Oliver Lung

Polymorphisms of COX-2 -765G > C and p53 codon 72 and risks of oral squamous cell carcinoma in a Taiwan population

The association between polymorphisms of COX-2 -765G>C and p53 codon 72, and oral squamous cell carcinoma (OSCC) remains unclear. We investigated the associations between COX-2 and p53 polymorphisms, oral precancerous lesions (OPL), and OSCC. Demographic data and substance use (smoking, drinking, and betel quid chewing) data were collected from 297 patients with OSCC, 70 with oral leukoplakia (OL), 39 with oral submucosal fibrosis (OSF), and 280 healthy controls. COX-2 and p53 polymorphisms were determined by PCR-RFLP methods. A significantly higher proportion of OSCC and OPL patients were male, and frequent habitual users of the three substances. No association was found between p53 and COX-2 polymorphisms, ethnicity, and gender. Polymorphisms of p53 were not associated with OSCC development and malignant potential of OPL, OSF, and OL. The frequency of COX-2 -765G/G genotype was significantly higher in healthy controls (chi(2)=93.83, p<0.0001). After adjusting for possible confounding factors, COX-2 -765C allele vs. -765G/G genotype (OR=0.22, 95%CI=0.12-0.39) was a protective factor against OSCC development, but was a risk factor for malignant potential of OSF (OR=3.20, 95%CI=1.32-8.94) and OL (OR=6.73, 95%CI=2.84-19.87). We suggest that COX-2 -765G>C polymorphisms play a different role in OSCC development than in malignant potential of OSF and OL. However, p53 codon 72 polymorphisms show no such correlation.

Polymorphisms in the apoptosis-associated genes FAS and FASL and risk of oral cancer and malignant potential of oral premalignant lesions in a Taiwanese population.

BACKGROUND Our aim was to measure the relationship of FAS (-1377G>A and -670A>G), FASL (-844C>T) gene variants and risk of oral cancer. METHODS Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to determine the FAS and FASL polymorphisms in 294 oral squamous cell carcinoma (OSCC), 53 oral submucous fibrosis (OSF), and 84 oral leukoplakia (OL) patients, as well as in 333 healthy controls. A standardized questionnaire was applied to collect demographic data, and potential confounding factors. JMP statistical software was used to analyze the association. RESULTS FAS and FASL polymorphisms were not correlated with OSCC development or the malignant potential of OL by simple and multivariate logistic regression. However, a two- to fourfold difference in the risks of betel quid chewing, alcohol consumption, and smoking on OSCC development were observed between participants with different FAS polymorphisms. FAS polymorphisms were significantly correlated with the malignant potential of OSF. Multivariate logistic regression analysis indicated that FAS A(-1377)-G(-670) vs. G(-1377)-A(-670) haplotype (OR = 2.26, 95% CI = 1.16-4.41) was correlated with the malignant potential of OSF. CONCLUSIONS We suggest that FAS and FASL polymorphisms are not significantly correlated with OSCC development or malignant potential of OL. The impact of substance usage on OSCC development could be differentiated by FAS polymorphisms. FAS A(-1377)-G(-670) haplotype may play a role in the malignant potential of OSF.

Multiplex RT-PCR detection and microarray typing of vesicular disease viruses.

A vesicular disease multiplex reverse transcription (RT)-PCR with an accompanying microarray assay was developed for simultaneous detection and typing of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV), and for the detection of swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV). The multiplex RT-PCR successfully detected viral RNA from a collection of 49 strains of vesicular viruses, including multiple strains from all seven serotypes of FMDV and both serotypes of VSV. The multiplex RT-PCR was also able to produce amplified products from the RNA genome of all four viruses simultaneously in mixed samples. An indirect (post-PCR labelling) amplicon labelling method and a direct (concurrent labelling with PCR) amplicon labelling method were compared for the purpose of microarray detection and typing. Accurate detection and typing was achieved with all strains tested in the microarray assay which utilized 163 virus- and serotype-specific probes. It was observed that microarray increased detection for some samples compared to using multiplex RT-PCR alone. This was most likely due to signal amplification resulting from fluorescent labelling. The limit of detection of the microarray assay was as low as 4.6TCID(50)/mL for FMDV. No amplification products or microarray reactivity was observed with non-target livestock pathogens tested or with samples collected from healthy cattle, sheep and pigs. All FMDV and VSV serotypes were detected as early as 2 days post-inoculation from oral swabs obtained from cattle infected experimentally.

Multiplex PCR and Microarray for Detection of Swine Respiratory Pathogens

Summary Porcine respiratory disease complex (PRDC) is one of the most important health concerns for pig producers and can involve multiple viral and bacterial pathogens. No simple, single‐reaction diagnostic test currently exists for the simultaneous detection of major pathogens commonly associated with PRDC. Furthermore, the detection of most of the bacterial pathogens implicated in PRDC currently requires time‐consuming culture‐based methods that can take several days to obtain results. In this study, a novel prototype automated microarray that integrates and automates all steps of post‐PCR microarray processing for the simultaneous detection and typing of eight bacteria and viruses commonly associated with PRDC is described along with associated multiplex reverse transcriptase PCR. The user‐friendly assay detected and differentiated between four viruses [porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus, porcine circovirus type 2, porcine respiratory corona virus], four bacteria (Mycoplasma hyopneumoniae, Pasteurella multocida, Salmonella enterica serovar Choleraesuis, Streptococcus suis), and further differentiated between type 1 and type 2 PRRSV as well as toxigenic and non‐toxigenic P. multocida. The assay accurately identified and typed a panel of 34 strains representing the eight targeted pathogens and was negative when tested with 34 relevant and/or closely related non‐target bacterial and viral species. All targets were also identified singly or in combination in a panel of clinical lung samples and/or experimentally inoculated biological material.

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

Summary Foot‐and‐mouth disease (FMD) is a highly contagious viral disease of cloven‐hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot‐and‐mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field‐deployable assay would support local decision‐making during an FMDV outbreak. Here we report validation of a novel reverse transcription‐insulated isothermal PCR (RT‐iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT ™ analyser that automatically analyses data and displays ‘+’ or ‘−’ results. The FMDV RT‐iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro‐transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross‐reactivity with viruses causing similar clinical diseases in cloven‐hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory‐based real‐time RT‐PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco™ mini transportable magnetic bead‐based, automated extraction system was used. This assay provides a potentially useful field‐deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD‐free countries or for routine diagnostics in endemic countries with less structured laboratory systems.

Electronic microarray assays for avian influenza and Newcastle disease virus

Microarrays are suitable for multiplexed detection and typing of pathogens. Avian influenza virus (AIV) is currently classified into 16 H (hemagglutinin) and 9 N (neuraminidase) subtypes, whereas Newcastle disease virus (NDV) strains differ in virulence and are broadly classified into high and low pathogenicity types. In this study, three assays for detection and typing of poultry viruses were developed on an automated microarray platform: a multiplex assay for simultaneous detection of AIV and detection and pathotyping of NDV, and two separate assays for differentiating all AIV H and N subtypes. The AIV-NDV multiplex assay detected all strains in a 63 virus panel, and accurately typed all high pathogenicity NDV strains tested. A limit of detection of 10(1)-10(3) TCID(50)/mL and 200-400 EID(50)/mL was obtained for NDV and AIV, respectively. The AIV typing assays accurately typed all 41 AIV strains and a limit of detection of 4-200 EID(50)/mL was obtained. Assay validation showed that the microarray assays were generally comparable to real-time RT-PCR. However, the AIV typing microarray assays detected more positive clinical samples than the AIV matrix real-time RT-PCR, and also provided information regarding the subtype. The AIV-NDV multiplex and AIV H typing microarray assays detected mixed infections and could be useful for detection and typing of AIV and NDV.

Genome wide analysis of the evolution of Senecavirus A from swine clinical material and assembly yard environmental samples

Senecavirus A (SVA), previously known as Seneca Valley virus, was first isolated in the United States in 2002. SVA was associated with porcine idiopathic vesicular disease in Canada and the USA in 2007 and 2012, respectively. Recent increase in SVA outbreaks resulting in neonatal mortality of piglets and/or vesicular lesions in sows in Brazil, the USA and Canada point to the necessity to study the pathogenicity and molecular epidemiology of the virus. Here, we report the analysis of the complete coding sequences of SVA from 2 clinical cases and 9 assembly yard environmental samples collected in 2015 in Canada, along with 22 previously released complete genomes in the GenBank. With this combined data set, the evolution of the SVA over a 12-month period in 2015/2016 was evaluated. These SVA isolates were characterized by a rapid accumulation of genetic variations driven mainly by a high nucleotide substitution rate and purifying selection. The SVA sequences clustered in clearly defined geographical areas with reported cases of SVA infection. No transmission links were identified between assembly yards, suggesting that point source introductions may have occurred. In addition, 25 fixed non-synonymous mutations were identified across all analyzed strains when compared to the prototype SVA strain (SVV-001). This study highlights the importance of monitoring SVA mutations for their role in increased virulence and impact on SVA diagnostics.

Microarray for Identification of the Chiropteran Host Species of Rabies Virus in Canada

Species identification through genetic barcoding can augment traditional taxonomic methods, which rely on morphological features of the specimen. Such approaches are especially valuable when specimens are in poor condition or comprise very limited material, a situation that often applies to chiropteran (bat) specimens submitted to the Canadian Food Inspection Agency for rabies diagnosis. Coupled with phenotypic plasticity of many species and inconclusive taxonomic keys, species identification using only morphological traits can be challenging. In this study, a microarray assay with associated PCR of the mitochondrial cytochrome c oxidase subunit I (COI) gene was developed for differentiation of 14 bat species submitted to the Canadian Food Inspection Agency from 1985–2012 for rabies diagnosis. The assay was validated with a reference collection of DNA from 153 field samples, all of which had been barcoded previously. The COI gene from 152 samples which included multiple specimens of each target species were successfully amplified by PCR and accurately identified by the microarray. One sample that was severely decomposed failed to amplify with PCR primers developed in this study, but amplified weakly after switching to alternate primers and was accurately typed by the microarray. Thus, the chiropteran microarray was able to accurately differentiate between the 14 species of Canadian bats targeted. This PCR and microarray assay would allow unequivocal identification to species of most, if not all, bat specimens submitted for rabies diagnosis in Canada.

Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

Summary Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)–PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non‐specific reactions were observed with a panel of 23 non‐target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post‐inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID 50/ml for RPV. The novel microarray platform automates the entire post‐PCR steps of the assay and integrates electrophoretic‐driven capture probe printing in a single user‐friendly instrument that allows array layout and assay configuration to be user‐customized on‐site.

Reoccurrence of Suspected Human‐to‐Turkey Transmission of H1N1 Pandemic 2009 Virus in Turkey Breeder Flocks in Ontario and Manitoba, 2016

Soon after the emergence of 2009 pandemic H1N1, the first outbreaks in breeder turkey operations were reported that implicated human-to-turkey transmission. In the spring of 2016, the reoccurrence of 2009 pandemic H1N1 lineage viruses infecting breeder turkey flocks in Ontario and Manitoba, Canada, also implicated human-to-turkey transmission. In addition to raising concerns over biosecurity and vaccine failures, these cases once again raise the issue of whether turkeys have the potential to act as a bridge species to generate novel influenza A virus reassortants with public health implications.