Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer

Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Advances of Studies on the Viral Proteins of PRRSV

Porcine reproductive and respiratory syndrome( PRRS) is one of viral diseases with severe reproductive obstacle of pregnant sows and respiratory tract symptoms and higher mortality of piglets as characteristics,which is caused by porcine reproductive and respiratory syndrome virus( PRRSV). PRRS has brought great threats to swine industry in the world. The advances of studies on the viral proteins of PRRSV were reviewed from the genome,non-structural proteins and structural proteins of PRRSV.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

Acute hepatitis C in a chronically HIV-infected patient:Evolution of different viral genomic regions

AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV infected patient that seroconverted to anti HCV antibodies were sequenced, from the event of superinfection through a period of 17 months and in a late sample (42nd month). Hypervariable genomic regions of HIV (V3 loop of the gp120) and HCV (HVR-1 on the E2 glycoprotein gene) were studied. In order to analyze genomic regions involved in different biological functions and with the cellular immune response, HCV core and NS5A were also chosen to be sequenced. Amplification of the different regions was done by RT-PCR and directly sequenced. Confirmation of sequences was done on reamplified material. Nucleotide sequences of the different time points were aligned with CLUSTAL W 1.5, and the corresponding amino acid ones were deduced.RESULTS: Hypervariable genomic regions of both viruses (HVR1 and gp120 V3 loop) presented several nonsynonymous changes but, while in the gp120 V3 loop mutations were detected in the sample obtained right after HCV superinfection and maintained throughout, they occurred following a sequential and cumulative pattern in the HVR1. In the NS5A region of HCV, two amino acid changes were detected during the follow-up period, whereas the core region presented several amino acid replacements, once the HCV chronic infection had been established.CONCLUSION: During the HIV-HCV superinfection, each genomic region analyzed shows a different evolutionary pattem.Most of the nucleotide substitutions observed are nonsynonymous and clustered in previously described epitopes,thus suggesting an immune-driven evolutionary process.

Viral integration detection strategies and a technical update on Virus-Clip

Oncovirus infection is crucial in human malignancies.Certain oncoviruses can lead to structural variations in the human genome known as viral genomic integration,which can contribute to tumorigenesis.Existing viral integration detection tools differ in their underlying algorithms pinpointing different aspects or features of viral integration phenomenon.We discuss about major procedures in performing viral integration detection.More importantly,we provide a technical update on Virus-Clip to facilitate its usage on the latest human genome builds(hg19 and hg38)and the adoption of multi-thread mode for faster initial read alignment.By comparing the execution of Virus-Clip using single-thread and multi-thread modes of read alignment on targeted-panel sequencing data of HBV-associated hepatocellular carcinoma patients,we demonstrate the marked improvement of multi-thread mode in terms of significantly reduced execution time,while there is negligible difference in memory usage.Taken together,with the current update of Virus-Clip,it will continue supporting the in silico detection of oncoviral integration for better understanding of various human malignancies.

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

The present review describes the current status of multiplex quantitative real time polymerase chain reaction(q PCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex q PCR for the detection of hepatitis viruses, including hepatitis A virus(HAV), hepatitis B virus(HBV), hepatitis C virus(HCV), hepatitis D virus(HDV), and hepatitis E virus(HEV). In addition, multiplex q PCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex q PCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex q PCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.

Assessment of correlation between serum titers of hepatitis c virus and severity of liver disease

AIM:The significance of hepatitis C virus (HCV) serum titers has been examined in several clinical situations. There is much evidence that patients with a lower viral load have better response rates to anti-viral therapy compared to those with higher levels.Moreover,a direct association has been observed between serum titers of HCV and transmission rates of the virus.The aim of the present study was to determine if there was any correlation between HCV viral load and the severity of liver disease. METHODS:Fifty patients with HCV infection were included in the study.These comprised of 34 subjects with a history of alcohol use and 16 non-alcoholics.Quantitative serum HCV RNA assay was carried out using the branched DNA (bDNA) technique.Linear regression analysis was performed between serum viral titers and liver tests.In addition,for the purpose of comparison,the subjects were divided into two groups:those with low viral liters (≤50 genome mEq/mL) and high titers (>50 mEq/mL). RESULTS:All subjects were men,with a mean±SD age of 47±7.8 years.The mean HCV RNA level in the blood was 76.3×10~5±109.1 genome equivalents/mL.There was no correlation between HCV RNA levels and age of the patients (r=0.181),and the history or amount (g/d) of alcohol consumption (r=0.07).Furthermore,no correlation was observed between serum HCV RNA levels and the severity of liver disease as judged by the values of serum albumin (r=0.175),bilirubin (r=0.217),ALT (r=0.06) and AST (r=0.004) levels.Similarly,no significant difference was observed between patients with low viral titers and high liters with respect to any of the parameters. CONCLUSION:Our results indicate that the severity of liver disease is independent of serum levels of hepatitis C virus.These findings are important since they have a direct impact on the current debate regarding the role of direct cytopathic effect of hepatitis C virus versus immune-mediated injury in the pathogenesis of HCV-related liver damage.

Pitfalls of restriction enzyme analysis in identifying, characterizing,typing, and naming viral pathogens in the era of whole genome data, as illustrated by HAdV type 55

Restriction endonuclease analysis(REA),or restriction fragment length polymorphism(RFLP),was useful for identifying and determining the relatedness and putative identities of microbial strains(Tang et al.,1997)and for characterizing and discriminating large numbers of samples inexpensively in the past。

Virus genomes and virus-host interactions in aquaculture animals

Over the last 30 years,aquaculture has become the fastest growing form of agriculture production in the world,but its development has been hampered by a diverse range of pathogenic viruses.During the last decade,a large number of viruses from aquatic animals have been identified,and more than 100 viral genomes have been sequenced and genetically characterized.These advances are leading to better understanding about antiviral mechanisms and the types of interaction occurring between aquatic viruses and their hosts.Here,based on our research experience of more than 20 years,we review the wealth of genetic and genomic information from studies on a diverse range of aquatic viruses,including iridoviruses,herpesviruses,reoviruses,and rhabdoviruses,and outline some major advances in our understanding of virus–host interactions in animals used in aquaculture.

Genetic diversity of RNA viruses infecting invertebrate pests of rice

Invertebrate species are a natural reservoir of viral genetic diversity,and invertebrate pests are widely distributed in crop fields.However,information on viruses infecting invertebrate pests of crops is limited.In this report,we describe the deep metatranscriptomic sequencing of 88 invertebrate samples covering all major invertebrate pests in rice fields.We identified 296 new RNA viruses and 13 known RNA viruses.These viruses clustered within 31 families,with many highly divergent viruses constituting potentially new families and genera.Of the identified viruses,13 RNA viruses clustered within the Fiersviridae family of bacteriophages,and 48 RNA viruses clustered within families and genera of mycoviruses.We detected known rice viruses in novel invertebrate hosts at high abundances.Furthermore,some novel RNA viruses have genome structures closely matching to known plant viruses and clustered within genera of several plant virus species.Fortyfive potential insect pathogenic RNA viruses were detected in invertebrate species.Our analysis revealed that host taxonomy plays a major role and geographical location plays an important role in structuring viral diversity.Cross-species transmission of RNA viruses was detected between invertebrate hosts.Newly identified viral genomes showed extensive variation for invertebrate viral families or genera.Together,the large-scale metatranscriptomic analysis greatly expands our understanding of RNA viruses in rice invertebrate species,the results provide valuable information for developing efficient strategies to manage insect pests and virus-mediated crop diseases.

Longitudinal virological changes and underlying pathogenesis in hospitalized COVID-19 patients in Guangzhou,China

Prolonged viral RNA shedding and recurrence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)in coronavirus disease 2019(COVID-19)patients have been reported.However,the clinical outcome and pathogenesis remain unclear.In this study,we recruited 43 laboratory-confirmed COVID-19 patients.We found that prolonged viral RNA shedding or recurrence mainly occurred in severe/critical patients(P<0.05).The average viral shedding time in severe/critical patients was more than 50 days,and up to 100 days in some patients,after symptom onset.However,chest computed tomography gradually improved and complete absorption occurred when SARS-CoV-2 RT-PCR was still positive,but specific antibodies appeared.Furthermore,the viral shedding time significantly decreased when the A1,430G or C12,473T mutation occurred(P<0.01 and FDR<0.01)and increased when G227A occurred(P<0.05 and FDR<0.05).High IL1R1,IL1R2,and TNFRSF21 expression in the host positively correlated with viral shedding time(P<0.05 and false discovery rate<0.05).Prolonged viral RNA shedding often occurs but may not increase disease damage.Prolonged viral RNA shedding is associated with viral mutations and host factors.

Prediction of pandemic risk for animal-origin coronavirus using a deep learning method

Background:Coronaviruses can be isolated from bats,civets,pangolins,birds and other wild animals.As an animalorigin pathogen,coronavirus can cross species barrier and cause pandemic in humans.In this study,a deep learning model for early prediction of pandemic risk was proposed based on the sequences of viral genomes.Methods:A total of 3257 genomes were downloaded from the Coronavirus Genome Resource Library.We present a deep learning model of cross-species coronavirus infection that combines a bidirectional gated recurrent unit network with a one-dimensional convolution.The genome sequence of animal-origin coronavirus was directly input to extract features and predict pandemic risk.The best performances were explored with the use of pre-trained DNA vector and attention mechanism.The area under the receiver operating characteristic curve(AUROC)and the area under precision-recall curve(AUPR)were used to evaluate the predictive models.Results:The six specifc models achieved good performances for the corresponding virus groups(1 for AUROC and 1 for AUPR).The general model with pre-training vector and attention mechanism provided excellent predictions for all virus groups(1 for AUROC and 1 for AUPR)while those without pre-training vector or attention mechanism had obvi‑ously reduction of performance(about 5–25%).Re-training experiments showed that the general model has good capabilities of transfer learning(average for six groups:0.968 for AUROC and 0.942 for AUPR)and should give reason‑able prediction for potential pathogen of next pandemic.The artifcial negative data with the replacement of the coding region of the spike protein were also predicted correctly(100%accuracy).With the application of the Python programming language,an easy-to-use tool was created to implements our predictor.Conclusions:Robust deep learning model with pre-training vector and attention mechanism mastered the features from the whole genomes of animal-origin coronaviruses and could predict the risk of cross-species infection for early warning of next pandemic.