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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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Porcine sapelovirus (PSV) is an emerging swine enteric virus that can cause various disorders including acute diarrhea, respiratory distress, reproductive failure, and polioencephalomyelitis in pigs. In this study, we isolated a PSV strain HNHB-01 from a clinical porcine deltacoronavirus- (PDCoV-) positive intestinal content of a diarrheic piglet. PSV was first identified using the small RNA deep sequencing and assembly, and further identified by the electron microscopic observation and the immunofluorescence assay. Subsequently, this virus was serially passaged in swine testis (ST) cells, and the complete genomics of PSV HNHB-01 passage 5 (P5), P30, P60, and P100 were sequenced and analyzed. 9 nucleotide mutations and 7 amino acid changes occurred in the PSV HNHB-01 P100 strain when compared with the PSV HNHB-01 P5. Pathogenicity investigation showed that orally inoculation of PSV HNHB-01 P30 could cause obvious clinical symptoms and had broad tissue tropism in 5-day-old piglets. Epidemiological investigation revealed that PSV infections and the coinfections of diarrhea coronaviruses were highly prevalent in swine herds. The complete genomes of 8 representative PSV epidemic strains were sequenced and analyzed. Phylogenetic analysis revealed that the PSV epidemic strains were closely related to other PSV reference strains that located in the Chinese clade. Furthermore, recombination analysis revealed that the recombination events were occurred in downstream of the 2C region in our sequenced PSV HNNY-02/CHN/2018 strain. Our results provided theoretical basis for future research studies of the pathogenic mechanism, evolutionary characteristics, and the development of vaccines against PSV.
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Over the past two and a half years the world has seen a desperate scramble to find a treatment for SARS-CoV-2 and COVID. In that regard, nucleosides have long served as the cornerstone to antiviral treatments due to their resemblance to the naturally occurring nucleosides that are involved in numerous biological processes. Unlike other viruses however, it was found early on during the search for drugs to treat SARS-1 and later MERS, that the coronaviruses possess a unique repair enzyme, an exonuclease (ExoN)[3] which rendered nucleoside analogues useless, thus negating their use.[4] During the current outbreak however, as both well-known and new nucleoside analogues were investigated or reinvestigated as a possible cure for SARS-CoV-2, several novel and/or lesser-known mechanisms of action were uncovered. This review briefly describes these mechanisms.
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Digital data objects on viruses have played a pivotal role in the fight against COVID-19, leading to healthcare innovation such as new diagnostics, vaccines, and societal intervention strategies. To effectively achieve this, scientists access viral data from online communities (OCs). The social-interactionist view on generativity, however, has put little emphasis on data. We argue that generativity on data depends on the number of data instances, data timeliness, and completeness of data classes. We integrated and analyzed eight OCs containing SARS-CoV-2 nucleotide sequences to explore how community structures influence generativity, revealing considerable differences between OCs. By assessing provided data classes from user perspectives, we found that generativity was limited in two important ways: When required data classes were either insufficiently collected or not made available by OC providers. Our findings highlight that OC providers control generativity of data objects and provide guidance for scientists selecting OCs for their research. © 2022 IEEE Computer Society. 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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A method to find a probability that a given bias of mutations occur naturally is proposed to test whether a newly detected virus is a product of natural evolution or a product of non-natural process such as genetic manipulation. The probability is calculated based on the neutral theory of molecular evolution and binominal distribution of non-synonymous (N) and synonymous (S) mutations. Though most of the conventional analyses, including dN/dS analysis, assume that any kinds of point mutations from a nucleotide to another nucleotide occurs with the same probability, the proposed model takes into account the bias in mutations, where the equilibrium of mutations is considered to estimate the probability of each mutation. The proposed method is applied to evaluate whether the Omicron variant strain of SARS-CoV-2, whose spike protein includes 29 N mutations and only one S mutation, can emerge through natural evolution. The result of binomial test based on the proposed model shows that the bias of N/S mutations in the Omicron spike can occur with a probability of 2.0 × 10−3 or less. Even with the conventional model where the probabilities of any kinds of mutations are all equal, the strong N/S mutation bias in the Omicron spike can occur with a probability of 3.7 × 10−3, which means that the Omicron variant is highly likely a product of non-natural process including artifact. © 2022 Information Processing Society of Japan.
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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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Bats are important reservoir hosts of emerging viruses. Recent viral outbreaks and pandemics have resulted in an increased research focus on the genetic diversity, population structure, and distribution of bat species. Lyle’s flying fox (Pteropus lylei) is widely distributed throughout central Thailand, with most colonies congregating in temples within proximity to humans. A lack of knowledge regarding the genetic connectivity among different colonies hinders the investigation of zoonotic disease epidemiology and wildlife management. In this study, we hypothesized that genetic material may be exchanged between Lyle’s flying fox colonies that live in proximity. We assessed the mitochondrial displacement loop and cytochrome b nucleotide sequences of samples collected from 94 individuals from ten colonies across different roosting sites and detected limited genetic differentiation but increased nucleotide divergence within colonies. This suggests that genetic connectivity among Lyle’s flying fox colonies has experienced frequent and recent gene flow. These findings indicate that this species has maintained demographic equilibrium in a stable population, with a slight expansion event in certain populations. These data provide insights into the dynamics of bat populations, and the genetic knowledge gained presents opportunities for the improved monitoring of bat population structure.
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Nucleic acid amplification detection is one of the most widely used molecular diagnostic techniques in recent years, which can rapidly and efficiently amplify the characteristic nucleotide sequences of pathogenic bacteria in the diagnosis of infectious diseases, it has been widely used in clinical diagnosis, disease screening and other fields. In this work, we report a micro-cavity digital PCR for rapid detection of pathogens on a silicon-based microfluidic chip. The device has the advantages of high flux, no pumping, rapid reaction, quantification and high sensitivity. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current global COVID-19 pandemic, in which millions of lives have been lost. Understanding the zoonotic evolution of the coronavirus may provide insights for developing effective vaccines, monitoring the transmission trends, and preventing new zoonotic infections. Homopolymeric nucleotide repeats (HP), the most simple tandem repeats, are a ubiquitous feature of eukaryotic genomes. Yet the HP distributions and roles in coronavirus genome evolution are poorly investigated. In this study, we characterize the HP distributions and trends in the genomes of bat and human coronaviruses and SARS-CoV-2 variants. The results show that the SARS-CoV-2 genome is abundant in HPs, and has augmented HP contents during evolution. Especially, the disparity of HP poly-(A/T) and ploy-(C/G) of coronaviruses increases during the evolution in human hosts. The disparity of HP poly-(A/T) and ploy-(C/G) is correlated to host adaptation and the virulence level of the coronaviruses. Therefore, we propose that the HP disparity can be a quantitative measure for the zoonotic evolution levels of coronaviruses. Peculiarly, the HP disparity measure infers that SARS-CoV-2 Omicron variants have a high disparity of HP poly-(A/T) and ploy-(C/G), suggesting a high adaption to the human hosts. © 2022 Changchuan Yin, published by De Gruyter.
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Introduction: Coronavirus disease 2019 (COVID-19) has brought countless infections and deaths worldwide. COVID-19 patients demonstrated a great diversity in clinical manifestation and disease severity, but factors for these diversions are yet to be determined. Genetic variations such as the single nucleotide polymorphisms (SNPs) might be responsible for COVID-19 severity. Methods: Genomic DNA was extracted from the peripheral blood of 215 recovered COVID-19 patients with different disease severities (asymptomatic, mild, and severe). Their personal information such as gender, age, BMI, and comorbidities was recorded. Global Screening Array (GSA) was performed to search for SNPs linked with the COVID-19 severity. Potential SNPs were further genotyped using real-time PCR for validation, and their risk association was assessed. Results: Hypertension and obesity were the most prevalent comorbidities in severe COVID-19 patients. The GSA showed three potential SNPs (rs923147, rs409017, and rs17062791) that were significantly associated with COVID-19 severity. Further analyses on these SNPs revealed that male, older (>60 years), and overweight subjects who carried an A allele in the rs923147 were protective against severe COVID-19. In contrast, male patients who inherited a G allele had an increased risk of severe COVID-19. For the rs409017 SNP, the presence of an A allele significantly increased the risk of subjects developing severe COVID-19. Besides, obese patients who carried a T allele in the rs17062791 SNP had a significantly lower risk of developing mild symptoms. Conclusion: This study suggests SNPs' potential role that is linked with the COVID-19 severity. These data are useful in predicting the disease severity of COVID-19 patients.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) relies on the central molecular machine RNA-dependent RNA polymerase (RdRp) for the viral replication and transcription. Remdesivir at the template strand has been shown to effectively inhibit the RNA synthesis in SARS-CoV-2 RdRp by deactivating not only the complementary UTP incorporation but also the next nucleotide addition. However, the underlying molecular mechanism of the second inhibitory point remains unclear. In this work, we have performed molecular dynamics simulations and demonstrated that such inhibition has not directly acted on the nucleotide addition at the active site. Instead, the translocation of Remdesivir from +1 to-1 site is hindered thermodynamically as the post-Translocation state is less stable than the pre-Translocation state due to the motif B residue G683. Moreover, another conserved residue S682 on motif B further hinders the dynamic translocation of Remdesivir due to the steric clash with the 1′-cyano substitution. Overall, our study has unveiled an alternative role of motif B in mediating the translocation when Remdesivir is present in the template strand and complemented our understanding about the inhibitory mechanisms exerted by Remdesivir on the RNA synthesis in SARS-CoV-2 RdRp. © 2022 Chinese Physical Society.
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The pandemic of Coronavirus Disease 2019 (COVID19) has compelled scientists to create highly reliable diagnostic tools quickly in order to successfully and properly diagnose this pathology and thereby prevent infection transmission. Even though structural and molecular properties of the severe acute respiratory syndrome coronavirus 2 (SARSCoV2) were previously unknown, private research institutes and biomedical firms quickly developed numerous diagnostic procedures beneficial for making a correct detection of COVID19. Rapid antigen or antibody testing, immunoenzymatic serological tests, and RT-PCR based molecular assays are the most frequently used and validated procedures now available. The PCR has grown in popularity in molecular diagnostics to the point where it is still considered the gold standard for finding nucleotides from a variety of sources becoming an indispensable tool in the research lab. Because of its improved speed, sensitivity, reproducibility, and lower likelihood of carry-over contamination, real-time PCR has gained greater popularity. Currently, five different chemistries are employed to detect PCR product during real-time PCR. The selffluorescing amplicons, DNA binding fluorophores, 5' endonuclease, neighbouring linear and hairpin oligoprobes, and self-fluorescing amplicons are all detailed in depth. We also go through the problems that have hampered the development of multiplex real-time PCR and the importance of real-time PCR in nucleic acid quantification.
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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.
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Coronavirus consists of single-stranded, enveloped and RNA virus, largest genome among all RNA viruses and has 4 proteins i.e. envelope, spike, nucleocapsid and membrane. Coronaviruses are classified into 4 genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus and Deltacoronavirus. Betacoronavirus most probably originated from bats and the virus may have jumped to avian species and evolved as Deltacoronavirus group. The avian coronaviruses jumped among other avian species, giving rise to Gammacoronavirus from Deltacoronavirus, while Betacoronavirus may have given rise to Alphacoronavirus. It is known that SARS-CoV-2 belongs to Betacoronavirus. This most similar virus is verified in bat and Malayan Pangolin. Analysis showed that SARS-CoV-2 most probably originated by recombination of both bat and pangolin viruses. Viral protein seroconversion and viral specific nucleotide positive documented in all COVID-19 patients tested provides confirmation of a link between the presence of this virus and the disease.
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A new virus, the coronavirus (COVID-19), is causing serious respiratory infections in humans. Rapid, specific, and sensitive diagnostic techniques for early-stage detection of SARS-CoV-2 viral protein are developing as a necessary response for effective smart diagnostics, treatment optimization, and exploration of therapeutics with better effectiveness in the fight against the COVID-19 pandemic. Keeping the considerations mentioned above, we propose a new modeling graphene nanocomposite-based biosensing device for detecting COVID-19 at the site of the epidemic as the best way to manage the pandemic. It is important to address the problems of COVID-19 management. With the challenges and aspects of COVID-19 management in mind, we present in this review a collective approach involving electrochemical COVID-19 biosensing required for early-stage COVID-19 diagnosis and the direct interaction with viral surface glycoproteins and metal nanoparticles that can enter cells and neutralize viruses by interacting directly with the viral genome (ribonucleic acid), which identifies the COVID-19 spike protein and antiviral procedure including virus inactivation, host cell receptor inactivation, electrostatic entrapment, and physicochemical destruction of viral species by nucleotide ring opening. The interactions between the graphene composite and virus may be boosted by functionalization of the carbon surface and decoration of metallic components that enhance these interactions. Our proposed new modeling molecular dynamic simulation-based neutralizing mechanism and real-time detection of COVID-19 on graphene nanocomposite-based biosensors are suitable for point-of-care diagnostic applications, and this sensing platform can be modified for the early diagnosis of severe viral infections using real samples. For the potential application, the suggested one is the chemical reaction and bond breaking between the metallic component and molecule of COVID19 with computer simulation data.
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Cyclic adenosine diphosphate ribose (cADPR) is a cyclic nucleotide involved in the Ca2+ homeostasis. In its structure, the northern ribose, bonded to adenosine through an N1 glycosidic bond, is connected to the southern ribose through a pyrophosphate bridge. Due to the chemical instability at the N1 glycosidic bond, new bioactive cADPR derivatives have been synthesized. One of the most interesting analogues is the cyclic inosine diphosphate ribose (cIDPR), in which the hypoxanthine replaced adenosine. The efforts for synthesizing new linear and cyclic northern ribose modified cIDPR analogues led us to study in detail the inosine N1 alkylation reaction. In the last few years, we have produced new flexible cIDPR analogues, where the northern ribose has been replaced by alkyl chains. With the aim to obtain the closest flexible cIDPR analogue, we have attached to the inosine N1 position a 2″,3″-dihydroxypentyl chain, possessing the two OH groups in a ribose-like fashion. The inosine alkylation reaction afforded also the O6-alkylated regioisomer, which could be a useful intermediate for the construction of new kinds of cADPR mimics.
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In recent years, it has become clear that RNA molecules are involved in almost all vital cellular processes and pathogenesis of human disorders. The functional diversity of RNA comes from its structural richness. Although composed of only four nucleotides, RNA molecules present a plethora of secondary and tertiary structures critical for intra and intermolecular contacts with other RNAs and ligands (proteins, small metabolites, etc.). In order to fully understand RNA function it is necessary to define its spatial structure. Crystallography, nuclear magnetic resonance and cryogenic electron microscopy have demonstrated considerable success in determining the structures of biologically important RNA molecules. However, these powerful methods require large amounts of sample. Despite their limitations, chemical synthesis and in vitro transcription are usually employed to obtain milligram quantities of RNA for structural studies, delivering simple and effective methods for large-scale production of homogenous samples. The aim of this paper is to provide an overview of methods for large-scale RNA synthesis with emphasis on chemical synthesis and in vitro transcription. We also present our own results of testing the efficiency of these approaches in order to adapt the material acquisition strategy depending on the desired RNA construct.
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Objective To analyze the genomics characteristics and nucleic acid detection results of the severe Acute respiratory syndrome coronavirus 2（SARS-CoV-2） in 2 297 clinical samples collected in January and February, 2020 in Laboratory of Microbiology of Changsha Municipal Center for Disease Control and Prevention. Methods Viral RNA of throat swabs or respiratory tract specimens of coronavirus disease 2019（COVID-19） suspected cases from January 19, 2020 to February 29, 2020 was extracted and SARS-CoV-2 nucleic acid was detected by real-time reverse transcription polymerase chain reaction.The full length genome of SARS-CoV-2 in positive samples was enriched by using viral genome capture kit and sequenced on Illumina MiSeq platform.The raw reads were mapped and aligned with SPAdes software v 3.13.0.Reference SARS-CoV-2 sequences were obtained from GISAID（https://www.gisaid.org） andviral genetic evolution and antigen variation were analyzed. Results A total of 215 SARS-Co V2-nucleic acid positive samples were identified from 2 297 clinical samples.Among the SARS-Co V2-positive samples, 110 were males and 105 were from females.The male to female ratio was 1.05∶1.The highest positive rate was among 40-<60 years old people（11.35%） and the lowest positive rate was in children under 6 years old（5.49%）.The peak of newly confirmed cases was in the 5 th week（January 26 to February 1, 2020） and then decreased.There was no newly positive case after February 25, 2020.Five SARS-Co V2-whole genome sequences were obtained and there were 4 to 6 nucleotide mutations compared to the Wuhan reference strain, and the homology was more than 99.90%.Most mutations occurred only once except C8782 T and T28144 C, indicating random mutations.Phylogenetic analysis revealed that the 5 sequences belonged to the L/B or S/A lineages and were highly homologous with strains prevalent in other provinces of China at the same time. Conclusions With the quick nucleic acid tests and quarantine measures, the SARS-Co V2-positive cases in Changsha began to decline after a 2-week increasing period, and there was no new confirmed cases 6 weeks later.The genomes of SARS-Co V-2 prevalent in Changsha are highly homology with the Wuhan strains in the early 2020 and no obvious mutation is found in the local pandemic period. Reset
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Leishmaniasis is an emerging, uncontrolled, and neglected zoonotic disease. Climate change is contributing to its ongoing global expansion. The dog is the main reservoir; hence the importance of implementing effective treatment, prevention, and control measures in this animal species to protect public health. However, although the standard treatment for canine leishmaniosis (CanL) is effective, it does not provide full parasitological clearance, and side effects and drug resistance have been described. The host's immune system plays a key role in the establishment and evolution of leishmaniasis. Dietary nucleotides modulate the immune response and, given their reported efficacy and safety in sick and clinically healthy Leishmania-infected dogs and because they represent a sustainable option with no associated side effects or resistance, they could be included within the prevention, treatment, and control strategies for leishmaniasis. This article briefly summarizes the scientific literature on CanL management, including unresolved issues, and reviews the scientific evidence on immunomodulatory effects of dietary nucleotides in different animal species. It also proposes a CanL management algorithm, including nucleotides. It is concluded that nutritional modulation of the immune response with nucleotides can contribute to better management of leishmaniasis following a One Health approach, especially in the COVID-19 era.