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.

Hepatitis B virus (HBV) infection is the major public health problem worldwide. In clinical practice, serological and molecular assays are the most commonly used diagnostic methods to detect HBV infection in clinical practices. Here we present a rapid and sensitive recombinase aided amplification assay (RAA) to detect HBV at 39.0 °C for 30 min without DNA extraction from serum samples. The analytical sensitivity of RAA assay was 100 copies per reaction and showed no cross reaction with human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The universality of RAA assay was validated by testing of 41 archived serum samples with predefined HBV genotypes (B, C and D). A total of 130 archived suspected HBV infected serum samples were detected by commercial qPCR with DNA extraction and RAA assay without DNA extraction (heat-treatment). Compared with qPCR assay as a reference, the RAA assay obtained 95.7% sensitivity and 100% specificity and a kappa value of 0.818. We developed a rapid, convenient, highly sensitive and specific method to detect HBV without DNA extraction in clinical samples. This RAA method of HBV detection is very suitable for clinical testing.

Diarrhea is a major source of morbidity and mortality among young children in low-income and middle-income countries. Human adenoviruses (HAdV), particular HAdV species F (40, 41) has been recognized as important causal pathogens, however limited data exist on molecular epidemiology of other HAdV associated with acute gastroenteritis. In the present preliminary study, we performed a case-control study involving 273 children who presented diarrheal disease and 361 healthy children matched control in Children’s hospital of Hebei Province (China) to investigate the relationship between non-enteric HAdV and diarrhea. HAdV were detected and quantified using quantitative real-time PCR (qPCR) and serotyped by sequencing and phylogenetic analysis. Odds ratio (OR) was used to assess the risk factor of HAdV. HAdV were detected in 79 (28.94%) of 273 children with diarrhea including 7 different serotypes (HAdV 40, 41, 3, 2,1,5 and 57) with serotypes 40, 41 and 3 being the most dominant and in 26 (7.20%) of 361 healthy children containing 9 serotypes (HAdV 40, 41, 3, 2,1,5,57,6 and 31). A majority (91.14%) of HAdV positives occurred in diarrhea children and 65.38% in controls< 3 years of age. No significant difference in the viral load was found between case and control groups or between Ad41-positive patients and healthy controls. In addition to HAdV 40 and 41, HAdV 3 was also associated with diarrhea (OR = 17.301, adjusted OR = 9.205, p < 0.001). Our results demonstrate a high diversity of HAdV present among diarrhea and healthy children and implicate that non-enteric HAdV3 may lead to diarrhea.

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.