Meiying Yan

Molecular Evolution Analysis and Geographic Investigation of Severe Acute Respiratory Syndrome Coronavirus-Like Virus in Palm Civets at an Animal Market and on Farms

ABSTRACT Massive numbers of palm civets were culled to remove sources for the reemergence of severe acute respiratory syndrome (SARS) in Guangdong Province, China, in January 2004, following SARS coronavirus detection in market animals. The virus was identified in all 91 palm civets and 15 raccoon dogs of animal market origin sampled prior to culling, but not in 1,107 palm civets later sampled at 25 farms, spread over 12 provinces, which were claimed to be the source of traded animals. Twenty-seven novel signature variation residues (SNVs) were identified on the spike gene and were analyzed for their phylogenetic relationships, based on 17 sequences obtained from animals in our study and from other published studies. Analysis indicated that the virus in palm civets at the live-animal market had evolved to infect humans. The evolutionary starting point was a prototype group consisting of three viral sequences of animal origin. Initially, seven SNV sites caused six amino acid changes, at positions 147, 228, 240, 479, 821, and 1080 of the spike protein, to generate low-pathogenicity viruses. One of these was linked to the first SARS patient in the 2003-2004 period. A further 14 SNVs caused 11 amino acid residue changes, at positions 360, 462, 472, 480, 487, 609, 613, 665, 743, 765, and 1163. The resulting high-pathogenicity groups were responsible for infections during the so-called early-phase epidemic of 2003. Finally, the remaining six SNVs caused four amino acid changes, at positions 227, 244, 344, and 778, which resulted in the group of viruses responsible for the global epidemic.

SARS-CoV infection in a restaurant from palm civet.

Contact with food animals was associated with SARS-CoV infection in the People’s Republic of China.

Genetic Diversity of Toxigenic and Nontoxigenic Vibrio cholerae Serogroups O1 and O139 Revealed by Array-Based Comparative Genomic Hybridization

Toxigenic serogroups O1 and O139 of Vibrio cholerae may cause cholera epidemics or pandemics. Nontoxigenic strains within these serogroups also exist in the environment, and also some may cause sporadic cases of disease. Herein, we investigate the genomic diversity among toxigenic and nontoxigenic O1 and O139 strains by comparative genomic microarray hybridization with the genome of El Tor strain N16961 as a base. Conservation of the toxigenic O1 El Tor and O139 strains is found as previously reported, whereas accumulation of genome changes was documented in toxigenic El Tor strains isolated within the 40 years of the seventh pandemic. High phylogenetic diversity in nontoxigenic O1 and O139 strains is observed, and most of the genes absent from nontoxigenic strains are clustered together in the N16961 genome. By comparing these toxigenic and nontoxigenic strains, we observed that the small chromosome of V. cholerae is quite conservative and stable, outside of the superintegron region. In contrast to the general stability of the genome, the superintegron demonstrates pronounced divergence among toxigenic and nontoxigenic strains. Additionally, sequence variation in virulence-related genes is found in nontoxigenic El Tor strains, and we speculate that these intermediate strains may have pathogenic potential should they acquire CTX prophage alleles and other gene clusters. This genome-wide comparison of toxigenic and nontoxigenic V. cholerae strains may promote understanding of clonal differentiation of V. cholerae and contribute to an understanding of the origins and clonal selection of epidemic strains.

Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.

Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) “hot spot” in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.

Laboratory-Based Surveillance of Non-typhoidal Salmonella Infections in Guangdong Province, China

Salmonella is one of the most common foodborne pathogens in humans. Laboratory-based surveillance for non-typhoidal Salmonella infection was conducted in Guangdong Province, China to improve understanding about the disease burden and detection of dispersed outbreaks. Salmonella isolated from patients with diarrhea were sent from 16 sentinel hospitals to local public health laboratories for confirmation, serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). PFGE patterns were analyzed to identify clusters representing potential outbreaks. Between September 2009 and October 2010, 352 (4%) Salmonella isolates were obtained from 9167 stool specimens. Salmonella enterica serotype Typhimurium (45%) and Salmonella enterica serotype Enteritidis (13%) were the most common serotypes, and multidrug resistance was high, especially in Salmonella Typhimurium isolates. PFGE patterns of obtained Salmonella isolates were found to be diverse, but a unique PFGE pattern comprising 53 Salmonella Typhimurium isolates were found to occur almost exclusively in infants. Epidemiologic studies are ongoing to determine whether a common exposure is the source of the Salmonella Typhimurium strain frequently isolated from infants.

Pleiotropic effects of the twin-arginine translocation system on biofilm formation, colonization, and virulence in Vibrio cholerae.

BackgroundThe Twin-arginine translocation (Tat) system serves to translocate folded proteins, including periplasmic enzymes that bind redox cofactors in bacteria. The Tat system is also a determinant of virulence in some pathogenic bacteria, related to pleiotropic effects including growth, motility, and the secretion of some virulent factors. The contribution of the Tat pathway to Vibrio cholerae has not been explored. Here we investigated the functionality of the Tat system in V. cholerae, the etiologic agent of cholera.ResultsIn V. cholerae, the tatABC genes function in the translocation of TMAO reductase. Deletion of the tatABC genes led to a significant decrease in biofilm formation, the ability to attach to HT-29 cells, and the ability to colonize suckling mouse intestines. In addition, we observed a reduction in the output of cholera toxin, which may be due to the decreased transcription level of the toxin gene in tatABC mutants, suggesting an indirect effect of the mutation on toxin production. No obvious differences in flagellum biosynthesis and motility were found between the tatABC mutant and the parental strain, showing a variable effect of Tat in different bacteria.ConclusionThe Tat system contributes to the survival of V. cholerae in the environment and in vivo, and it may be associated with its virulence.

The Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Salmonella enterica serovar Typhi

Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol.

The emergence and outbreak of multidrug-resistant typhoid fever in China

Typhoid fever remains a severe public health problem in developing countries. The emergence of resistant typhoid, particularly multidrug-resistant typhoid infections, highlights the necessity of monitoring the resistance characteristics of this invasive pathogen. In this study, we report a typhoid fever outbreak caused by multidrug-resistant Salmonella enterica serovar Typhi strains with an ACSSxtT pattern. Resistance genes conferring these phenotypes were harbored by a large conjugative plasmid, which increases the threat of Salmonella Typhi and thus requires close surveillance for dissemination of strains containing such genes.

A Large-Scale Community-Based Outbreak of Paratyphoid Fever Caused by Hospital-Derived Transmission in Southern China.

Background Since the 1990s, paratyphoid fever caused by Salmonella Paratyphi A has emerged in Southeast Asia and China. In 2010, a large-scale outbreak involving 601 cases of paratyphoid fever occurred in the whole of Yuanjiang county in China. Epidemiological and laboratory investigations were conducted to determine the etiology, source and transmission factors of the outbreak. Methodology/Principal Findings A case-control study was performed to identify the risk factors for this paratyphoid outbreak. Cases were identified as patients with blood culture–confirmed S. Paratyphi A infection. Controls were healthy persons without fever within the past month and matched to cases by age, gender and geography. Pulsed-field gel electrophoresis and whole-genome sequencing of the S. Paratyphi A strains isolated from patients and environmental sources were performed to facilitate transmission analysis and source tracking. We found that farmers and young adults were the populations mainly affected in this outbreak, and the consumption of raw vegetables was the main risk factor associated with paratyphoid fever. Molecular subtyping and genome sequencing of S. Paratyphi A isolates recovered from improperly disinfected hospital wastewater showed indistinguishable patterns matching most of the isolates from the cases. An investigation showed that hospital wastewater mixed with surface water was used for crop irrigation, promoting a cycle of contamination. After prohibition of the planting of vegetables in contaminated fields and the thorough disinfection of hospital wastewater, the outbreak subsided. Further analysis of the isolates indicated that the origin of the outbreak was most likely from patients outside Yuanjiang county. Conclusions This outbreak is an example of the combined effect of social behaviors, prevailing ecological conditions and improper disinfection of hospital wastewater on facilitating a sustained epidemic of paratyphoid fever. This study underscores the critical need for strict treatment measures of hospital wastewater and the maintenance of independent agricultural irrigation systems in rural areas.

Optimization of Pulsed-Field Gel Electrophoresis Protocols for Salmonella Paratyphi A Subtyping

Salmonella enterica serovar Paratyphi A infection has caused public health problems in some countries in recent years. Pulsed-field gel electrophoresis (PFGE) has been used for the subtyping and epidemiological investigations of some serotypes of Salmonella, mainly in outbreaks caused by non-typhoidal Salmonella. In this study, different restriction endonucleases and electrophoresis parameters were compared for the PFGE subtyping by using Salmonella Paratyphi A strain panels. Two protocols for the enzymes SpeI and XbaI showed higher discriminatory power, which may facilitate epidemiological analysis for more accurate case definition, and clonality study of Salmonella Paratyphi A.