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Our study optimized METex14 skipping mutation detection by analyzing 223 Oncomine™ Focus Assay-positive cases using Pan Lung Cancer PCR Panel and reverse transcription (RT)-PCR. Among the 11 METex14 skipping mutation-positive cases (average read counts: 1390), 2 with Oncomine™ Focus Assay read counts of 2540 and 10,177 were positive on all platforms. Those with Oncomine™ Focus Assay read counts ranging from 179 to 612 tested negative elsewhere. Specimens with low ratios (average ratio: 0.12% for nine cases) may yield false-positive results. Our results suggested that monitoring read counts and ratios and validating the results with RT-PCR are crucial to prevent false positives. [ABSTRACT FROM AUTHOR]

Hop stunt viroid (HSVd), a small, single stranded, circular, non-coding infectious RNA known to cause infection in various economically important crop plants. In the present investigation, a study was conducted in the southern part of Karnataka districts of India to detect the possible association of HSVd infection in mulberry plants. A total of 41 mulberry plants showing typical viroid-like symptoms along with asymptomatic samples were collected and screened using conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) using a specific set of HSVd-Fw/ HSVd-Re primers. Out of 41 samples, the study confirmed the presence of HSVd in six samples of mulberry collected from Ramanagara (1 sample), Chikkaballapur (3 samples) and Doddaballapura (2 samples) regions with an expected HSVd amplicon size of ∼ 290–300 nucleotides. The mechanical transmission of HSVd was also confirmed on cucumber (cv. Suyo) seedlings through bioassay, which was reconfirmed by RT-PCR. The amplicons were cloned, sequenced, and the representative nucleotide sequences were deposited in the NCBI GenBank. Subsequently, molecular phylogenetic analysis showed that HSVd mulberry isolates from this study were most closely related to grapevine isolates, indicating a common origin. On the other hand, it was shown to belong to a different group from mulberry isolates so far reported from Iran, Italy, Lebanon, and China. The secondary structure analysis of HSVd mulberry Indian isolates exhibited substitutions in the terminal left, pathogenicity, and variable regions compared to those of the Indian grapevine isolates. As far as this study is concerned, HSVd was detected exclusively in some mulberry plants with viral-like symptoms, but the pathogenesis and symptom expression needs to be further investigated to establish the relationship between HSVd and the disease symptoms in the mulberry plants. [ABSTRACT FROM AUTHOR]

BACKGROUND & AIMS: Metabolism supports cell proliferation and growth. Surprisingly, the tumor suppressor miR-22 is induced by metabolic stimulators like bile acids. Thus, this study examines whether miR-22 could be a metabolic silencer. METHODS: The relationship between miR-22 and the expression of fibroblast growth factor 21 (FGF21) and its receptor FGFR1 was studied in cells and fatty livers obtained from patients and mouse models. We evaluated the effect of an miR-22 inhibitor alone and in combination with obeticholic acid (OCA) for the treatment of steatosis. RESULTS: The levels of miR-22 were inversely correlated with those of FGF21, FGFR1, and PGC1α in human and mouse fatty livers, suggesting that hepatic miR-22 acts as a metabolic silencer. Indeed, miR-22 reduced FGFR1 by direct targeting and decreased FGF21 by reducing the recruitment of PPARα and PGC1α to their binding motifs. In contrast, an miR-22 inhibitor increases hepatic FGF21 and FGFR1, leading to AMPK and ERK1/2 activation, which was effective in treating alcoholic steatosis in mouse models. The farnesoid x receptor-agonist OCA induced FGF21 and FGFR1, as well as their inhibitor miR-22. An miR-22 inhibitor and OCA were effective in treating diet-induced steatosis, both alone and in combination. The combined treatment was the most effective at improving insulin sensitivity, releasing glucagon-like peptide 1, and reducing hepatic triglyceride in obese mice. CONCLUSION: The simultaneous induction of miR-22, FGF21 and FGFR1 by metabolic stimulators may maintain FGF21 homeostasis and restrict ERK1/2 activation. Reducing miR-22 enhances hepatic FGF21 and activates AMPK, which could be a novel approach to treat steatosis and insulin resistance. LAY SUMMARY: This study examines the metabolic role of a tumor suppressor, miR-22, that can be induced by metabolic stimulators such as bile acids. Our novel data revealed that the metabolic silencing effect of miR-22 occurs as a result of reductions in metabolic stimulators, which likely contribute to the development of fatty liver. Consistent with this finding, an miR-22 inhibitor effectively reversed both alcohol- and diet-induced fatty liver; miR-22 inhibition is a promising therapeutic option which could be used in combination with obeticholic acid.

VHH antibodies or nanobodies, which are antigen-binding domains of heavy chain antibodies from camelid species, have several advantageous characteristics, including compact molecular size, high productibility in bacteria and easy engineering for functional improvement. Focusing on these advantages of VHHs, we attempted to establish an immunoassay system for detection of Legionella , the causative pathogen of Legionnaires' disease. A VHH phage display library was constructed using cDNA from B cells of alpacas immunized with Legionella pneumophila serogroup1 (LpSG1). Through biopanning, two specific VHH clones were isolated and used to construct a Legionella detection system based on the latex agglutination assay. After engineering the VHHs and improving the assay system, the sensitive detection system was successfully established for the LpSG1 antigen. The immunoassay developed in this study should be useful in easy and sensitive detection of Legionella , the causative agent of Legionnaires' disease, which is a potentially fatal pneumonia. [ABSTRACT FROM AUTHOR]

Digital PCR (dPCR) enables absolute quantitation of nucleic acid without calibration using a standard curve, and is promising for quantitation of SARS-CoV-2 viral load. However, dPCR suffers from the need for complicated and expensive instruments. We previously reported a dPCR system using a poly(dimethylsiloxane) (PDMS) microwell array (MWA) chip and common laboratory tools. This dPCR system had been applied to DNA quantitation. In this paper, application of this dPCR system to RNA quantitation through one-step reverse transcription PCR (RT-PCR) is described. As a model template, SARS-CoV-2 genetic RNA was selected. Artificial standard samples of SARS-CoV-2 N gene were mixed with RT-PCR reagents. The resulting mixture was introduced into the microwells by the power-free pumping technique utilizing degassed PDMS. Thermal cycling and image acquisition were carried out using basic instruments such as a thermal cycler and an inverted fluorescence microscope. The fluorescence images showed distinctive difference between bright (positive) microwells and dark (negative) microwells. The number of the positive microwells was used for estimation of the template concentration in the sample based on the Poisson distribution theory. The estimated template concentrations exhibited excellent agreement with the input template concentrations in the range from 1.0 copy/μL to 10,000 copies/μL. The RT step in the thermal program was confirmed to be indispensable for the accurate quantitation. These results may open up the possibility of facile digital RT-PCR experiments for gene expression analysis and molecular diagnosis without the need for expensive specialized instruments. • Digital reverse transcription PCR was carried out using a simple poly(dimethylsiloxane) microfluidic chip. • The experiments were performed using only common laboratory instruments such as a thermal cycler and a fluorescence microscope. • Standard samples of SARS-CoV-2 RNA were successfully quantified in the range from 1.0 copy/μL to 10,000 copies/μL. • These results may open up the possibility of facile and inexpensive digital RT-PCR experiments for gene expression analysis and molecular diagnosis in a broad range of laboratories. [ABSTRACT FROM AUTHOR]

Copyright of Revista Chapingo Serie Ciencias Forestales is the property of Universidad Autonoma Chapingo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

We report a superspreading event of severe acute respiratory syndrome coronavirus 2 infection initiated at a bar in Vietnam with evidence of symptomatic and asymptomatic transmission, based on ministry of health reports, patient interviews, and whole-genome sequence analysis. Crowds in enclosed indoor settings with poor ventilation may be considered at high risk for transmission.

We assessed the relationship between antigen and reverse transcription PCR (RT-PCR) test positivity and successful virus isolation. We found that antigen test results were more predictive of virus recovery than RT-PCR results. However, virus was isolated from some antigen-negative and RT-PCR‒positive paired specimens, providing support for the Centers for Disease Control and Prevention antigen testing algorithm. [ABSTRACT FROM AUTHOR]

Broad-range PCR targeting 28S D1-D2 ribosomal DNA (rDNA) identifies numerous fungi but has limited sensitivity in clinical specimens. Ribosomal RNA (rRNA) vastly outnumbers rDNA, suggesting reverse transcription (RT)-PCR could improve detection. Among contrived samples, RT-PCR decreased 28S PCR cycle threshold values by 10--12 cycles and lowered the limit of detection > 2000-fold. Among 32 bronchoalveolar lavage specimens, RT-PCR detected 12/15 (80%) fungal PCR- or culture-positive specimens, versus 6/12 (50%) by 28S PCR, 9/12 (75%) by any fungal PCR, and 13/15 (87%) by culture. RT-PCR newly identified fungi in 4/17 (24%) PCR- and culture-negative specimens. RT substantially increased 28S PCR sensitivity overall. Lay summary Fungal infection remains difficult to diagnose in the laboratory. Here, we have shown that detecting ribosomal RNA and DNA, rather than only ribosomal DNA, in a broad range fungal assay results in a significant enhancement in the ability to detect and identify fungal pathogens in clinical samples. [ABSTRACT FROM AUTHOR]

Purpose: Sunlight‐induced p53 mutations are known to contribute towards increased risk of ocular surface squamous neoplasia (OSSN). Stratifin (14‐3‐3σ)/HEM (human epithelial marker) is a p53‐mediated inhibitor of cell cycle progression and has been shown to be a target of epigenetic deregulation in various carcinomas. In the present study, Stratifin expression, its promoter methylation status as well as expression of mutant p53 in early and advanced AJCC stages (8th edition) of OSSN, was evaluated. Methods: Sixty‐four OSSN [20 conjunctival intraepithelial neoplasia (CIN) and 44 squamous cell carcinoma (SCC)] patients were registered for this study, and they were followed up for 36–58 months (mean 48 ± 3.6). Immunoexpression of Stratifin and mutant p53 protein, mRNA expression of Stratifin by reverse transcription polymerase chain reaction (PCR) and methylation status of Stratifin by methylation‐specific PCR, was undertaken. Results: Hypermethylation of Stratifin promoter in 63% (40/64), loss of Stratifin expression in 75% (48/64) and downregulation of Stratifin mRNA in 61% (39/64) were observed. Stratifin hypermethylation was significantly associated with reduced disease‐free survival in both early and advanced T stage SCC cases. Expression of mutant p53 expression was seen in 48% (31/64) OSSN cases. Of the 31 patients with mutant p53 expression, 87% (27/31) also demonstrated loss of Stratifin immunoexpression. A significant association was seen between mutant p53 expression and Stratifin loss (p = 0.01) in advanced T stage SCC cases. Conclusions: Hypermethylation of Stratifin gene and its reduced mRNA expression both are potential biomarkers for identifying high‐risk OSSN patients. Aberrant methylation of Stratifin and simultaneous mutant p53 expression implicates involvement of p53‐Stratifin mediated signalling pathway in the pathogenesis of OSSN. [ABSTRACT FROM AUTHOR]

We aimed to generate an unbiased estimate of the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 4 urban counties in Utah, USA. We used a multistage sampling design to randomly select community-representative participants >12 years of age. During May 4-June 30, 2020, we collected serum samples and survey responses from 8,108 persons belonging to 5,125 households. We used a qualitative chemiluminescent microparticle immunoassay to detect SARS-CoV-2 IgG in serum samples. We estimated the overall seroprevalence to be 0.8%. The estimated seroprevalence-to-case count ratio was 2.5, corresponding to a detection fraction of 40%. Only 0.2% of participants from whom we collected nasopharyngeal swab samples had SARS-CoV-2-positive reverse transcription PCR results. SARS-CoV-2 antibody prevalence during the study was low, and prevalence of PCR-positive cases was even lower. The comparatively high SARS-CoV-2 detection rate (40%) demonstrates the effectiveness of Utah's testing strategy and public health response. [ABSTRACT FROM AUTHOR]

ABSTRACT Rapid antigen tests are simple to perform and provide results within 15 min. We describe our implementation and assess performance of the BinaxNOW COVID-19 Antigen Test (Abbott Laboratories) in 6,099 adults at a self-referred walk-up testing site. Participants were grouped by self-reported COVID-19 exposure and symptom status. Most (89%) were asymptomatic, of whom 17% reported potential exposure. Overall test sensitivity compared with reference laboratory reverse-transcription [RT] PCR testing was 81% (95% confidence interval [CI] 75%, 86%). It was higher in symptomatic (87%; 95% CI 80%, 91%) than asymptomatic (71%; 95% CI 61%, 80%) individuals. Sensitivity was 82% (95% CI 66%, 91%) for asymptomatic individuals with potential exposure and 64% (95% CI 51%, 76%) for those with no exposure. Specificity was greater than 99% for all groups. BinaxNOW has high accuracy among symptomatic individuals and is below the FDA threshold for emergency use authorization in asymptomatic individuals. Nonetheless, rapid antigen testing quickly identifies positive among those with symptoms and/or close contact exposure and could expedite isolation and treatment. IMPORTANCE The BinaxNOW rapid antigen COVID-19 test had a sensitivity of 87% in symptomatic and 71% asymptomatic individuals when performed by health care workers in a high-throughput setting. The performance may expedite isolation decisions or referrals for time-sensitive monoclonal antibody treatment in communities where timely COVID PCR tests are unavailable.

BACKGROUND: Induced resistance against several plant pathogens was reported using different beneficial plant growth‐promoting microorganisms. The potential of five fungal isolates, Trichoderma harzianum GT 3‐2, Fusarium equiseti GF 18‐3, F. equiseti GF 19‐1, Phoma sp. GS 10‐1 and Phoma sp. GS 14‐1, to stimulate tomato growth and resistance against bacterial speck disease caused by Pseudomonas syringae pathovar (pv.) tomato DC3000 was evaluated. RESULTS: Based on the results of disease severity and growth promotion experiments, GF 18‐3 exhibited the best results among all fungal isolates. Treatment with barley grain inocula (BGI) and culture filtrate (CF) of the isolates promoted tomato growth and suppressed the pathogen in pot trials. Furthermore, expressions of the pathogenesis‐related genes (PR‐1, β‐1,3‐glucanase A, β‐1,3‐glucanase B and LOX) were relatively higher than the control in the leaves of tomato plants treated with both BGI and CF. The transcription levels remained consistently higher than the control plants for 6 days post‐inoculation with pathogen. CONCLUSION: Taken together, the results indicate that the tested fungal isolates have the potential to promote tomato growth and induce systemic resistance against the bacterial speck disease. Analysis of certain PR gene expression revealed significant activation in both BGI and CF treatments, leading to stimulated resistance against the pathogen. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The purpose of systemic treatment in patients with breast cancer is based largely on the molecular characteristics of the primary tumor, but many clinical recommendations suggest also the study of metastatic nodes with an assessment of their receptor status (estrogen receptor ER, progesterone receptor RP, human epidermal growth factor receptor 2 Her2/neu). This is due to the fact that according to numerous studies, the discrepancy between the status of the primary tumor and the secondary nodes can reach high rates: 3–54 % for ER, 5–78 % for RP, and 0–34 % for Her2/neu. At the same time, more and more data actively demonstrate the imperfection of immunohistochemical analysis and the need to study additional parameters to improve the quality of diagnosis of patients with breast cancer. Material and methods. A morphological and immunohistochemical study of the tumor tissue of the primary node and axillary lymph nodes was performed in 199 patients with breast cancer (T1-3N0-3M0) using standard methods, and RT-PCR was also studied with the expression of 24 genes. Results. The incidence of differences between the molecular phenotypes of the main tumor and metastatic axillary lymph nodes was 26 (26 %) of 99 cases. Most often, differences were noted in cases of breast cancer with luminal A type – 13 cases (50 %). According to the results of a comparative PCR analysis of tissue samples from the primary tumor and metastatic regional lymph nodes, only the expression of the CD68, ERSR1, GRB7 and MMD11 receptors was statistically significant. Conclusion. The results indicate the need for an integrated approach and additional methods for the diagnosis of breast cancer, which will undoubtedly improve the quality of planning and the effectiveness of systemic treatment in patients with breast cancer.

During 2011–2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome.

Hand, Foot, and Mouth disease (HFMD) is a mild exanthematous and febrile disease occurs in children aged ≤10 years old. The present study highlights clinical, epidemiological characteristics, distribution of enterovirus (EV) types, and sub genotypes in HFMD cases reported during 2017 to 2018 in Western India. A total of 93 clinical samples collected from 68 HFMD cases were included. The presence of EV‐RNA was determined by 5'UTR based nested reverse transcription polymerase chain reaction followed by molecular typing, sub genotyping by VP1/2A junction or VP1, full VP1 gene amplification, and phylogenetic analysis. The study reports 80.64% (75/93) EV positivity and 94.66% (71/75) typing rate, with a predominant circulation of CVA16 and CVA6 strains. Sequence analysis revealed the presence of coxsackievirus (CV)A16 (57.7%), CVA6 (40.8%), and Echo1 (1.4%) strains. EV infections were predominantly observed in children aged 1 to 3 years old (43.9%). Although cases were reported throughout the year, peaked in July (15.8%) and August (24.6%) months and persisted till September (19.3%). All the CVA16 and CVA6 positive strains were genotyped using full VP1 gene amplification. All CVA16 Indian strains (n = 41) were clustered with rarely reported B1c sub genotype and CVA6 strains (n = 29) with E2 sub‐lineage. The study highlights the genetic characteristics of circulating CVA16, CVA6, and Echo1 strains in HFMD cases from Western India. The emergence of CVA16 B1c genotype and sub‐lineage E2 of CVA6 strains and their constant circulation further demands systemic surveillance studies on HFMD from different parts of India to facilitate the rapid diagnosis of CVA16 and CVA6 strains using the molecular and serological based approach and for intervention strategies. Highlights: Predominance of CVA16 and CVA6 strains in HFMD cases from western India. Circulation of CVA16 B1c sub genotype strains and CVA6 sub lineage E2 strains in HFMD cases, western India. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 pandemic has presented new challenges to food manufacturers. During the early phase of the pandemic, several large outbreaks of coronavirus disease 2019 (COVID-19) occurred in food manufacturing plants resulting in deaths and economic loss, with approximately 15% of personnel diagnosed as asymptomatic for COVID-19. Spread by asymptomatic and presymptomatic individuals has been implicated in large outbreaks of COVID-19. In March 2020, we assisted in implementation of environmental monitoring programs for SARS-CoV-2 in zones 3 and 4 of 116 food production facilities. All participating facilities had already implemented measures to prevent symptomatic personnel from coming to work. During the study period, from 17 March to 3 September 2020, 1.23% of the 22,643 environmental samples tested positive for SARS-CoV-2, suggesting that infected individuals were actively shedding virus. Virus contamination was commonly found on frequently touched surfaces such as doorknobs, handles, table surfaces, and sanitizer dispensers. Most processing plants managed to control their environmental contamination when they became aware of the positive findings. Comparisons of positive test results for plant personnel and environmental surfaces in one plant revealed a close correlation. Our work illustrates that environmental monitoring for SARS-CoV-2 can be used as a surrogate for identifying the presence of asymptomatic and presymptomatic personnel in workplaces and may aid in controlling infection spread. Environmental contamination by SARS-CoV-2 was found in food processing plants. Of 22,643 environmental swabs, 278 (1.23%) were positive for SARS-CoV-2. Frequently touched surfaces had the most contamination. Doorknobs, computer devices, tabletops, and sanitizers had the most contamination. Surface testing may indicate the presence of asymptomatic virus carriers. [ABSTRACT FROM AUTHOR]

Virus shedding in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can occur before onset of symptoms; less is known about symptom progression or infectiousness associated with initiation of viral shedding. We investigated household transmission in 5 households with daily specimen collection for 5 consecutive days starting a median of 4 days after symptom onset in index patients. Seven contacts across 2 households implementing no precautionary measures were infected. Of these 7, 2 tested positive for SARS-CoV-2 by reverse transcription PCR on day 3 of 5. Both had mild, nonspecific symptoms for 1-3 days preceding the first positive test. SARS-CoV-2 was cultured from the fourth-day specimen in 1 patient and from the fourth- and fifth-day specimens in the other. We also describe infection control measures taken in the households that had no transmission. Persons exposed to SARS-CoV-2 should self-isolate, including from household contacts, wear a mask, practice hand hygiene, and seek testing promptly. [ABSTRACT FROM AUTHOR]

Copyright of China Tropical Medicine is the property of China Tropical Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Of 103 serum samples collected from dogs in South Korea, 3 (2.9%) were positive for severe fever with thrombocytopenia syndrome virus (SFTSV) and 22 (21.4%) were positive for antibodies against SFTSV. A dog-derived isolate of SFTSV clustered with many South Korea SFTSV strains in the Japanese clade.

During 2017-2018, Barmah Forest virus was recovered from mosquitoes trapped in military training areas in Australia and from a soldier infected at 1 of these areas. Phylogenies of the nucleotide sequences of the envelope glycoprotein gene E2 and the 3' untranslated region suggest that 2 lineages are circulating in eastern Australia. [ABSTRACT FROM AUTHOR]

Before 2001, all serosurveys for morbilliviruses in sea otters (Enhydra lutris) in California, Washington, and Alaska, US, documented a 0% seroprevalence. The first published serologic detections of morbillivirus in sea otters occurred in 2001–02 in live-captured Washington sea otters, with a documented 80% seroprevalence. We conducted a retrospective study of sea otter cases from 1989 to 2010 compiled at the US Geological Survey, National Wildlife Health Center to identify cases of morbilliviral disease in Washington sea otters and to characterize the disease using immunohistochemistry, reverse transcription (RT)-PCR, genetic sequencing, virus isolation, and serology. We identified six cases of morbilliviral disease and 12 cases of morbilliviral infection in this population of sea otters during 2000–10. Significant histologic findings included inflammation in the white and gray matter of the brain characterized by lymphoplasmacytic perivascular cuffing, neuronal necrosis, and satellitosis in gray matter and by spongiosis, myelin degeneration, spheroids, and gemistocytes in white matter. Intranuclear and intracytoplasmic viral inclusion bodies were found in neurons, Purkinje cells, and glia. Immunohistochemistry for canine distemper virus (CDV) showed positive staining in neurons, glial cells, and cell processes. A pan-morbillivirus RT-PCR with subsequent restriction endonuclease digestion or sequencing identified CDV. Virus isolation was not successful. Two sea otters with morbilliviral encephalitis showed greater antibody titers to CDV than phocine distemper virus. Histologic changes were confined to the central nervous system and resembled neurologic canine distemper in domestic dogs. Cases of sea otters with morbilliviral infection without histologic changes could represent early infections or incompletely cleared sublethal infections. We found that morbillivirus was present in the Washington sea otter population as early as 2000, and we provide a description of the pathology of canine distemper in sea otters. [ABSTRACT FROM AUTHOR]

Among 11 patients in Thailand infected with severe acute respiratory syndrome coronavirus 2, we detected viral RNA in upper respiratory specimens a median of 14 days after illness onset and 9 days after fever resolution. We identified viral co-infections and an asymptomatic person with detectable virus RNA in serial tests. We describe implications for surveillance. [ABSTRACT FROM AUTHOR]

One mosquito opsin gene, NYD-OP7, has been cloned from Culex pipiens pallens. An open reading frame (ORF) of 1116 bp was found to encode a putative 371 amino acids protein which exhibits high identity with opsins from Aedes and Anopheles mosquitoes. Transcript expression of NYD-OP7 was determined by real-time PCR in all life stages of deltamethrin-susceptible and -resistant strains of the Culex mosquito. The results demonstrated that this gene is expressed at all developmental stages, and it is expressed predominantly at the pupae and adult stages. Meanwhile, in pupae and adults, NYD-OP7 is overexpressed in deltamethrin-resistant strain than in -susceptible strain. Importantly, stable expression of NYD-OP7 in the mosquito C6/36 cells can confer moderate deltamethrin resistance. Our study provides the first direct evidence that increased expression of an opsin gene may play some role in the development of deltamethrin resistance in Cx. pipiens pallens. © 2006 Elsevier Inc. All rights reserved.

We sequenced 10% of imported severe acute respiratory syndrome coronavirus 2 infections detected in travelers to Hong Kong and revealed the genomic diversity of regions of origin, including lineages not previously reported from those countries. Our results suggest that international or regional travel hubs might be useful surveillance sites to monitor sequence diversity. [ABSTRACT FROM AUTHOR]

Quantitative monitoring of BCR-ABL1IS gene using reverse transcription quantitative-PCR (RT-qPCR) is an important method for evaluating the treatment effects in patients with chronic myeloid leukemia (CML). Digital-PCR (dPCR) can be applied to detect the BCR-ABL1 gene with high sensitivity. In the present study, the results of the Clarity™ dPCR system were compared with those of the RT-qPCR in order to determine whether dPCR can be applied in the clinical setting. A total of 83 patients were included in the present study, and they were divided into two groups according to the results of BCR-ABL1IS during ongoing monitoring. A total of 43 patients with undetectable BCR-ABL1IS where enrolled in group A. BCR-ABL1 testing was performed using the dPCR system on the same peripheral blood samples of patients from group A, and the association between dPCR results and relapse was analyzed. The RT-qPCR platform and dPCR system were used simultaneously to detect the BCR-ABL1 gene of another 40 patients who achieved either partial cytogenetic response (PCyR) or further response. Among patients with undetectable BCR-ABL1IS, patients with dPCR-positive disease (BCR-ABL1 >0.1%) were more likely to undergo molecular relapse (P=0.018). The results of dPCR detection of BCR-ABL1% were consistent with the RT-qPCR results (R2=0.9510) in patients who achieved PCyR or further response. For samples with BCR-ABL1IS <1.0%, the consistency of the dPCR and RT-qPCR results was better than that of BCR-ABL1IS >1.0% (R2=0.9488 vs. R2=0.9264 for BCR-ABL1IS). The detection results of the BCR-ABL1 gene in patients with CML using dPCR matched well with those from the RT-qPCR. To conclude, the results of the dPCR system can be applied as a supplement to the RT-qPCR platform, particularly for those with BCR-ABL1IS <1.0%. [ABSTRACT FROM AUTHOR]

The present study highlights pathogenesis and molecular aspects of Coxsackie virus A‐16 (CVA‐16) strains isolated from hand, foot, and mouth disease (HFMD) cases from India using a neonatal mice model. ICR mice were intraperitoneally inoculated with CVA‐16/311 strain isolated from HFMD cases. Mice developed hind and forelimb paralysis on day 3 of post infection. Histopathological observations of hind limb muscles showed necrosis, dissolution of muscle fiber cells, infiltration of inflammatory cells, marked dilated ventricle, hemorrhages, and neuronal degeneration in the brain. Immunohistochemical studies revealed high expression of CVA‐16/311–specific viral antigen in limb muscles, brain, heart from day 3 till day 7 of post‐infection. VP1 gene‐based reverse transcription polymerase chain reaction conducted in RNA samples of different tissue organs of infected mice followed by sequencing of the positive amplimers revealed presence of CVA‐16/311–specific viral sequences. Phylogenetic analysis based on the VP1 gene showed the presence of B1c sub genotype of CVA‐16/311 strain in targeted tissue organs. Sequence analysis revealed major genetic changes in heart, skeletal muscle tissues at the nucleotide and amino acid levels. Genetic changes occurred in organs of mice might predict some potential targets and might act as markers of virulence for neuronal tropism. Pathogenesis and molecular studies of CVA‐16 strains isolated from HFMD cases using neonatal mice model was conducted for the first time from India. Highlight: Pathological and molecular aspects of CVA‐16/311 strain from HFMD using a neonatal mice model.Reported for the first time from India. [ABSTRACT FROM AUTHOR]

During 2011-2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome. [ABSTRACT FROM AUTHOR]

The corticotropin-releasing factor (CRF) gene family includes the three urocortins (UCN1, 2 and 3) and the two receptors (CRFR1 and 2), which play a significant role in the physiology of various organs. The expression of the CRF family of genes and its receptors are shown to participate in the pathogenesis of inflammation and even tumorigenesis. However, data regarding the human urinary tract, especially the bladder, are scarce. To the best of our knowledge, no studies are currently available on the CRF system and bladder cancer. The primary goal of the present study was to investigate the mRNA expression of the CRF family members in bladder cancer. The secondary aim was to analyze the differences with the expression of the same mRNAs in normal bladders. From August 2018 to July 2021, 43 recruited patients were divided into three groups. Group A included healthy patients, group B included patients with bladder cancer and group C included patients with a history of cancer from whom samples were taken from the normal bladder mucosa. Detection of mRNA of the CRF family of genes was performed using reverse transcription-quantitative PCR. The mRNA of the three urocortins, CRF and the two receptors were predominantly expressed in all three groups of patients. Statistical analysis using the Kruskal-Wallis test showed that UCN1 was downregulated in patients with bladder cancer and those with possible cancer compared with the healthy group (mean rank group A=24.3 vs. mean rank group B=12.58; P=0.006) and (mean rank group A=24.3 vs. mean rank group C=8.88; P=0.001). The present experiments showed that mRNA of the CRF family of genes was amplified in normal and cancer bladder tissues. Downregulation of the UCN1 gene may be associated with bladder cancer, contributing to the prognosis, diagnosis or therapy of urothelial malignancies. [ABSTRACT FROM AUTHOR]

To validate the specimen-pooling strategy for real-time reverse transcription PCR detection of severe acute respiratory syndrome coronavirus 2, we generated different pools including positive specimens, reflecting the distribution of cycle threshold values at initial diagnosis. Cumulative sensitivities of tested pool sizes suggest pooling of <6 specimens for surveillance by this method. [ABSTRACT FROM AUTHOR]

During the coronavirus disease pandemic in Spain, from April 10-24, 2020, a total of 5,869 persons were screened for severe acute respiratory syndrome coronavirus 2 at nursing homes. Among residents, 768 (23.9%) tested positive; among staff, 403 (15.2%). Of those testing positive, 69.7% of residents and 55.8% of staff were asymptomatic. [ABSTRACT FROM AUTHOR]

We report an asymptomatic child who was positive for a coronavirus by reverse transcription PCR in a stool specimen 17 days after the last virus exposure. The child was virus positive in stool specimens for at least an additional 9 days. Respiratory tract specimens were negative by reverse transcription PCR. [ABSTRACT FROM AUTHOR]

The aim of this 42-day study was to examine the effects of Chlorella vulgaris on immunorelated factors and their expression in Penaeus vannamei. Results showed that C. vulgaris significantly enhanced the activities of phenolic oxidase (PO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in the serum and hepatopancreas of P. vannamei (p<0.05, p<0.01), increased the expression levels of PO, SOD, GSH-Px, CAT, and IMD genes in the serum of P. vannamei (p<0.05, p<0.01), and significantly increased the expression level of Toll receptor genes in the hepatopancreas of P. vannamei (p<0.05, p<0.01) and the expression level of IMD gene. In conclusion, C. vulgaris regulated the immune function of P. vannamei through the Toll receptor gene and the IMD pathways. We speculated the Chorella polysaccharides were the main active ingredients of C. vulgaris. [ABSTRACT FROM AUTHOR]

Most eukaryotic genes contain introns, which are noncoding sequences that are removed during premRNA processing. Introns are usually preserved across evolutionary time. However, the sizes of introns vary greatly. In Arabidopsis, some introns are longer than 10 kilo base pairs (bp) and others are predicted to be shorter than 10 bp. To identify the shortest intron in the genome, we analyzed the predicted introns in annotated version 10 of the Arabidopsis thaliana genome and found 103 predicted introns that are 30 bp or shorter, which make up only 0.08% of all introns in the genome. However, our own bioinformatics and experimental analyses found no evidence for the existence of these predicted introns. The predicted introns of 30–39 bp, 40–49 bp, and 50–59 bp in length are also rare and constitute only 0.07%, 0.2%, and 0.28% of all introns in the genome, respectively. An analysis of 30 predicted introns 31–59 bp long verified two in this range, both of which were 59 bp long. Thus, this study suggests that there is a limit to how small introns in A. thaliana can be, which is useful for the understanding of the evolution and processing of small introns in plants in general. [ABSTRACT FROM AUTHOR]

Rotaviruses (RVs), a common cause of viral gastroenteritis in humans and animals, are classified into 9 established groups/species (RVA–RVI). Although RVB has been found in several countries, genetic variation among RVB field strains remains poorly characterized. RVB strains can be classified into G genotypes based on a nucleotide (nt) homology that exceeds a cutoff value of 80% for the gene that encodes the structural protein VP7. In this study, we determined the VP7 nt and deduced amino acid sequences of one RVB strain (RB62) identified in a diarrheic fecal sample obtained from a piglet in Brazil in 2012. Comparative analysis of this strain and the strains of the other 21 previously identified VP7 ge­notypes showed that the highest nt identity (71.2%) was found with the porcine PB-70-H5 strain within the G4 genotype. However, when compared with the nonclassified Vietnamese RVB G genotype 14177_18 strain, the nt sequence identity was of 82.9%. These results led us to conclude that the Brazilian strain BR62 and the Vietnamese strain 14177_18 belong to a novel G genotype (G22). [ABSTRACT FROM AUTHOR]

Human noroviruses (HuNoVs) are one of the leading global causes of diarrhoeal diseases and are transmitted mainly from person to person but also through contaminated food, water and fomites. The possible zoonotic nature of NoVs has occasionally been discussed, although the viruses are generally considered to be host-species- specific. We investigated whether wild birds and rodents could serve as carriers of HuNoVs, thereby transmitting the virus to humans directly or indirectly by contaminating foods. All samples, 115 avian and 100 rat faeces collected in springs 2009- 2013 from dump sites, and 85 faeces from yellow-necked mice trapped in late autumn 2008 and 2009 after the rodents entered human settlements due to the first night frosts, were screened for HuNoV using real-time reverse transcription PCR. HuNoVs were detected in 31 (27%) faecal samples of wild birds, in two (2%) faecal samples of rats and in no samples of mice. Most (25) of the positive bird samples and both rat samples contained genogroup II, and six positive bird samples contained genogroup I HuNoV. The avian species shedding faeces containing HuNoVs were identified as gulls and crows using DNA barcoding. Our results show that wildlife, birds and rats in particular, is capable of spreading HuNoVs in the environment. [ABSTRACT FROM AUTHOR]

Raspberries have lately caused several human norovirus (HuNoV) outbreaks in Europe. In this study, we developed and evaluated for HuNoV reverse transcription (RT)-PCR detection in frozen raspberries extraction methods that have equal sensitivity but are less time-consuming than widely used methods based on polyethylene glycol (PEG) precipitation and chloroform–butanol purification. One method was applied to stored frozen raspberries linked to previous HuNoV outbreaks and berries on sale. In the virus elution-based Method 1, sparkling water eluted viruses most efficiently from the berries. Method 2, based on direct nucleic acid extraction with minor PEG supplement, yielded the highest number of positive findings (4 out of 9) at low virus concentration level of 100 genome copies HuNoV genogroup II per 25 g raspberries. Both methods showed approximately equal sensitivity to a method including PEG precipitation and chloroform–butanol purification. Two naturally contaminated berry samples linked to HuNoV outbreaks in 2006 and 2009 were still positive for HuNoV genogroup I, but all berry products purchased from a local store remained negative for HuNoV. In conclusion, this study presents two efficient and rapid methods which can be used in urgent HuNoV outbreak investigations, since the results of the virus analysis are available in a few hours. [ABSTRACT FROM AUTHOR]

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Most of the eukaryotic genes contain introns, which are removed from the pre-RNA during RNA processing. In contrast to the introns in animals, which are usually several kilo base pairs (kb), those in plants generally are very small, which are mostly from dozens of base pairs (bp) to a few hundred bp. According to annotation version 10.0 of the genome of Arabidopsis thaliana, there are 127,854 introns in the nuclear genes; 99.23% of them are less than 1 kb, and only 16 introns are annotated to be larger than 5 kb, which are extremely large introns (ELI) in Arabidopsis. To learn whether these introns are true introns or not and how large introns could be in Arabidopsis, RT-PCR analysis of genes containing these ELIs were carried out. The results indicated that some of these putative introns are indeed ELIs. These ELIs are mainly composed of transposons or transposable elements (TE), excepting one, whose counterparts are also very long in diverse plant species. Thus, this study confirms the existence of introns larger than 5 kb or even 10 kb in Arabidopsis. [ABSTRACT FROM AUTHOR]

A variety of molecular assays have been developed for CTCs detection and molecular characterization. Molecular assays are based on the nucleic acid analysis in CTCs and are based on total RNA isolation and subsequent mRNA quantification of specific genes, or isolation of genomic DNA that can be for DNA methylation studies and mutation analysis. This review is mainly focused on gene expression and methylation studies in CTCs in various types of cancer. [ABSTRACT FROM AUTHOR]

Background: Hepatitis G virus (HGV) is newly identified virus, transmitted by infected blood and blood products. Effect of HGV infection on liver diseases is not well known. Aims: Co-infection of HGV with hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has been reported however; very limited data is available from India. Therefore, we have performed a pilot study for the presence of co-infection of HGV in chronic liver disease patients. Setting and Design: The study was performed in research laboratory at P.D. Hinduja National hospital and Medical research center, Mahim, Mumbai. Prospective study was designed. Methods and Materials: Forty HBV, HCV related chronic liver disease patients were studied. Forty randomly selected voluntary healthy blood donors visiting our blood bank were included as controls. Serum bilirubin, alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were estimated. HGV infection was detected by using reverse transcriptase molony murine leukemia virus (M-MLV) with the help of HGV 340/625IC kit (Sacace, Italy). Results and Conclusion: One HCV positive patient had infection with HGV among 40 HBV/HCV chronic liver disease patients.

Nucleic acid (NA)-targeted tests detect and quantify viral DNA and RNA (collectively xNA) to support epidemiological surveillance and, in individual patients, to guide therapy. They commonly use polymerase chain reaction (PCR) and reverse transcription PCR. Although these all have rapid turnaround, they are expensive to run. Multiplexing would allow their cost to be spread over multiple targets, but often only with lower sensitivity and accuracy, noise, false positives, and false negatives; these arise by interactions between the multiple nucleic acid primers and probes in a multiplexed kit. Here we offer a multiplexed assay for a panel of respiratory viruses that mitigates these problems by combining several nucleic acid analogs from the emerging field of synthetic biology: (i) self-avoiding molecular recognition systems (SAMRSs), which facilitate multiplexing, and (ii) artificially expanded genetic information systems (AEGISs), which enable low-noise PCR. These are supplemented by “transliteration” technology, which converts standard nucleotides in a target to AEGIS nucleotides in a product, improving hybridization. The combination supports a multiplexed Luminex-based respiratory panel that potentially differentiates influenza viruses A and B, respiratory syncytial virus, severe acute respiratory syndrome coronavirus (SARS), and Middle East respiratory syndrome (MERS) coronavirus, detecting as few as 10 MERS virions in a 20-μl sample. [ABSTRACT FROM AUTHOR]

Highlights: [•] Electropositive membrane can adsorb influenza virus in water sample efficiently. [•] Genome RNA of influenza virus can be directly extracted from electropositive membrane. [•] Enriching the influenza virus in water can improve the detection efficiency of RT-PCR. [•] This method is simple and efficient for monitoring of influenza virus in water. [ABSTRACT FROM AUTHOR]