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Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.
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Intro: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, an enveloped RNA of the coronavirus family. The advancement in molecular technology and biochemistry has accelerated the development of diagnostic reagents and assays. Much attention has been focused on the S protein, but the high mutation rate in this region could lead to false negative results. Thus, a better target protein for diagnostic application is needed for accurate detection. Method(s): Nucleotide sequences encoded for membrane (M) glycoprotein gene region of SARS-CoV-2 from Malaysian isolates were extracted from GISAID, aligned, and selected accordingly. The DNA plasmid was commercially synthesized with codon optimization for Escherichia coli (E. coli), and the presence of the M gene was confirmed by PCR. The plasmid was then transformed into E. coli. Later, the expression of M glycoprotein was induced, separated on an SDS-PAGE gel, and transferred onto a nitrocellulose membrane, followed by immunostaining. Finding(s): The analysis of the M glycoprotein against the Omicron strains demonstrated that the amino acid is conserved (99.5%). The M glycoprotein was successfully expressed and detected with antibodies from SARS-CoV-2 infected patients at ~26 kDa. The protein is currently upscale for the generation of monoclonal Ab (Mab). Discussion(s): The M protein of SARS-CoV-2 is more conserved among the virus and also has been reported to confer antigenic properties. Selection of M protein perhaps a better option compared to current detection assays that use spike (S) protein, which could lead to false negative results, as this gene region particularly the ribosome-binding domain (RBD) rapidly undergoes mutations. The utilization of M protein potentially improves negative predictive value (NPV) of the diagnostic test. Conclusion(s): Further development of diagnostic reagents is needed to improve the assay's specificity. The newly developed M protein and the MAb can be used to generate a more accurate viral detection assay.Copyright © 2023
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Intro: Delta and Omicron variants of SARS-CoV-2 are highly contagious, currently dominating the globe and recognized as variant of concern (VOC). The transmissibility efficiency of viruses, disease symptoms, and severity of COVID- 19 is highly heterogeneous. Therefore, testing at the community level is essential to identify the infected people at an early stage-carrying VOC to reduce the spread of virus and combat the pandemic. Method(s): In this study, we analysed thousands of genome sequences representing 30 different SARS-CoV-2 variants and identified Delta and Omicron variants specific nucleic acid signatures in the spike gene. Based on the variant specific nucleic acid sequences we synthesized different oligos and optimized a mPCR assay that can specifically differentiate the Delta and Omicron variants. We further translated our work into a dipstick assay (Tohoku Bio-array, Japan) by adding tag linker sequence to 5' end of the forward primer and adding biotin in 3' end of the oligos. Finding(s): A total of 250 samples were subjected to WGS using MiSeq platform and these confirmed samples were processed for validation of our specific designed probes using PCR assay and the readout was found to be 100% specific to Delta, BA.1 & BA.2 of SARS-CoV-2 variants which were further confirmed by Sanger sequencing. The dipstick was used to screen these samples, and specific signals were observed. WGS and Sanger sequencing were used to validate our PCR and dipstick assay results, and the readout was found to be 100% specific. The results can be visualised by the naked eye and interpreted easily. Conclusion(s): This study developed a rapid point-of-care test of SARS-CoV-2 patients, which can differentiate Delta, BA.1 and BA.2 variants at the same time of confirmation of the infection in patient. The current nucleic-acid chromatography-based dipstick assay is highly specific and can work even in the case of low viral load as well.Copyright © 2023
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OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.
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Introduction: Covid-19 epidemic results from an infection caused by SARS-CoV2. Evolution-based analyses on the nucleotide sequences show that SARS-CoV2 is a member of the genus Beta-coronaviruses and its genome consists of a single-stranded RNA, encoding 16 proteins. Among the structural proteins, the nucleocapsid is the most abundant protein in virus structure, highly immunogenic, with sequence conservatory. Due to a large number of mutations in the spike protein, the aim of this study was to investigate bioinformatics, expression of nucleocapsid protein and evaluate its immunogenicity as an immunogenic candidate Material(s) and Method(s): B and T cell epitopes of nucleocapsid protein were examined in the IEDB database. The PET28a-N plasmid was transferred to E. coli BL21(DE3) expression host, and IPTG induced recombinant protein expression. The protein was purified using Ni-NTA column affinity chromatography, and the Western blotting method was utilized to confirm it. Finally, mice were immunized with three routes of purified protein. Statistical analysis of the control group injection and test results was carried out by t-test from SPSS software. Result(s): The optimized gene had a Codon adaptation index (CAI) of 0/97 Percentage of codons having high-frequency distribution was improved to 85%. Expression of recombinant protein in E.coli led to the production of BoNT/B-HCC with a molecular weight of 45 kDa. The total yield of purified protein was 43 mg/L. Immunization of mice induced serum antibody response. Statistical analysis showed that the antibody titer ratio was significantly different compared to the control sample and the antibody titer was acceptable up to a dilution of 1.256000 Conclusion(s): According to the present study results, the protein can be used as an immunogenic candidate for developing vaccines against SARS-CoV2 in future research.Copyright © 2022, Semnan University of Medical Sciences. All rights reserved.
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The pandemic spread of African swine fever (ASF) has caused serious effects on the global pig industry. Virus genome sequencing and genomic epidemiology analysis play an important role in tracking the outbreaks of the disease and tracing the transmission of the virus. Here we obtained the full-length genome sequence of African swine fever virus (ASFV) in the first outbreak of ASF in China on August 3rd, 2018 and compared it with other published genotype II ASFV genomes including 9 genomes collected in China from September 2018 to October 2020. Phylogenetic analysis on genomic sequences revealed that genotype II ASFV has evolved into different genetic clusters with temporal and spatial correlation since being introduced into Europe and then Asia. There was a strong support for the monophyletic grouping of all the ASFV genome sequences from China and other Asian countries, which shared a common ancestor with those from the Central or Eastern Europe. An evolutionary rate of 1.312 × 10−5 nucleotide substitutions per site per year was estimated for genotype II ASFV genomes. Eight single nucleotide variations which located in MGF110-1L, MGF110-7L, MGF360-10L, MGF505-5R, MGF505-9R, K145R, NP419L, and I267L were identified as anchor mutations that defined genetic clusters of genotype II ASFV in Europe and Asia. This study expanded our knowledge of the molecular epidemiology of ASFV and provided valuable information for effective control of the disease.
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Whole-genome sequencing of upper respiratory tract specimens from patients with confirmed COVID-19 in Henan Province was performed to compare the performance of the Illumina and Oxford Nanopore sequencing platforms, thus providing a reference for whole-genome monitoring of the novel coronavirus (SARS-CoV-2). Ten samples from COVID-19 cases in Henan Province from June 2021 to January 2022 were collected and sequenced with Illumina and Nanopore high-through-put sequencing technology to obtain full genome sequences of the novel coronavirus, which were compared with the Wuhan reference sequence (Wuhan-Hu-1). Bioinformatics software (CLC) was used for sequence alignment analysis. Three of the ten samples were Omicron (BA.1) variants with 55,61 nucleotide variation sites. One sample was an Alpha (B.1.1.7) variant with 41 nucleotide variation sites. Six samples were Delta (8.1.617.2) variants with 35,42,47 nucleotide variation sites. The sequence identity of mutation sites in six samples was 100%, and the mutation sites in the S genome segment of seven samples were consistent. For samples with a Ct value < 33, both next-generation and third-generation sequencing achieved high genome coverage and sequencing depth. A significant difference in coverage was observed between second-generation sequencing and third-generation sequencing (t=-2.037, P < 0.06). However, the coverage at different time points of the third-generation sequencing did not significantly differ (F=2.498, P > 0.05). The needs for SARS-CoV-2 mutant detection could be met through use of either high-throughput sequencing platform. The identification of mutations in the novel coronavirus through Illumina high-throughput sequencing was more accurate, whereas Nanopore high-throughput sequencing technology could be used for rapid detection and typing of different novel coronaviruses.
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Genome sequence analysis of Atlantic salmon bafinivirus (ASBV) revealed a small open reading frame (ORF) predicted to encode a Type I membrane protein with an N-terminal cleaved signal sequence (110 aa), likely an envelope (E) protein. Bioinformatic analyses showed that the predicted protein is strikingly similar to the coronavirus E protein in structure. This is the first report to identify a putative E protein ORF in the genome of members of the Oncotshavirus genus (subfamily Piscavirinae, family Tobaniviridae, order Nidovirales) and, if expressed would be the third family (after Coronaviridae and Arteriviridae) within the order to have the E protein as a major structural protein.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
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Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
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BACKGROUND: Gammacoronaviruses, which are single-stranded, positive-sense RNA viruses, are responsible for a wide variety of existing and emerging diseases in birds. The Gammacoronaviruses primarily infect avian hosts. OBJECTIVE(S): This study aimed to investigate the genetic diversity of Gammacoronaviruses in quail population in Iran. METHOD(S): In the period from 2016 to 2018, samples from 47 quail flocks with or without enteric signs, were collected from four provinces in Iran. RESULT(S): Gammacoronavirus was detected in samples of 4 flocks by using RT-PCR and characterized by N gene sequencing. The isolates formed a distinct group from other Gamma- coronaviruses groups CONCLUSION(S): The finding suggests the existence of a novel Gammacoronavirus circulating in quail farms. The phylogenetic relationship of the isolates concerning different sequences and geographical regions displayed complexity and diversity. The present study is the first detection of Gammacoronavirus in quail farms in Iran. Further studies are required and should include the isolation and experimental studies of Gammacoronaviruses in Iran.Copyright © 2019.
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The emergence of new variants of SARS-CoV-2 associated with varying infectivity, pathogenicity, diagnosis, and effectiveness against treatments challenged the overall management of the COVID-19 pandemic. Wastewater surveillance (WWS), i.e., monitoring COVID-19 infections in communities through detecting viruses in wastewater, was applied to track the emergence and spread of SARS-CoV-2 variants globally. However, there is a lack of comprehensive understanding of the use and effectiveness of WWS for new SARS-CoV-2 variants. Here we systematically reviewed published articles reporting monitoring of different SARS-CoV-2 variants in wastewater by following the PRISMA guidelines and provided the current state of the art of this study area. A total of 80 WWS studies were found that reported different monitoring variants of SARS-CoV-2 until November 2022. Most of these studies (66 out of the total 80, 82.5%) were conducted in Europe and North America, i.e., resource-rich countries. There was a high variation in WWS sampling strategy around the world, with composite sampling (50/66 total studies, 76%) as the primary method in resource-rich countries. In contrast, grab sampling was more common (8/14 total studies, 57%) in resource-limited countries. Among detection methods, the reverse transcriptase polymerase chain reaction (RT-PCR)-based sequencing method and quantitative RT-PCR method were commonly used for monitoring SARS-CoV-2 variants in wastewater. Among different variants, the B1.1.7 (Alpha) variant that appeared earlier in the pandemic was the most reported (48/80 total studies), followed by B.1.617.2 (Delta), B.1.351 (Beta), P.1 (Gamma), and others in wastewater. All variants reported in WWS studies followed the same pattern as the clinical reporting within the same timeline, demonstrating that WWS tracked all variants in a timely way when the variants emerged. Thus, wastewater monitoring may be utilized to identify the presence or absence of SARS-CoV-2 and follow the development and transmission of existing and emerging variants. Routine wastewater monitoring is a powerful infectious disease surveillance tool when implemented globally.
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Aptamers are single-stranded DNA or RNA oligonucleotides that can selectively bind to a specific target. They are generally obtained by SELEX, but the procedure is challenging and time-consuming. Moreover, the identified aptamers tend to be insufficient in stability, specificity, and affinity. Thus, only a handful of aptamers have entered the practical use stage. Recently, computational approaches have demonstrated a significant capacity to assist in the discovery of high-performance aptamers. This review discusses the advances achieved in several aspects of computational tools in this field, as well as the new progress in machine learning and deep learning, which are used in aptamer identification and optimization. To illustrate these computationally aided processes, aptamer selections against SARS-CoV-2 are discussed in detail as a case study. We hope that this review will aid and motivate researchers to develop and utilize more computational techniques to discover ideal aptamers effectively. Copyright © 2022 Elsevier B.V.
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Literature review on the technologies available for the development, production and testing of a messenger RNA vaccine, starting with the establishment of the optimal sequence elements, methods for production and purification of nucleic acids and delivery systems used in vaccination and gene therapy studies. The mRNA sequence, especially the 5' and 3' untranslated regulatory elements are essential to increase stability and ensure a high level of antigen expression in vitro and in vivo. Reactogenicity and decrease in antigen expression level due to the activation of in vivo antiviral response mechanisms can be diminished by introducing additional purification steps aimed at eliminating DNA, double-stranded RNA and other contaminants resulting from the in vitro transcription processes.
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Purpose: To study the clinical application of metagenomic next-generation sequencing (mNGS) in the detection of viral infections in kidney transplant recipients (KTRs) during the COVID-19 pandemic. Method(s): Using mNGS techniques, 50 human fluid samples of KTRs were detected in Henan Province People's Hospital between May 2020 to May 2021, including 20 bronchoalveolar lavage fluid (BALF) samples, 21 urine samples and 9 blood samples. The detected nucleic acid sequences were compared and analyzed with the existing viral nucleic acid sequences in the database, and the virus infection spectrum of KTRs was drawn. Result(s): The viral nucleic acids of 15 types of viruses were detected in 96.00% (48/50) of the samples, of which 11 types of viruses were in BALF (95.00%, 19/20), and the dominant viruses were torque teno virus (TTV) (65.00%;13/20), cytomegalovirus (CMV) (45.00%;9/20) and human alphaherpesvirus 1 (25.00%;5/20). 12 viruses (95.24%, 20/21) were detected in the urine, and the dominant viruses were TTV (52.38%;11/21), JC polyomavirus (52.38%;11/21), BK polyomavirus (42.86%;9/21), CMV (33.33%;7/21) and human betaherpesvirus 6B (28.57%;6/21). 7 viruses were detected in the blood (100.00%, 9/9), and the dominant virus was TTV (100.00%;9/9). Four rare viruses were detected in BALF and urine, including WU polyomavirus, primate bocaparvovirus 1, simian virus 12, and volepox virus. Further analysis showed that TTV infection with high reads indicated a higher risk of acute rejection (P<0.05). Conclusion(s): mNGS detection reveals the rich virus spectrum of infected persons after kidney transplantation, and improves the detection rate of rare viruses. TTV may be a new biomarker for predicting rejection. (Figure Presented).
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Objective To understand the genomic characteristics of SARS-CoV-2 from 40 imported cases with confirmed COVID-19 in Sichuan during January and March 2022. Methods Total viral RNA was extracted from respiratory samples of 182 confirmed COVID-19 cases who entered China through Chendu International Airport from January to March 2022.Mutation nucleic acid detection kit was used to identify the mutant strains and Illumina sequencing platform was applied for whole genome sequence（WGS） of virus.SARS-CoV-2 reference sequences were downloaded from NCBI database for genetic evolution and antigen variation analysis.The Nextclade and Pangolin online virus analysis platform were used to determine the virus family and type,and to analyze the mutation loci of the virus.The phylogenetic tree was constructed,along with the epidemiological data of cases to analyze the source and correlation of viruses. Results Among 182 imported COVID-19 cases,B.1.617.2 mutations were identified in 3 cases and B.1.1.529 mutations were detected in 57 cases.A total of 40 SARS-CoV-2 whole genome sequences with coverage>95% were obtained in this study.Nextclade typing analysis showed that 3 sequences belonged to 21J（Delta）,5 sequences belonged to 21K（Omicron）and the remaining 32 sequences belonged to 21L（Omicron）.Pangolin typing analysis showed that the 3 sequences of 21J（Delta）belonged to AY.4,AY.109and B.1.617.2,the 5sequences of 21K（Omicron）all belonged to BA.1.1,and the remaining 32 sequences of 21L（Omicron）belonged to BA.2.Our sequence results were99.7% consistency with the Omicron variants sequences in current GISAID database.Compared with the reference sequence strain Wuhan-Hu-1（NC＿045512.2）,45,47and 42nucleotide variation sites and 36,25 and 36amino acid variation sites were found in the 3 sequences of 21J（Delta）.There were average 59（26-64）nucleotide mutation sites and 48（10-53）amino acid mutation sites in the 5sequences of 21K（Omicron）.The median number of nucleotide mutation sites of 71（66-76）and amino acid mutation sites of 53（40-56）were identified in the 32sequences of 21L（Omicron）.Phylogenetic tree analysis showed that 40SARS-CoV-2WGSs were all related to the current variants of concern（VOC）. Conclusions Continuous sequencing of SARS-CoV-2whole genome from imported cases with confirmed COVID-19is of great significance for the prevention and control of the outbreak and prevalence of local epidemic caused by imported viruses in Sichuan.
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In the Canadian Maritimes, many beekeepers rent honey bee, Apis mellifera Linnaeus (Hymenoptera: Apidae), hives to growers of lowbush blueberry, Vaccinium angustifolium (Ericaceae), for pollination services. Anecdotally, hives have less vigour following pollination, potentially due to higher Nosema spp. (Nosematidae) spore loads, the microsporidian causing nosemosis. We undertook a study to determine whether sending honey bee hives to lowbush blueberry fields for pollination (blueberry hives) results in higher Nosema spp. spore loads relative to hives remaining in apiaries (home hives). Nosema spp. spore loads were quantified using light microscopy. Nosema apis and Nosema ceranae were differentiated using polymerase chain reaction and sequencing. Nosema spp. spore loads were greatest in April and May and declined to low levels from June to September. Ninety-eight per cent of Nosema detections were positive for N. ceranae. In April, blueberry hives had a lower spore load than home hives did;however, in June, spore loads were significantly higher in blueberry hives. No other differences in Nosema spp. spore loads were observed between hive types. We conclude that Nosema ceranae is the dominant Nosema species in the Canadian Maritimes and that using hives for lowbush blueberry pollination does not appear to influence long-term Nosema spp. spore loads.
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Surveillance of municipal wastewater for RNA of SARS-CoV-2, the causative agent of coronavirus disease (COVID-19), is well documented around the world. However, unlike most countries where wastewater surveillance was initially employed during 2020, New Zealand was in the fortunate position of having very few COVID-19 cases, generally confined to Managed Isolation and Quarantine facilities. As such, the prevalence of SARS-CoV-2 RNA in wastewater was likely much lower than seen in other countries. A nine-week pilot study was undertaken to assess the feasibility of detecting SARSCoV-2 RNA in wastewater in New Zealand. Wastewater from 18 catchments across New Zealand was monitored, including six that contained Managed Isolation and Quarantine facilities. Testing both in regions known to have COVID-19 cases and regions where detection was not expected (catchments not containing Managed Isolation and Quarantine facilities) allowed the sensitivity and specificity of detection methods to be assessed. SARS-CoV-2 RNA was detected in seven out of the nine weeks of this study in the Auckland South Western Interceptor catchment, which contained a dedicated isolation facility to which confirmed cases from Auckland, Hamilton and Rotorua were transferred. In weeks two and three of sampling, SARS-CoV-2 RNA was detected in the Christchurch catchment. This coincided with up to 14 COVID-19 cases likely to be shedding high levels of virus (PCR Cq value < 20) in the Managed Isolation and Quarantine facilities. Samples from the seven other weeks were negative despite up to 35 infected cases present at any one time. However, on any of these test dates eight cases or fewer had a PCR Cq value < 30 and were within 10 days of symptom onset or positive PCR test date. Sample inhibition and non-specificity were not observed to be issues. The results of this pilot study underpinned recommendations that wastewater monitoring for SARS-CoV-2 RNA be incorporated as a surveillance tool in New Zealand's COVID-19 response.
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A trim distance between two positions in the set of nucleotide sequences is a tree-based distance between the trimmed phylogenetic trees at two positions, each of which is obtained by applying the label-based closest-neighbor trimming method to the relabeled phylogenetic tree at the position that the index as a label of leaves is relabeled to the nucleotide occurring at the position. In this paper, as a tree-based distance, we adopt a label histogram distance and a depth histogram distance. Then, we introduce new trim distances that a label trim distance and a depth trim distance, respectively. Finally, by using the nucleotide sequences and the reconstructed phylogenetic tree from them provided from NCBI, we investigate the trim distances between the positions in the nucleotide sequences for structural proteins of spike, envelope, membrane and nucleocapsid proteins of SARS-CoV-2. © 2021 IEEE.