Yongfeng Hu

Virome Analysis for Identification of Novel Mammalian Viruses in Bat Species from Chinese Provinces

ABSTRACT Bats are natural hosts for a large variety of zoonotic viruses. This study aimed to describe the range of bat viromes, including viruses from mammals, insects, fungi, plants, and phages, in 11 insectivorous bat species (216 bats in total) common in six provinces of China. To analyze viromes, we used sequence-independent PCR amplification and next-generation sequencing technology (Solexa Genome Analyzer II; Illumina). The viromes were identified by sequence similarity comparisons to known viruses. The mammalian viruses included those of the Adenoviridae, Herpesviridae, Papillomaviridae, Retroviridae, Circoviridae, Rhabdoviridae, Astroviridae, Flaviridae, Coronaviridae, Picornaviridae, and Parvovirinae; insect viruses included those of the Baculoviridae, Iflaviridae, Dicistroviridae, Tetraviridae, and Densovirinae; fungal viruses included those of the Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae; and phages included those of the Caudovirales, Inoviridae, and Microviridae and unclassified phages. In addition to the viruses and phages associated with the insects, plants, and bacterial flora related to the diet and habitation of bats, we identified the complete or partial genome sequences of 13 novel mammalian viruses. These included herpesviruses, papillomaviruses, a circovirus, a bocavirus, picornaviruses, a pestivirus, and a foamy virus. Pairwise alignments and phylogenetic analyses indicated that these novel viruses showed little genetic similarity with previously reported viruses. This study also revealed a high prevalence and diversity of bat astroviruses and coronaviruses in some provinces. These findings have expanded our understanding of the viromes of bats in China and hinted at the presence of a large variety of unknown mammalian viruses in many common bat species of mainland China.

Survey of enterovirus infections from hand, foot and mouth disease outbreak in china, 2009

BackgroundIn China, a rapid expansion of Hand, foot, and mouth disease (HFMD) outbreaks has occurred since 2004 and HFMD has become an important issue for China. However, people are still only concerned with human enterovirus 71(HEV-71) and coxsackie virus A16 (CV-A16). Much of what is known about the other enterovirus infections relies on fractional evidence and old epidemic data, with little knowledge concerning their distribution. To alert potential threatens of the other enteroviruses, our study genetically characterized specimens from different regions of China and yielded novel information concerning the circulating and phylogenetic characteristics of enteroviral strains from HFMD cases.FindingsA total of 301 clinical throat swabs were randomly obtained from patients suffering from HFMD from the southern, northern and central regions of China during outbreaks in 2009. 266 of 301 (88.4%) HFMD cases were found positive for HEV and seven genotypes, HEV-71, CV-A16, -B5, -A4, -A6, -A10, and -A12, were detected.ConclusionsThe HFMD pathogen compositions in the different regions of China were significantly different. HFMD epidemics might persist for a long time in China due to the multiple pathogen compositions, the enteroviral characteristic of recombination and co-infection, the ever-increasing travel and migration and the deficiency of effective vaccine. Our study deserves the attention on HFMD control and vaccine development.

Unbiased Parallel Detection of Viral Pathogens in Clinical Samples by Use of a Metagenomic Approach

ABSTRACT Viral infectious diseases represent a major threat to public health and are among the greatest disease burdens worldwide. Rapid and accurate identification of viral agents is crucial for both outbreak control and estimating regional disease burdens. Recently developed metagenomic methods have proven to be powerful tools for simultaneous pathogen detection. Here, we performed a systematic study of the capability of the short-read-based metagenomic approach in the molecular detection of viral pathogens in nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections (n = 16). Using the high-throughput capacity of ultradeep sequencing and a dedicated data interpretation method, we successfully identified seven species of known respiratory viral agents from 15 samples, a result that was consistent with results of conventional PCR assays. We also detected a coinfected case that was missed by regular PCR testing. Using the metagenomic data, 11 draft genomes of the abundantly detected viruses in the samples were reconstructed with 21.84% to 98.53% coverage. Our results show the power of the short-read-based metagenomic approach for accurate and parallel screening of viral pathogens. Although there are some inherent difficulties in applying this approach to clinical samples, including a lack of controls, limited specimen quantity, and high contamination rate, our work will facilitate further application of this unprecedented high-throughput method to clinical samples.

Complete Genome Analysis of Coxsackievirus A2, A4, A5, and A10 Strains Isolated from Hand-Foot-and-Mouth Disease Patients in China Revealing Frequent Recombination of Human Enterovirus A

ABSTRACT Coxsackievirus (CV) strains CVA2, CVA4, CVA5, and CVA10 were isolated from patients with hand, foot, and mouth disease during a 2009 outbreak in China. Full genome sequences for four representative strains, CVA2/SD/CHN/09 (A2SD09), CVA4/SZ/CHN/09 (A4SZ09), CVA5/SD/CHN/09 (A5SD09), and CVA10/SD/CHN/09 (A10SD09), were determined. Phylogenetic and recombination analyses of the isolates by comparison with human enterovirus A prototype strains revealed that genetic recombination occurred during cocirculation of the viruses. The A2SD09 and A4SZ09 strains were most closely related to their corresponding prototype strains in the capsid region but shared noncapsid sequences with each other. Similarly, strains A5SD09 and A10SD09 had serotype-specific homology for the capsid proteins but shared noncapsid sequences with each other. Phylogenetic analyses of the four isolates with homotypic strains showed that CVA2 strains were divided into five genotypes. The A2SD09 strain clustered with Mongolia strains isolated in 2003, forming genotype V. The A4SZ09 strain and other isolates from mainland China and Taiwan clustered with genotype III strains and are likely related to strains that circulated in Europe and Mongolia. The A5SD09 strain is closely related to other Chinese strains isolated in 2008. The A10SD09 isolate, together with other Chinese strains isolated since 2004, formed a distinct lineage that was likely imported from Japan and South Korea. This study shows that natural recombination is a frequent event in human enterovirus A evolution. More comprehensive surveillance of enteroviruses that focus not only on EV71 or CVA16 is needed for us to understand the molecular epidemiology of enteroviruses and to track recombination events which may ultimately affect the virulence of viruses during outbreaks.

Epidemic characteristics of hand, foot, and mouth disease in southern China, 2013: Coxsackievirus A6 has emerged as the predominant causative agent

vere community-acquired pneumonia: a systematic review. Crit Care 2008;12(3):R76. 8. Salomon R, Hoffmann E, Webster RG. Inhibition of the cytokine response does not protect against lethal H5N1 influenza infection. Proc Natl Acad Sci U S A 2007;104(30): 12479e81. 9. Walsh KB, Teijaro JR, Wilker PR, Jatzek A, Fremgen DM, Das SC, et al. Suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus. Proc Natl Acad Sci U S A 2011;108(29):12018e23.

Comparative study of the cytokine/chemokine response in children with differing disease severity in enterovirus 71-induced hand, foot, and mouth disease.

Background Enterovirus 71 (EV71) infection can lead to a rapidly progressing, life-threatening, and severe neurological disease in young children, including the development of human hand, foot, and mouth disease (HFMD). This study aims to further characterize the specific immunological features in EV71–mediated HFMD patients presenting with differing degrees of disease severity. Methodology Comprehensive cytokine and chemokine expression were broadly evaluated by cytokine antibody array in EV71–infected patients hospitalized for HFMD compared to Coxsackievirus A16-infected patients and age-matched healthy controls. More detailed analysis using Luminex-based cytokine bead array was performed in EV71–infected patients stratified into diverse clinic outcomes. Additionally, immune cell frequencies in peripheral blood and EV71–specific antibodies in plasma were also examined. Principal Findings Expression of several cytokines and chemokines were significantly increased in plasma from EV71–infected patients compared to healthy controls, which further indicated that: (1) GM-CSF, MIP-1β, IL-2, IL-33, and IL-23 secretion was elevated in patients who rapidly developed disease and presented with uncomplicated neurological damage; (2) G-CSF and MCP-1 were distinguishably secreted in EV71 infected very severe patients presenting with acute respiratory failure; (3) IP-10, MCP-1, IL-6, IL-8, and G-CSF levels were much higher in cerebrospinal fluid than in plasma from patients with neurological damage; (4) FACS analysis revealed that the frequency of CD19+HLADR+ mature B cells dynamically changed over time during the course of hospitalization and was accompanied by dramatically increased EV71–specific antibodies. Our data provide a panoramic view of specific immune mediator and cellular immune responses of HFMD and may provide useful immunological profiles for monitoring the progress of EV71–induced fatal neurological symptoms with acute respiratory failure.

Enterovirus Coinfection During an Outbreak of Hand, Foot, and Mouth Disease in Shandong, China

During an Outbreak of Hand, Foot, and Mouth Disease in Shandong, China To the Editor—Hand, foot, and mouth disease (HFMD) is caused by human enteroviruses, most frequently human enterovirus 71 (HEV-71) and coxsackievirus A16 (CV-A16). Other viruses (CV-A4 to A7, A9, A10, A24, and B2 to B5; echoviruses 1, 4, 11, and 18; and HEV-18) may also be associated with HFMD outbreaks or sporadic cases. HFMD is generally a benign febrile exanthematous childhood disease, excluding a small proportion of HEV-71 infections associated with severe complications, including encephalitis, aseptic meningitis, pulmonary edema or hemorrhage, and acute flaccid paralysis. CV-B5 has been reported to cause more serious neurologic symptoms such as encephalitis [1–3]. Currently, HEV-71 and CV-A16 can be routinely detected from throat swab or stool samples in hospitalized cases in China, allowing physicians to make a diagnosis and predict disease progression and prognosis. In China, a rapid expansion of HFMD outbreaks has occurred since 2004 [4]. From April to May 2009, a total of 110 HFMD children were brought to Linyi Hospital in Shandong, China; all patients were identified according to Ministry of Health diagnostic criteria (http://www.moh.gov.cn/publicfiles/ business/htmlfiles/mohyzs/s3586/201004/ 46884.htm). We collected clinical throat swabs and serum from patients and found that 97/110 (88.2%) swab samples were positive for HEV by seminested reverse-transcription polymerase chain reaction (RT-PCR) with general enterovirus primers. HEV-71 constituted 60/97 (61.9%) of positive cases, and CV-A16 constituted 16/97 (16.5%) of typed strains. We simultaneously detected four other HEVs: CV-B5 (14.4%), CV-A6 (3.1%), CV-A10 (1%), and CVA12 (3.1%). Following further cloning and sequencing of RT-PCR products of the CV-A16–positive swabs, 4 cases (Table 1) also carried another 1–2 types of enterovirus (CV-B5 and -A6). Although

Analysis of the Secretome and Identification of Novel Constituents from Culture Filtrate of Bacillus Calmette-Guérin Using High-resolution Mass Spectrometry

Tuberculosis (TB) is an infectious bacterial disease that causes morbidity and mortality, especially in developing countries. Although its efficacy against TB has displayed a high degree of variability (0%–80%) in different trials, Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been recognized as an important weapon for preventing TB worldwide for over 80 years. Because secreted proteins often play vital roles in the interaction between bacteria and host cells, the secretome of mycobacteria is considered to be an attractive reservoir of potential candidate antigens for the development of novel vaccines and diagnostic reagents. In this study, we performed a proteomic analysis of BCG culture filtrate proteins using SDS-PAGE and high-resolution Fourier transform mass spectrometry. In total, 239 proteins (1555 unique peptides) were identified, including 185 secreted proteins or lipoproteins. Furthermore, 17 novel protein products not annotated in the BCG database were detected and validated by means of RT-PCR at the transcriptional level. Additionally, the translational start sites of 52 proteins were confirmed, and 22 proteins were validated through extension of the translational start sites based on N-terminus-derived peptides. There are 103 secreted proteins that have not been reported in previous studies on the mycobacterial secretome and are unique to our study. The physicochemical characteristics of the secreted proteins were determined. Major components from the culture supernatant, including low-molecular-weight antigens, lipoproteins, Pro-Glu and Pro-Pro-Glu family proteins, and Mce family proteins, are discussed; some components represent potential predominant antigens in the humoral and cellular immune responses.

Sensitive and rapid detection of viruses associated with hand foot and mouth disease using multiplexed MALDI-TOF analysis

BACKGROUND Human enterovirus (HEV) is the major cause of hand foot and mouth disease (HFMD). A powerful method for detecting HEVs associated with HFMD can provide results in a clinically relevant time frame. However, the limitations of the current enterovirus test make it difficult to identify multiple genotypes on the first pass. OBJECTIVE To develop a more sensitive and easy applicable assay for detecting 18 HFMD-associated HEV serotypes in multiplex PCR products. STUDY DESIGN : A total of 241 clinical specimens were collected from HFMD patients during the 2010 outbreak in China. These samples were tested by DNA sequencing and MassARRAY analysis, respectively. RESULTS Analysis of a dilution series of plasmids revealed the detection limit per PCR reaction for the MassARRAY method was one copy for the tested HEVs. We compared results from 241 samples to those of the sequence analysis of the VP1 gene. The MassARRAY method detected all samples found positive by consensus PCR and sequencing method. Comparison of the results of MassARRAY and the DNA sequencing method found concordant results for 225 (93.4%) of the 241 samples. In 14 (5.8%) samples, the MassARRAY method detected multiple types, whereas the DNA sequencing method detected a single type. In another 2 (0.8%) samples, the MassARRAY method detected single types, whereas the DNA sequencing method detected no HEV. CONCLUSIONS The MassARRAY assay is a highly sensitive and accurate method for the type-specific detection of 18 HEVs in HFMD and is a powerful complement to current detection methods.

Full Genome of Influenza A (H7N9) Virus Derived by Direct Sequencing without Culture

An epidemic caused by influenza A (H7N9) virus was recently reported in China. Deep sequencing revealed the full genome of the virus obtained directly from a patient’s sputum without virus culture. The full genome showed substantial sequence heterogeneity and large differences compared with that from embryonated chicken eggs.