Multiplex Reverse transcription-PCR Research Articles

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Molecular and biological characterization of pepino mosaic virus isolates occurring in the main tomato-producing areas in Mexico

Pepino mosaic virus (PepMV) has caused major epidemics in tomatoes (Solanum lycopersicum L.) worldwide. Five genotypes have been identified: Peruvian (LP), European (EU), American (US1), Chilean (CH2), and Southern Peruvian (PES). The aim of this study was to determine the current distribution of PepMV in the main tomato-producing areas of Mexico, as well as to identify the predominant genotypes and their biological behavior in alternative hosts and indicator species. A total of 77 samples of tomato plants showing PepMV symptoms-like with leaf mottling, mosaic, and chlorosis symptoms, as well as marbled fruit, were analyzed by multiplex reverse transcription PCR (RT-PCR). Sequencing data confirmed the identification of PepMV genotypes in 15 samples from six states. Six samples with single CH2 genotype infection, two with LP genotype, two with EU genotype, and five LP + CH2 co-infections were detected. Moreover, a high prevalence of ToBRFV was found, either in single infection or in co-infection with PepMV. Five PepMV sources (three CH2, one EU, and one with LP + CH2 co-infection) were used in the biological indexing. All isolates induced systemic infection in Solanum lycopersicum, Datura stramonium, Nicotiana occidentalis, and Solanum melongena. In tomato, CH2 isolates induced mild mosaics and chlorosis, whereas the EU isolate caused chlorotic mosaics typical of PepMV. In D. stramonium and S. melongena, chlorosis, interveinal yellowing, and mosaics were observed. Nicotiana occidentalis showed symptoms with all isolates, particularly with the CH2-MXM01 isolate, which caused necrotic lesions. Overall, PepMV is present in at least six states in Mexico, with the CH2 genotype and mixed LP + CH2 infection prevailing. US1 and PES genotypes were not detected. Interestingly, an EU isolate was characterized as an aggressive variant, causing mosaics and severe chlorosis.

A sensitive mass spectrometry-based method to identify common respiratory pathogens in children.

Public health threats posed by emerging respiratory infections are a significant concern, particularly in children and infants. Traditional culture-based detection methods are time-consuming and typically require 1-3 days. Herein, we developed and evaluated a 23-plex common respiratory pathogen mass spectrometry assay that enables the simultaneous detection of 18 common respiratory pathogens in children. This assay combines matrix-assisted laser desorption/ionization time of flight mass spectrometry with multiplex reverse transcription-PCR and targets 11 bacterial and 7 viral pathogens (including 10 subtypes), and two internal controls. The detection limit of the common respiratory pathogen mass spectrometry assay was as low as 1 copy/µL, with no cross-reactivity with other organisms. We assessed the clinical performance of the common respiratory pathogen mass spectrometry assay using respiratory samples from 450 children. The total 450 clinical specimens underwent analysis via matrix-assisted laser desorption/ionization time of flight mass spectrometry, and the outcomes were juxtaposed with those derived from real-time reverse-transcriptase PCR conducted concurrently. The concordance between these methods was 96.0%, and the multiple infection identification rate was 7.1%. This innovative approach enables the simultaneous analysis of numerous outcomes from a solitary examination across 192 specimens within a timeframe of approximately 7 hours, with a dramatically reduced sample use and cost. In summary, the common respiratory pathogen mass spectrometry assay is a sensitive, accurate, and cost-effective method for detecting common respiratory pathogens in children and has the potential to revolutionize the diagnosis of respiratory tract infections. IMPORTANCE This study aimed to present and evaluate a novel co-detection method that enables the simultaneous identification of 11 bacterial and 7 viral pathogens in about 7 hours using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Our approach utilizes a combination of multiplex reverse transcription-PCR and matrix-assisted laser desorption/ionization time of flight mass spectrometry, which overcomes the limitations of conventional assays, which include a long assessment time, technical difficulty, and high costs. As a screening method for common respiratory pathogens in children, common respiratory pathogen mass spectrometry assay has the potential to revolutionize the diagnosis of respiratory tract infections by providing an accurate etiological diagnosis. The common respiratory pathogen mass spectrometry assay is expected to be a critical tool for the diagnosis of respiratory infections in children, offering a more efficient, cost-effective, and accurate approach for the detection of common respiratory pathogens.

Retinal inflammation in murine models of type 1 and type 2 diabetes with diabetic retinopathy

Aims/hypothesisThe loss of pericytes surrounding the retinal vasculature in early diabetic retinopathy underlies changes to the neurovascular unit that lead to more destructive forms of the disease. However, it is unclear which changes lead to loss of retinal pericytes. This study investigated the hypothesis that chronic increases in one or more inflammatory factors mitigate the signalling pathways needed for pericyte survival.MethodsLoss of pericytes and levels of inflammatory markers at the mRNA and protein levels were investigated in two genetic models of diabetes, Ins2Akita/+ (a model of type 1 diabetes) and Leprdb/db (a model of type 2 diabetes), at early stages of diabetic retinopathy. In addition, changes that accompany gliosis and the retinal vasculature were determined. Finally, changes in retinal pericytes chronically incubated with vehicle or increasing amounts of IFNγ were investigated to determine the effects on pericyte survival. The numbers of pericytes, microglia, astrocytes and endothelial cells in retinal flatmounts were determined by immunofluorescence. Protein and mRNA levels of inflammatory factors were determined using multiplex ELISAs and quantitative reverse transcription PCR (qRT-PCR). The effects of IFNγ on the murine retinal pericyte survival-related platelet-derived growth factor receptor β (PDGFRβ) signalling pathway were investigated by western blot analysis. Finally, the levels of cell death-associated protein kinase C isoform delta (PKCδ) and cleaved caspase 3 (CC3) in pericytes were determined by western blot analysis and immunocytochemistry.ResultsThe essential findings of this study were that both type 1 and 2 diabetes were accompanied by a similar progression of retinal pericyte loss, as well as gliosis. However, inflammatory factor expression was dissimilar in the two models of diabetes, with peak expression occurring at different ages for each model. Retinal vascular changes were more severe in the type 2 diabetes model. Chronic incubation of murine retinal pericytes with IFNγ decreased PDGFRβ signalling and increased the levels of active PKCδ and CC3.Conclusions/interpretationWe conclude that retinal inflammation is involved in and sustains pericyte loss as diabetic retinopathy progresses. Moreover, IFNγ plays a critical role in reducing pericyte survival in the retina by reducing activation of the PDGFRβ signalling pathway and increasing PKCδ levels and pericyte apoptosis.Graphical

Streptococcus pneumoniae nasal carriage patterns with and without common respiratory virus detections in households in Seattle, WA, USA before and during the COVID-19 pandemic.

Respiratory viruses might influence Streptococcus pneumoniae nasal carriage and subsequent disease risk. We estimated the association between common respiratory viruses and semiquantitative S. pneumoniae nasal carriage density in a household setting before and during the COVID-19 pandemic. From November 2019-June 2021, we enrolled participants in a remote household surveillance study of respiratory pathogens. Participants submitted weekly reports of acute respiratory illness (ARI) symptoms. Mid-turbinate or anterior nasal swabs were self-collected at enrollment, when ARI occurred, and, in the second year of the study only, from household contacts after SARS-CoV-2 was detected in a household member. Specimens were tested using multiplex reverse-transcription PCR for respiratory pathogens, including S. pneumoniae, rhinovirus, adenovirus, common human coronavirus, influenza A/B virus, respiratory syncytial virus (RSV) A/B, human metapneumovirus, enterovirus, and human parainfluenza virus. We estimated differences in semiquantitative S. pneumoniae nasal carriage density, estimated by the inverse of S. pneumoniae relative cycle threshold (Crt) values, with and without viral detection for any virus and for specific respiratory viruses using linear generalized estimating equations of S. pneumoniae Crt values on virus detection adjusted for age and swab type and accounting for clustering of swabs within households. We collected 346 swabs from 239 individuals in 151 households that tested positive for S. pneumoniae (n = 157 with and 189 without ≥1 viruses co-detected). Difficulty breathing, cough, and runny nose were more commonly reported among individuals with specimens with viral co-detection compared to without (15%, 80% and 93% vs. 8%, 57%, and 51%, respectively) and ear pain and headache were less commonly reported (3% and 26% vs. 16% and 41%, respectively). For specific viruses among all ages, semiquantitative S. pneumoniae nasal carriage density was greater with viral co-detection for enterovirus, RSV A/B, adenovirus, rhinovirus, and common human coronavirus (P < 0.01 for each). When stratified by age, semiquantitative S. pneumoniae nasal carriage density was significantly greater with viral co-detection among children aged <5 (P = 0.002) and 5-17 years (P = 0.005), but not among adults aged 18-64 years (P = 0.29). Detection of common respiratory viruses was associated with greater concurrent S. pneumoniae semiquantitative nasal carriage density in a household setting among children, but not adults.

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A genomic DNA-based NGS method for the simultaneous detection of multiple fusion genes in pediatric leukemia.

Fusion genes are products of chromosomal translocations that generate either a dysregulated partner gene or a chimeric fusion protein with new properties, and contribute significantly to leukemia development and clinical risk stratification. However, simultaneous detection of several hundreds of fusion genes has always been a challenge in a clinical laboratory setting. In the present study, a total of 182 pediatric patients with leukemia were screened for fusion genes by employing a novel genomic DNA-, instead of RNA-, based next-generation sequencing (NGS) method. This involved the comparison of the multiply targeted capture sequencing method with a detection panel of 270 fusion genes (MTCS-270) with an RNA-based multiplex reverse transcription-PCR technique with a detection panel of 57 fusion genes (MRTP-57). MRTP-57 has been well established in the clinical lab at Beijing Hightrust Diagnostics, Co. (Beijing, China) for an up-front leukemia diagnosis and served as the control technique in the present study. In the series, MTCS-270 and MRTP-57 yielded a positive fusion gene detection rate of 50.0% (91/182) and 41.8% (76/182), respectively, indicating an advantage of MTCS-270 over MRTP-57 in overall detection sensitivity. Specifically, all the fusion genes detected by MRTP-57 were also identified by MTCS-270, clearly signifying the respectable detection accuracy of MTCS-270. Notably, across the patients screened, MTCS-270 identified more samples with fusion genes than MRTP-57, illustrating a broader fusion gene detection coverage by MTCS-270. The present study provides solid evidence that this DNA-based NGS approach can be used as a potential detection tool together with other well-established molecular cytogenetic methods for leukemia management, and to the best of our knowledge, represents the largest leukemia fusion gene identification analysis by genomic NGS.

Enterovirus D68 in Hospitalized Children, Barcelona, Spain, 2014-2021.

To determine molecular epidemiology and clinical features of enterovirus D68 (EV-D68) infections, we reviewed EV-D68–associated respiratory cases at a hospital in Barcelona, Spain, during 2014–2021. Respiratory samples were collected from hospitalized patients or outpatients with symptoms of acute respiratory tract infection or suggestive of enterovirus infection. Enterovirus detection was performed by real-time multiplex reverse transcription PCR and characterization by phylogenetic analysis of the partial viral protein 1 coding region sequences. From 184 patients with EV-D68 infection, circulating subclades were B3 (80%), D1 (17%), B2 (1%), and A (<1%); clade proportions shifted over time. EV-D68 was detected mostly in children (86%) and biennially (2016, 2018, 2021). In patients <16 years of age, the most common sign/symptom was lower respiratory tract infection, for which 11.8% required pediatric intensive care unit admission and 2.3% required invasive mechanical ventilation; neurologic complications developed in 1. The potential neurotropism indicates that enterovirus surveillance should be mandatory.

Detection of Newcastle disease virus and H5-subtype Influenza virus in swiftlet houses by Multiplex reverse transcription PCR assay

The purpose of this study was to investigate the presence of Avian paramyxovirus 1 (APMV-1), also known as Newcastle disease virus (NDV), and Avian influenza type A, especially H5 subtype (AIV-H5) in swiftlet houses and swiftlet nest by multiplex RT-PCR (mRT-PCR). The assay used two specific primer pairs designed to detect the conserved sequence of the F gene of NDV and the HA gene of the AIV-H5, with product sizes of 282 bp and 420 bp, respectively. The mRT-PCR was established with detection limit of 25 copies/reaction for each target virus. The thermal cycle was optimized as follows: cDNA synthesis at 45ºC for 20 min, an initial denaturation at 95ºC for 5 min, followed by 35 cycles of amplification encompassing denaturation at 95ºC for 30 sec, annealing at 58ºC for 30 sec, and extension at 72ºC for 45 sec, ending by a final extension step at 72ºC for 7 min. Eighty-eight field samples including feces and swabs of the nest surface were examined and all samples were confirmed to be negative for these two viruses. The results of this study indicated that the swiftlet nests and the environment of swiftlet houses were not contaminated with NDV or AIV-H5 viruses. Moreover, the established mRT-PCR protocol had good specificity, detection limit, and can apply for routine veterinary diagnosis.

PB1891: VARIANT PHILADELPHIA CHROMOSOME IN CML

Background: Chronic myeloid leukemia is a clonal malignant disorder of a pluripotent hematopoietic stem cell characterized by the Philadelphia chromosome and the reciprocal translocation t(9:22)(q34;q11). Almost 5 to 10 percent of chronic myeloid leukemia cases demonstrate variant Philadelphia translocations. Aims: To contribute to the description of patients’ clinical characteristics and prognosis with variant Ph chromosome-positive leukemia, we report a Ph chromosome-positive CML in the chronic phase characterized by an unusual variant Ph chromosome. Methods: An elderly male Libyan patient was suspected to have chronic myeloid leukemia. Cytogenetic and molecular exploration were conducted using bone marrow and blood samples. Cytogenetic analysis was performed on lymphocytes and bone marrow cells after short-term culture (48 hours). The cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Multiplex reverse transcription-PCR (RT-PCR) was used to identify the BCR-ABL1 transcript. RT-PCR was performed according to European recommendations, as described previously. Results: Clinical assessment confirmed the first chronic phase of a chronic myeloid leukemia. Chromosomal formula showed an homogeneous structural translocation: t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Molecular investigation confirmed the Ph nature of the chromosomal abnormality by showing the BCR-ABL1 transcript. The patient was treated by using Imatinib molecule with a favorable therapeutic response. Summary/Conclusion: A new variant Ph chromosome-positive CML in the chronic phase is reported. It is a four-way Ph translocation t(1;9;12;22)(p35-36;q34;p13;q11) involving four chromosomal regions, 1p35-36, 9q34, 12p13 and 22q11.2. Imatinib, the first-generation TKI treatment was efficient.

Clinicopathological and molecular characteristics of RSPO fusion-positive colorectal cancer.

RSPO fusions that lead to WNT pathway activation are potential therapeutic targets in colorectal cancer (CRC), but their clinicopathological significance remains unclear. We screened 1019 CRCs for RSPO fusions using multiplex reverse transcription-PCR. The RSPO fusion-positive tumours were subjected to whole-exome sequencing (WES). Our analysis identified 29 CRCs with RSPO fusions (2.8%), consisting of five with an EIF3E-RSPO2 fusion and 24 with PTPRK-RSPO3 fusions. The patients were 17 women and 12 men. Thirteen tumours (45%) were right-sided. Histologically, approximately half of the tumours (13/29, 45%) had a focal or extensive mucinous component that was significantly more frequent than the RSPO fusion-negative tumours (13%; P = 8.1 × 10-7). Four tumours (14%) were mismatch repair-deficient. WES identified KRAS, BRAF, and NRAS mutations in a total of 27 tumours (93%). In contrast, pathogenic mutations in major WNT pathway genes, such as APC, CTNNB1 and RNF43, were absent. RSPO fusion status did not have a statistically significant influence on the overall or recurrence-free survival. These clinicopathological and genetic features were also confirmed in a pooled analysis of previous studies. RSPO fusion-positive CRCs constitute a rare subgroup of CRCs with several characteristic clinicopathological and genetic features.

Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes

Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.

Evaluation and simultaneous determination of rectal mucosa markers by multiplex reverse transcription-PCR for biological evidence of sexual assault with anal penetration

Sexual assault with anal penetration is closely related to child sexual abuse or male victims. However, it is difficult to prove such an act by using biological samples collected from the surface of a suspected object because procedures for identifying rectal mucosa have not been developed sufficiently. Therefore, for the specific identification of rectal mucosa, mRNA markers reported to be characteristically expressed in the rectum were screened and a multiplex RT-PCR procedure was developed for the simultaneous determination of those candidate markers. The detectability and specificity of rectal mucosa candidate markers were evaluated using rectal mucosa samples and forensically relevant body fluids. Diluted or mixed samples were also tested to evaluate the applicability of this procedure for forensic casework. As a result, simultaneous amplification and determination of the selected candidates (PHGR1, MUC13, CLCA1, MEP1A, CDX1, and ZG16) and reference gene were successfully performed using a multiplex RT-PCR assay combined with capillary electrophoresis and fragment analysis. Applying the cutoff values, none of the other body fluids cross-reacted with rectal mucosa candidate markers. Because the low sensitivity and detectability of some candidate markers could be compensated for by their simultaneous detection, all six candidate markers were considered to be applicable as rectal mucosa markers. Besides, the developed assay should not be performed on suspicious fecal samples directly because these markers could be positive in the fecal samples themselves. The developed multiplex RT-PCR procedure might not be suitable for minute or diluted samples; however, it might be resistant to contamination with sexual assault-related body fluids. In conclusion, the simultaneous determination of selected rectal mucosa markers with a biological sample collected from the surface of a suspected object could be beneficial for criminal investigation of sexual assault with anal penetration.

Prevalence and Strain Composition of Potato virus Y Circulating in Potato Fields in China's North-Central Province of Shanxi.

Potato is an important crop in Shanxi province, located in north-central China. In 2019 to 2020, 319 potato leaf samples were collected from eight locations distributed in three major potato production areas in Shanxi. BioChip testing revealed the presence of several potato viruses, of which Potato virus Y (PVY) was the most common, reaching an incidence of 87.8% of all symptomatic samples. Immunocaptured multiplex reverse transcription (RT) PCR was used to identify strains for all 280 PVY-positive samples, unveiling 242 samples infected with a single strain of PVY (86.4%) and 38 (13.6%) with a mixed infection. Of samples with a single-strain infection, PVY-SYR-II accounted for 102 (42.1%), followed by PVYN-Wi (33, 13.6%), PVY-SYR-I (28, 11.6%), 261-4 (22, 9.1%), PVYNTNa (20, 8.3%), PVYNTNb (19, 7.9%), and PVY-SYR-III (18, 7.4%). Seven isolates representing different recombinants were selected for whole genome sequencing. Phylogenetic and recombination analyses confirmed the RT-PCR-based strain typing for all seven strains of PVY found in Shanxi. SXKL-12 is the first SYR-III strain from potato reported from China. However, unlike that in other known SYR-III isolates, the region positioned from 1,764 to 1,902 nt in SXKL-12 shared the highest sequence identity of 82.2% with an uncharacterized PVY isolate, JL-23, from China. Interestingly, PVYN-Wi isolate SXZY-40 also possessed a more divergent sequence for the region positioned from 6,156 to 6,276 nt than other N-Wi isolates known to date, sharing the highest identity of 86.6% with an uncharacterized Chinese PVY isolate, JL-11. Pathogenicity analysis of dominant strains PVY-SYR-II and PVYN-Wi in six local popular potato cultivars revealed that 'Kexin 13', 'Helan 15', and 'Jizhangshu 12' were susceptible to these two strains, with mild mottling or mosaic symptom expression, and three cultivars, 'Jinshu 16', 'Qingshu 9', and 'Xisen 6', were fully resistant.

Detection of Borrelia miyamotoi and Powassan Virus Lineage II (Deer Tick Virus) from Odocoileus virginianus Harvested Ixodes scapularis in Oklahoma

Odocoileus virginianus (white-tailed deer) is the primary host of adult Ixodes scapularis (deer tick). Most of the research into I. scapularis has been geographically restricted to the northeastern United States, with limited interest in Oklahoma until recently as the I. scapularis populations spread due to climate change. Ticks serve as a vector for pathogenic bacteria, protozoans, and viruses that pose a significant human health risk. To date, there has been limited research to determine what potential tick-borne pathogens are present in I. scapularis in central Oklahoma. Using a one-step multiplex real-time reverse transcription-PCR, I. scapularis collected from white-tailed deer was screened for Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, and deer tick virus (DTV). Ticks (n = 394) were pooled by gender and life stage into 117 samples. Three pooled samples were positive for B. miyamotoi and five pooled samples were positive for DTV. This represents a minimum infection rate of 0.8% and 1.2%, respectively. A. phagocytophilum, B. burgdorferi, and B. microti were not detected in any samples. This is the first report of B. miyamotoi and DTV detection in Oklahoma I. scapularis ticks. This demonstrates that I. scapularis pathogens are present in Oklahoma and that further surveillance of I. scapularis is warranted.

A multiplex RT-PCR assay for detection of emergent pepper Tsw resistance-breaking variants of tomato spotted wilt virus in South Korea

Tomato spotted wilt virus (TSWV) is a highly destructive virus for pepper. Introgression of the resistance gene Tsw in pepper is used to manage TSWV worldwide; however, the occurrence of Tsw resistance-breaking (RB) variants threatens the pepper industry. Here, we developed a multiplex reverse-transcription PCR assay for detection of recently emerged Tsw RB variants in South Korea with high specificity and sensitivity.

A Simple Multiplex Reverse Transcription-PCR Method for the Diagnosis of L-A and M Totiviruses in Saccharomyces cerevisiae.

Killer yeasts and their toxins have many potential applications in environmental, medical, and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait are usually performed phenotypically based on their toxin production and immunity. In this study, we describe a simple and specific reverse transcription (RT) multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated with the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primer addition to the RT reaction. Using a mixture of specific primers at the PCR step, this multiplex RT-PCR protocol provided an accurate diagnosis of both L-A and M totivirus in all its known variants, L-A-1/M1, L-A-2/M2, L-A-28/M28, and L-A-lus/Mlus, found in infected killer yeasts. Using this method, the expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified but, importantly, asymptomatic L-A-2/M2 infected cells were found in addition to unexpected L-A-lus/M2 totiviral associations. IMPORTANCE The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, the development of simple methods for their detection significantly facilitates their study. The simplified multiplex RT-PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depended on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provided valuable insights into L-A/M viral associations and their coadaptational events in nature.

Genetic diversity of rotavirus strains circulating in Norway before and after the introduction of rotavirus vaccination in children.

Globally, rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in young children under 5 years of age. Implementation of RV vaccination is expected to result in fewer cases of RV in the target population, but it is unknown if this also results in vaccine-induced virus strain replacement. Rotarix, a monovalent vaccine based on G1P[8] RV, was introduced in Norway in the children's immunization program in September 2014. The main aim of this study was to describe the diversity of RV circulating pre and post introduction of the RV vaccine in Norway and investigate changes in genotype distribution during the first 4 years after implementation. A total of 1108 samples were collected from children under 5 years enrolled with AGE from five large hospitals in Norway and were analyzed for RV by enzyme immunoassay (EIA). All positive results were genotyped by multiplex semi-nested reverse transcription PCR for identification of G and P types. In total, 487 of the 1108 (44%) samples, collected from the enrolled children, were positive for RV by EIA method which were further genotyped. G1P[8] was found to be the most common type of RV pre and post RV vaccine implementation followed by G9P[8]. There were neither geographical nor temporal differences in genotype dominance. Also, no apparent changes were shown in the genotype distribution in the postvaccine era for years from 2015 to 2018. In 21.4% of the cases, vaccine strains were detected. Continuous RV genotype surveillance is vital for assessing the effectiveness of a vaccine program and monitoring for any emergence of vaccine-escape strains. Genotyping is also necessary to detect vaccine strains to avoid reporting false-positive cases of active RV infection in newly vaccinated cases.

The feasibility of field collected pig oronasal secretions as specimens for the virologic surveillance of Japanese encephalitis virus.

Virologic surveillance of Japanese encephalitis virus (JEV) relies on collecting pig blood specimens and adult mosquitoes in the past. Viral RNAs extracted from pig blood specimens suffer from low detecting positivity by reverse transcription PCR (RT-PCR). The oronasal transmission of the virus has been demonstrated in experimentally infected pigs. This observation suggested oronasal specimens could be useful source in the virus surveillance. However, the role of this unusual route of transmission remains unproven in the operational pig farm. In this study, we explore the feasibility of using pig oronasal secretions collected by chewing ropes to improve the positivity of detection in commercial pig farms. The multiplex genotype-specific RT-PCR was used in this study to determine and compare the positivity of detecting JEV viral RNAs in pig’s oronasal secretions and blood specimens, and the primary mosquito vector. Oronasal specimens had the overall positive rate of 6.0% (95% CI 1.3%–16.6%) (3/50) to 10.0% (95% CI 2.1%–26.5%) (3/30) for JEV during transmission period despite the negative results of all blood-derived specimens (n = 2442). Interestingly, pig oronasal secretions and female Culex tritaeniorhynchus mosquito samples collected from the same pig farm showed similar viral RNA positive rates, 10.0% (95% CI 2.1%–26.5%) (3/30) and 8.9% (95% CI 2.5%–21.2%) (4/45), respectively (p> 0.05). Pig oronasal secretion-based surveillance revealed the seasonality of viral activity and identified closely related genotype I virus derived from the mosquito isolates. This finding indicates oronasal secretion-based RT-PCR assay can be a non-invasive, alternative method of implementing JEV surveillance in the epidemic area prior to the circulation of virus-positive mosquitoes.