High-throughput sequencing for species authentication and contamination detection of 63 cell lines.

Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency's National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvß6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

Molecular Characterization of Southern African Territories 2 (SAT2) Serotype of Foot-and-Mouth Disease Virus from Nigeria in 2017 to 2018.

This report describes the nucleotide sequences of eight Southern African Territories 2 (SAT2) serotype foot-and-mouth disease virus strains from 2017 to 2018 outbreaks in cattle in Nigeria. These viruses belong to topotype VII of SAT2 and were closely related to previous isolates from Nigeria and other West African countries.

Differential pathogenesis between Andes virus strains CHI-7913 and Chile-9717869in Syrian Hamsters.

Hantavirus cardiopulmonary syndrome (HCPS) is a severe respiratory disease caused by orthohantaviruses in the Americas with a fatality rate as high as 35%. In South America, Andes orthohantavirus (Hantaviridae, Orthohantavirus, ANDV) is a major cause of HCPS, particularly in Chile and Argentina, where thousands of cases have been reported since the virus was discovered. Two strains of ANDV that are classically used for experimental studies of the virus are Chile-9717869, isolated from the natural reservoir, the long-tailed pygmy rice rat, and CHI-7913, an isolate from a lethal human case of HCPS. An important animal model for studying pathogenesis of HCPS is the lethal Syrian golden hamster model of ANDV infection. In this model, ANDV strain Chile-9717869 is uniformly lethal and has been used extensively for pathogenesis, vaccination, and therapeutic studies. Here we show that the CHI-7913 strain, despite having high sequence similarity with Chile-9717869, does not cause lethal disease in Syrian hamsters. CHI-7913, while being able to infect hamsters and replicate to moderate levels, showed a reduced ability to replicate within the tissues compared with Chile-9717869. Hamsters infected with CHI-7913 had reduced expression of cytokines IL-4, IL-6, and IFN-γ compared with Chile-9717869 infected animals, suggesting potentially limited immune-mediated pathology. These results demonstrate that certain ANDV strains may not be lethal in the classical Syrian hamster model of infection, and further exploration into the differences between lethal and non-lethal strains provide important insights into molecular determinants of pathogenic hantavirus infection.Importance:Andes orthohantavirus (ANDV) is a New World hantavirus that is a major cause of hantavirus cardiopulmonary syndrome (HCPS, also referred to as hantavirus pulmonary syndrome) in South America, particularly in Chile and Argentina. ANDV is one of the few hantaviruses for which there is a reliable animal model, the Syrian hamster model, which recapitulates important aspects of human disease. Here we infected hamsters with a human isolate of ANDV, CHI-7913, to assess its pathogenicity compared with the classical lethal Chile-9717869 strain. CHI-7913 had 22 amino acid differences compared with Chile-9717869, did not cause lethal disease in hamsters, and showed reduced ability to replicate in vivo Our data indicate potentially important molecular signatures for pathogenesis of ANDV infection in hamsters and may lead to insights into what drives pathogenesis of certain hantaviruses in humans.

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens.

Respiratory and enteric diseases continue to be two major causes of economic losses to the cattle industry worldwide. Despite their multifactorial etiology, the currently available diagnostic tests for bovine respiratory disease complex (BRDC) and bovine enteric disease (BED) are single-pathogen-tests. DNA microarray when combined with multiplex polymerase chain reaction (PCR) is a powerful tool in detection and differentiation of multiple pathogens in a single sample. This study reports development and initial validation of two independent highly sensitive and specific multiplex PCR-electronic microarray assays, one for the detection and differentiation of pathogens of the BRDC and the other for detection and differentiation of pathogens of the BED. The BRDC multiplex PCR-microarray assay was able to detect and differentiate four bacteria (Mannheimia haemolytica, Histophilus somni, Pasteurella multocida, and Mycoplasma bovis) and five viruses [bovine parainfluenza virus-3, bovine respiratory syncytial virus, bovine herpesvirus-1, bovine coronavirus (BCoV), and bovine viral diarrhea virus (BVDV)] associated with BRDC. The BED multiplex PCR- microarray- assay was able to detect and differentiate four bacteria (Clostridium perfringens, Escherichia coli, Salmonella enterica Dublin, and Salmonella enterica Typhimurium), three protozoa (Eimeria zuernii, Eimeria bovis, and Cryptosporidium parvum), and four viruses (bovine torovirus, bovine rotavirus, BCoV, and BVDV) associated with the BED. Both assays detected their respective targets individually or in combination when present. The limit-of-detection of each assay at the PCR amplification and DNA microarray levels was determined using previously titrated laboratory amplified target pathogens or using quantified synthetic nucleotides. Both assays showed very high analytical sensitivity and specificity, and were validated using a limited number of clinical samples. The BRDC and BED multiplex PCR- microarray-assays developed in this study, with further clinical validation, could be used in veterinary diagnostic laboratories for the rapid and simultaneous identification of pathogens to facilitate quick and accurate decision making for the control and treatment of these two economically important disease complexes. Furthermore, these assays could be very effective tools in epidemiological studies as well as for screening of healthy animals to identify carriers that may potentially develop BRDC or BED.

A Genome-Wide Association Study to Identify Diagnostic Markers for Human Pathogenic Campylobacter jejuni Strains.

Campylobacter jejuni is a leading human enteric pathogen worldwide and despite an improved understanding of its biology, ecology, and epidemiology, limited tools exist for identifying strains that are likely to cause disease. In the current study, we used subtyping data in a database representing over 24,000 isolates collected through various surveillance projects in Canada to identify 166 representative genomes from prevalent C. jejuni subtypes for whole genome sequencing. The sequence data was used in a genome-wide association study (GWAS) aimed at identifying accessory gene markers associated with clinically related C. jejuni subtypes. Prospective markers (n = 28) were then validated against a large number (n = 3,902) of clinically associated and non-clinically associated genomes from a variety of sources. A total of 25 genes, including six sets of genetically linked genes, were identified as robust putative diagnostic markers for clinically related C. jejuni subtypes. Although some of the genes identified in this study have been previously shown to play a role in important processes such as iron acquisition and vitamin B5 biosynthesis, others have unknown function or are unique to the current study and warrant further investigation. As few as four of these markers could be used in combination to detect up to 90% of clinically associated isolates in the validation dataset, and such markers could form the basis for a screening assay to rapidly identify strains that pose an increased risk to public health. The results of the current study are consistent with the notion that specific groups of C. jejuni strains of interest are defined by the presence of specific accessory genes.

Development and validation of a comparative genomic fingerprinting method for high-resolution genotyping of Campylobacter jejuni.

Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide. The need for molecular subtyping methods with enhanced discrimination in the context of surveillance- and outbreak-based epidemiologic investigations of Campylobacter spp. is critical to our understanding of sources and routes of transmission and the development of mitigation strategies to reduce the incidence of campylobacteriosis. We describe the development and validation of a rapid and high-resolution comparative genomic fingerprinting (CGF) method for C. jejuni. A total of 412 isolates from agricultural, environmental, retail, and human clinical sources obtained from the Canadian national integrated enteric pathogen surveillance program (C-EnterNet) were analyzed using a 40-gene assay (CGF40) and multilocus sequence typing (MLST). The significantly higher Simpson's index of diversity (ID) obtained with CGF40 (ID = 0.994) suggests that it has a higher discriminatory power than MLST at both the level of clonal complex (ID = 0.873) and sequence type (ID = 0.935). High Wallace coefficients obtained when CGF40 was used as the primary typing method suggest that CGF and MLST are highly concordant, and we show that isolates with identical MLST profiles are comprised of isolates with distinct but highly similar CGF profiles. The high concordance with MLST coupled with the ability to discriminate between closely related isolates suggests that CFG40 is useful in differentiating highly prevalent sequence types, such as ST21 and ST45. CGF40 is a high-resolution comparative genomics-based method for C. jejuni subtyping with high discriminatory power that is also rapid, low cost, and easily deployable for routine epidemiologic surveillance and outbreak investigations.

Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions.

BACKGROUND: The pan-genome of a bacterial species consists of a core and an accessory gene pool. The accessory genome is thought to be an important source of genetic variability in bacterial populations and is gained through lateral gene transfer, allowing subpopulations of bacteria to better adapt to specific niches. Low-cost and high-throughput sequencing platforms have created an exponential increase in genome sequence data and an opportunity to study the pan-genomes of many bacterial species. In this study, we describe a new online pan-genome sequence analysis program, Panseq. RESULTS: Panseq was used to identify Escherichia coli O157:H7 and E. coli K-12 genomic islands. Within a population of 60 E. coli O157:H7 strains, the existence of 65 accessory genomic regions identified by Panseq analysis was confirmed by PCR. The accessory genome and binary presence/absence data, and core genome and single nucleotide polymorphisms (SNPs) of six L. monocytogenes strains were extracted with Panseq and hierarchically clustered and visualized. The nucleotide core and binary accessory data were also used to construct maximum parsimony (MP) trees, which were compared to the MP tree generated by multi-locus sequence typing (MLST). The topology of the accessory and core trees was identical but differed from the tree produced using seven MLST loci. The Loci Selector module found the most variable and discriminatory combinations of four loci within a 100 loci set among 10 strains in 1 s, compared to the 449 s required to exhaustively search for all possible combinations; it also found the most discriminatory 20 loci from a 96 loci E. coli O157:H7 SNP dataset. CONCLUSION: Panseq determines the core and accessory regions among a collection of genomic sequences based on user-defined parameters. It readily extracts regions unique to a genome or group of genomes, identifies SNPs within shared core genomic regions, constructs files for use in phylogeny programs based on both the presence/absence of accessory regions and SNPs within core regions and produces a graphical overview of the output. Panseq also includes a loci selector that calculates the most variable and discriminatory loci among sets of accessory loci or core gene SNPs. AVAILABILITY: Panseq is freely available online at http://76.70.11.198/panseq. Panseq is written in Perl.

In silico genomic analyses reveal three distinct lineages of Escherichia coli O157:H7, one of which is associated with hyper-virulence.

BACKGROUND: Many approaches have been used to study the evolution, population structure and genetic diversity of Escherichia coli O157:H7; however, observations made with different genotyping systems are not easily relatable to each other. Three genetic lineages of E. coli O157:H7 designated I, II and I/II have been identified using octamer-based genome scanning and microarray comparative genomic hybridization (mCGH). Each lineage contains significant phenotypic differences, with lineage I strains being the most commonly associated with human infections. Similarly, a clade of hyper-virulent O157:H7 strains implicated in the 2006 spinach and lettuce outbreaks has been defined using single-nucleotide polymorphism (SNP) typing. In this study an in silico comparison of six different genotyping approaches was performed on 19 E. coli genome sequences from 17 O157:H7 strains and single O145:NM and K12 MG1655 strains to provide an overall picture of diversity of the E. coli O157:H7 population, and to compare genotyping methods for O157:H7 strains. RESULTS: In silico determination of lineage, Shiga-toxin bacteriophage integration site, comparative genomic fingerprint, mCGH profile, novel region distribution profile, SNP type and multi-locus variable number tandem repeat analysis type was performed and a supernetwork based on the combination of these methods was produced. This supernetwork showed three distinct clusters of strains that were O157:H7 lineage-specific, with the SNP-based hyper-virulent clade 8 synonymous with O157:H7 lineage I/II. Lineage I/II/clade 8 strains clustered closest on the supernetwork to E. coli K12 and E. coli O55:H7, O145:NM and sorbitol-fermenting O157 strains. CONCLUSION: The results of this study highlight the similarities in relationships derived from multi-locus genome sampling methods and suggest a "common genotyping language" may be devised for population genetics and epidemiological studies. Future genotyping methods should provide data that can be stored centrally and accessed locally in an easily transferable, informative and extensible format based on comparative genomic analyses.