular immunity might contribute to the increased transmissibility and disease severity associated with the new SARS-CoV-2 variants.

A lab-on-a-chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resource-limited settings

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL−1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings.

Diversity of Citrus tristeza virus Strains in the Upper Gulf Coast Area of Texas.

Citrus tristeza virus (CTV) in Texas was first reported in the 1950s and has since been sporadically reported in the residential areas in the Upper Gulf Coast region. Because the major rootstock for commercial citriculture in South Texas is sour orange, which is susceptible to CTV decline, the spread of CTV into South Texas can pose a great threat to Texas citrus industry. Thirty-six CTV-positive samples, collected during surveys conducted in the Upper Gulf Coast area of Texas from 2013 to 2018, were first analyzed by strain-specific real-time PCR (RT-PCR) targeting various regions of CTV Open reading frame (Orf) 1a and then by amplicon sequencing derived from p25 and p20 region of CTV genome. Among 36 samples, 33 were successfully genotyped by strain-specific RT-PCR and by amplicon sequencing followed by phylogenetic analysis. Variability in the detection of CTV strains was observed over a 6-year period. In 2013, T3 and T30 were the only strains detected in the Upper Gulf Coast of Texas, but in further surveys until 2018, additional strains were detected, including T36, VT, and RB. Mixed infections were also detected in 14 samples comprising about 42% of CTV samples examined in the study. Although genotyping mixed infection samples by targeting Orf 1a and full-length p25, residing in the 5' and 3' region of the CTV genome, respectively, confirmed the presence of multiple strains in these samples, incongruent genotyping data were observed. These findings suggested that the current status of CTV strain diversity in Texas Upper Gulf Coast region might have been established by multiple introductions of CTV-infected plant materials for propagation and with a potential recombination in planta.

COVID-19 screening test by using random oropharyngeal saliva.

An optimal clinical specimen for accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by minimizing the usage of consumables and reduce hazard exposure to healthcare workers is an urgent priority. The diagnostic performance of SARS‐CoV‐2 detection between healthcare worker‐collected nasopharyngeal and oropharyngeal (NP + OP) swabs and patient performed self‐collected random saliva was assessed. Paired NP + OP swabs and random saliva were collected and processed within 48 h of specimen collection from two cohort studies which recruited 562 asymptomatic adult candidates. Real‐time reverse‐transcription polymerase chain reaction targeting Open reading frame 1a (ORF1a) and nucleocapsid (N) genes was performed and the results were compared. Overall, 65 of 562 (28.1%) candidates tested positive for COVID‐19 based on random saliva, NP + OP swabs, or both testing techniques. The detection rate of SARS‐CoV‐2 was higher in random saliva compared to NP + OP testing (92.3%; 60/65 vs. 73.8%; 48/65; p < .05). The estimated sensitivity and specificity of random saliva were higher than NP + OP swabs (95.0; 99.9 vs. 72.2; 99.4). The C t values of ORF1a and N genes were significantly lower in random saliva compared to NP + OP swabs specimens. Our findings demonstrate that random saliva is an alternative diagnostic specimen for the detection of SARS‐CoV‐2. Self‐collected random oropharyngeal saliva is a valuable specimen that provides accurate SARS‐CoV‐2 surveillance testing of a community.

Genetic diversity and recombination between turnip yellows virus strains in Australia.

Disease outbreaks caused by turnip yellows virus (TuYV), a member of the genus Polerovirus, family Luteoviridae, regularly occur in canola and pulse crops throughout Australia. To understand the genetic diversity of TuYV for resistance breeding and management, genome sequences of 28 TuYV isolates from different hosts and locations were determined using high-throughput sequencing (HTS). We aimed to identify the parts of the genome that were most variable and clarify the taxonomy of viruses related to TuYV. Poleroviruses contain seven open reading frames (ORFs): ORF 0-2, 3a, and 3-5. Phylogenetic analysis based on the genome sequences, including isolates of TuYV and brassica yellows virus (BrYV) from the GenBank database, showed that most genetic variation among isolates occurred in ORF 5, followed by ORF 0 and ORF 3a. Phylogenetic analysis of ORF 5 revealed three TuYV groups; P5 group 1 and group 3 shared 45-49% amino acid sequence identity, and group 2 is a recombinant between the other two. Phylogenomic analysis of the concatenated ORFs showed that TuYV is paraphyletic with respect to BrYV, and together these taxa form a well-supported monophyletic group. Our results support the hypothesis that TuYV and BrYV belong to the same species and that the phylogenetic topologies of ORF 0, 3a and 5 are incongruent and may not be informative for species demarcation. A number of beet western yellow virus (BWYV)- and TuYV-associated RNAs (aRNA) were also identified by HTS for the first time in Australia.

Immunity, virus evolution, and effectiveness of SARS-CoV-2 vaccines.

Phylogenetic and pathogenesis studies of the severe acute respiratory syndrome-related coronaviruses (SARS-CoVs) strains have highlighted some specific mutations that could confer the RNA genome fitness advantages and immunological resistance for their rapid spread in the human population. The analyses of 30 kb RNA SARS-CoVs genome sequences, protein structures, and functions have provided us a perspective of how host-virus protein-protein complexes act to mediate virus infection. The open reading frame (ORF)1a and ORF1b translation yields 16 non-structural (nsp1-16) and 6 accessory proteins (p6, p7a, p8ab, p9b) with multiple functional domains. Viral proteins recruit over 300 host partners forming hetero-oligomeric complexes enabling the viral RNA synthesis, packing, and virion release. Many cellular host factors and the innate immune cells through pattern-recognition receptors and intracellular RNA sensor molecules act to inhibit virus entry and intracellular replication. However, non-structural ORF proteins hijack them and suppress interferon synthesis and its antiviral effects. Pro-inflammatory chemokines and cytokines storm leads to dysfunctional inflammation, lung injury, and several clinical symptoms in patients. During the global pandemic, COVID-19 patients were identified with non-synonymous substitution of G614D in the spike protein, indicating virus co-evolution in host cells. We review findings that suggest that host RNA editing and DNA repair systems, while carrying on recombination, mutation, and repair of viral RNA intermediates, may facilitate virus evolution. Understanding how the host cell RNA replication process may be driven by SARS-CoV-2 RNA genome fitness will help the testing of vaccines effectiveness to multiple independent mutated coronavirus strains that will emerge.