Xuejun Ma

VIP: an integrated pipeline for metagenomics of virus identification and discovery

Identification and discovery of viruses using next-generation sequencing technology is a fast-developing area with potential wide application in clinical diagnostics, public health monitoring and novel virus discovery. However, tremendous sequence data from NGS study has posed great challenge both in accuracy and velocity for application of NGS study. Here we describe VIP (“Virus Identification Pipeline”), a one-touch computational pipeline for virus identification and discovery from metagenomic NGS data. VIP performs the following steps to achieve its goal: (i) map and filter out background-related reads, (ii) extensive classification of reads on the basis of nucleotide and remote amino acid homology, (iii) multiple k-mer based de novo assembly and phylogenetic analysis to provide evolutionary insight. We validated the feasibility and veracity of this pipeline with sequencing results of various types of clinical samples and public datasets. VIP has also contributed to timely virus diagnosis (~10 min) in acutely ill patients, demonstrating its potential in the performance of unbiased NGS-based clinical studies with demand of short turnaround time. VIP is released under GPLv3 and is available for free download at: https://github.com/keylabivdc/VIP.

A fatal yellow fever virus infection in China: description and lessons

Yellow fever (YF) is a viral disease endemic to the tropical regions of Africa and South America. An outbreak of YF has been occurring in Angola, since the beginning of 2016. In March 2016, a 32-year-old Chinese man who returned from Angola was hospitalized and diagnosed with the first case of imported YF in China. Clinical observations, blood viral RNA detection, serological testing and treatments for the patient were performed daily. The virus was isolated in Vero cells, and the complete viral genome was sequenced and analyzed using the next-generation genomic sequencing platform. The patient presented with hemorrhagic fever, jaundice and oliguria at day 3 after onset, which rapidly progressed to multisystem organ failure with extremely elevated liver, pancreatic and myocardial enzymes. The patient died despite the intensive supportive treatments that were performed. A liver biopsy showed severe and multilobular necrosis. Viral RNA was detectable throughout the clinical course of the disease. Whole-genomic sequence analysis revealed that the virus belongs to the Angola71 genotype. Although the virus has been circulating in Angola for 45 years, only 14 amino-acid substitutions and no amino-acid changes were observed in the membrane and envelope proteins compared with the virus collected in 1971. The presence of this imported YF case in China indicated that with the increase in business travel among countries, YF outbreaks in Africa can lead to the international spread of the disease. The production and use of YF vaccines is, therefore, an urgent issue.

Etiology of Multiple Non-EV71 and Non-CVA16 Enteroviruses Associated with Hand, Foot and Mouth Disease in Jinan, China, 2009-June 2013.

Hand, foot, and mouth disease (HFMD) is an infectious disease caused by human enterovirus 71 (EV71), coxsackievirus A16 (CVA16) and other enteroviruses. It is of interest that other enteroviruses associated with HFMD in Jinan have been rarely reported. The aim of the present study is to detect and characterize the circulating serotypes of non-EV71 and non-CVA16 enteroviruses associated with HFMD in Jinan city, Shandong province, China. A total of 400 specimens were collected from clinically diagnosed HFMD cases in Jinan from January 2009 to June 2013. All specimens were infected with non-EV71 and non-CVA16 enteroviruses previously confirmed by RT-PCR or real-time PCR according to the protocols at that time. The GeXP-based multiplex RT-PCR assay (GeXP assay) was performed to investigate the pathogen spectrum of 15 enteroviruses (coxsackieviruses A4, A5, A6, A9, A10, A16; coxsackieviruses B1, B3, B5; Echoviruses 6, 7, 11, 13, 19 and EV71) infections associated with HMFD. For GeXP assay negative samples, reverse transcription nested PCR (nested RT-PCR) based on the 5’ -untranslated region (5’- UTR) sequence and phylogenetic analysis were conducted to further explore the etiology of multiple enteroviruses. The results showed that a total of twenty serotypes of enteroviruses (including EV71 and CVA16) were identified by GeXP assay and nested RT-PCR. The most circulating twelve serotypes of enteroviruses with HFMD in Jinan from 2009 to June 2013 were EV71, CVA16, CVA10, CVA6, CVA12, CVA2, Echo3, CVA4, CVA9, CVB1, CVB3 and Echo6. CVA10 and CVA6 were the most prevalent pathogens other than EV71 and CVA16 in Jinan and their most prevalent seasons were spring and summer, and a slight increase was observed in autumn and early winter. It should be noted that mixed-infections were identified by GeXP assay and the phylogenetic tree clearly discriminated the multiple pathogens associated with HFMD. Our results thus demonstrate that there was a clear lack of a reliable testing method for EV71 and CVA16 and multiple non-EV71 and non-CVA16 enteroviruses associated with HFMD were present in Jinan. The GeXP assay combined with nested RT-PCR based on 5’-UTR region could meet the need for the national surveillance of multiple enteroviruses or the investigation of epidemic outbreaks triggered by enteroviruses in the future.

Evaluation of a Direct Reverse Transcription Loop-Mediated Isothermal Amplification Method without RNA Extraction for the Detection of Human Enterovirus 71 Subgenotype C4 in Nasopharyngeal Swab Specimens

Human enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD) worldwide and has been associated with neurological complications which resulted in fatalities during recent outbreak in Asia pacific region. A direct reverse transcription loop-mediated isothermal amplification (direct RT-LAMP) assay using heat-treated samples without RNA extraction was developed and evaluated for the detection of EV71 subgenotype C4 in nasopharyngeal swab specimens. The analytical sensitivity and specificity of the direct RT-LAMP assay were examined. The detection limit of the direct RT-LAMP assays was 1.6 of a 50% tissue culture infective dose (TCID50) per reaction and no cross-reaction was observed with control viruses including Cosackievirus A (CVA) viruses (CVA2,4,5,7,9,10,14,16, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3,6,11, and 19). The direct RT-LAMP assay was evaluated and compared to both RT-LAMP and quantitative real-time PCR (qRT-PCR) in detecting EV71 infection with 145 nasopharyngeal swab specimens. The clinical performance demonstrated the sensitivity and specificity of direct RT-LAMP was reported to be 90.3% and 100% respectively, compared to RT-LAMP, and 86.83% and 100% respectively, compared to qRT-PCR. These data demonstrated that the direct RT-LAMP assay can potentially be developed for the point of care screening of EV71 infection in China.

Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the First Imported MERS-CoV Case in China

ABSTRACT On 26 May 2015, an imported Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in Guangdong Province, China, and found to be closely related to the MERS-CoV strain prevalent in South Korea. The full genome of the ChinaGD01 strain was sequenced and analyzed to investigate the epidemiology and evolution of MERS-CoV circulating in South Korea and China.

A high throughput multiplex PCR assay for simultaneous detection of seven aminoglycoside-resistance genes in Enterobacteriaceae

BackgroundThe aminoglycoside-resistance genes encoding aminoglycoside modifying enzymes and 16S rRNA methyltransferases are main factors contributing to increasing resistance to aminoglycosides. Characterization and distribution of antimicrobial resistance gene profiles provide important information on the potential difficulty of treatment of bacteria. Several molecular methods have been developed to investigate the prevalence of aminoglycoside-resistance genes. These existing methods are time-consuming, labor-intensive, expensive or limited sensitivity in the epidemiological investigation. Therefore, it is necessary to develop a rapid, less-costly and high throughput and sensitive method to investigate the distribution of antimicrobial resistance gene in clinical isolates.ResultsIn this study, we developed a GeXP analyzer-based multiplex PCR assay to simultaneously detect seven aminoglycoside-resistance genes, including aac(3)-II, aac(6′)-Ib, aac(6′)-II, ant(3″)-I,aph(3′)-VI,armA and rmtB, and to analyze the distribution of these genes in clinical Enterobacteriaceae isolates. Under optimized conditions, this assay achieved a limit-of-detection as low as 10 copies of each of the seven genes. The presented method was applied to analyze the distribution of aminoglycoside-resistance genes in 56 clinical Enterobacteriaceae isolates, and the results were compared with that of the conventional single PCR assay. Kappa values of the two methods for detecting each of the seven resistance genes were 0.831, 0.846, 0.810, 0.909, 0.887, 0.810 and 0.825, respectively.ConclusionThis GeXP assay is demonstrated to be a rapid, cost-effective and high throughput method with high sensitivity and specificity for simultaneously detecting seven common aminoglycoside-resistance genes.

Complete Genome Sequence of Zika Virus from the First Imported Case in Mainland China

ABSTRACT The first case of Zika virus infection was identified in a Chinese traveler returning from Venezuela in February 2016. This report describes the complete genome sequence of Zika virus from the first imported case in China. The genome sequence analysis showed that the Zika virus isolated in this case belongs to the Asian lineage.

A Novel Multiplex Tetra-Primer ARMS-PCR for the Simultaneous Genotyping of Six Single Nucleotide Polymorphisms Associated with Female Cancers

Background The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNPs, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer–and two cervical cancer risk–related SNPs. Methods A total of 24 T-ARMS-PCR primers, each 5′-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method. Results Of the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples. Conclusions This novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.

A Two-Tube Multiplex Reverse Transcription PCR Assay for Simultaneous Detection of Sixteen Human Respiratory Virus Types/Subtypes

There is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens. With an intended application in provincial Centers for Diseases Control and Prevention, in this study, we present a two-tube multiplex RT-PCR assay (two-tube assay) using automatic electrophoresis to simultaneously detect sixteen common respiratory viruses. The specificity and the sensitivity of the assay were tested. The assay could detect 20–200 copies per reaction when each viral type was assayed individually, 2000 copies with 9 premixed viral targets in the multiplexed assay in tube 1, and 200 copies with 8 premixed templates in tube 2. A total of 247 specimens were used to evaluate the two-tube assay, and the results were compared with those obtained from the Luminex xTAG RVP Fast assay. The discordant results were confirmed by sequencing or by the Seeplex RV15 ACE detection kit. There were no false positives, but six false negatives occurred with the two-tube assay. In conclusion, the two-tube assay is demonstrated to have great potential for routine surveillance of respiratory virus infection in China.

Human Coronaviruses HCoV-NL63 and HCoV-HKU1 in Hospitalized Children with Acute Respiratory Infections in Beijing, China

The human coronaviruses (HCoVs) HCoV-NL63 and HCoV-HKU1 are two recently discovered coronaviruses that circulate widely and are associated with acute respiratory infections (ARI). We detected HCoV-NL63 and HCoV-HKU1 in specimens collected from May 2008 to March 2010 from patients with ARI aged <7.75 years of age attending the Beijing Childrens Hospital. Thirty-two (8.4%) and 57 (14.9%) of 382 specimens tested positive for HCoV-NL63 and HCoV-HKU1, respectively, by real-time RT-PCR. Use of a Luminex xTAG RVP Fast kit showed that coinfection with respiratory syncytial virus and parainfluenza 3 virus was common among patients infected with either virus type. In HCoV-HKU1-infected patients, the predominant clinical symptoms were cough, fever, and expectoration. In HCoV-NL63-infected patients they were cough, fever, and rhinorrhea. Phylogenetic studies showed that the HCoV-HKU1 nucleoprotein gene was relatively conserved compared to NCBI reference sequences, while the 1ab gene of HCoV-NL63 showed more variation.