During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the ...Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.
Production and maintenance of virus‐free planting materials is pivotal for the control of viral diseases. The present study attempted to test exogenous application of melatonin for eradication of ...apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple cultivar Gala. Exogenous application of 15 μm melatonin to the shoot proliferation medium significantly increased the number of shoots and shoot length. The level of endogenous indole‐3‐acetic acid (IAA) was the highest in the shoots proliferating on the shoot proliferation medium containing 15 μm melatonin. Shoot regrowth levels were significantly higher in shoot tips of the virus‐infected shoots cultured for 4 weeks on this medium than the control. In addition, culture of shoot tips of the virus‐infected in vitro shoots proliferated for 4 weeks on this medium resulted in 95% of shoots being virus‐free, while no virus‐free shoots were obtained in shoot tips of the virus‐infected shoots cultured without melatonin. Analyses by microtissue direct RT‐PCR and RT‐qPCR showed that ASGV concentration decreased in shoot tips of the virus‐infected shoots proliferating on the medium containing 15 μm melatonin for 4 weeks. Virus localization showed that exogenous application of melatonin enlarged the virus‐free area in the virus‐infected shoot tips. These data provide explanations as to why exogenous application of melatonin can efficiently eradicate ASGV. Exogenous application of melatonin provides an alternative means for plant virus eradication and has the potential to produce virus‐free plants.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
To face the new Covid-19 pandemic, the need for early and accurate diagnosis of the disease among suspected cases quickly became obvious for effective management, and for better control of the spread ...of the disease in the population. Since the beginning of this disease epidemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), reverse transcriptase polymerase chain reaction (RT-PCR) has routinely been used to confirm diagnosis. However, several authors have pointed out the poor performance of this technique, particularly in terms of sensitivity.1,2 This article is protected by copyright. All rights reserved.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
In this study, we collected a total of 610 hospitalized patients from Wuhan between February 2, 2020, and February 17, 2020. We reported a potentially high false negative rate of real‐time ...reverse‐transcriptase polymerase chain reaction (RT‐PCR) testing for SARS‐CoV‐2 in the 610 hospitalized patients clinically diagnosed with COVID‐19 during the 2019 outbreak. We also found that the RT‐PCR results from several tests at different points were variable from the same patients during the course of diagnosis and treatment of these patients. Our results indicate that in addition to the emphasis on RT‐PCR testing, clinical indicators such as computed tomography images should also be used not only for diagnosis and treatment but also for isolation, recovery/discharge, and transferring for hospitalized patients clinically diagnosed with COVID‐19 during the current epidemic. These results suggested the urgent needs for the standard of procedures of sampling from different anatomic sites, sample transportation, optimization of RT‐PCR, serology diagnosis/screening for SARS‐CoV‐2 infection, and distinct diagnosis from other respiratory diseases such as fluenza infections as well.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA‐PRRSV2), highly pathogenic (HP‐PRRSV2) and NADC30‐like (NL‐PRRSV2)) are ...currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real‐time RT‐PCR assays using TaqMan‐MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real‐time RT‐PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real‐time RT‐PCR assays were further validated by comparing with a universal PRRSV conventional RT‐PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real‐time RT‐PCR assays and the conventional RT‐PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV‐positive, including 2 samples that were determined to be co‐infected with NL‐PRRSV2 and HP‐PRRSV2 isolates by the quadruplex real‐time RT‐PCR assay. ORF5 sequencing confirmed the real‐time RT‐PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA‐PRRSV2, NL‐PRRSV2 and HP‐PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Waterfowl are the main reservoir for most influenza A virus subtypes, and they can effectively transmit these viruses to other birds and humans. This study aims to identify two influenza virus ...subtypes, H5 and H7, in domestic geese and ducks in Basrah governorate, Southern Iraq. 310 cloacal swabs were obtained from 150 domestic geese and 160 domestic ducks from different geographical areas. The viruses were first detected by RT-PCR using a pair of universal primers. All positive samples underwent RT-PCR using gene-specific primers to identify H5 and H7 influenza virus subtypes. The results showed that the prevalence of influenza viruses detected through universal primers was 37.7%. Of these, 24.6% and 50% were positive for viruses in domestic geese and ducks, respectively. Regarding virus subtyping in geese, the infection rates with H5 and H7 were 43.2% and 29.7%, respectively, with 27% as a combination of the two, while in domestic ducks, the infection rate with H5 was 27.5%, and with H7 it was 15%. Interestingly, ducks had a high concurrent infection rate for both H5 and H7 subtypes, accounting for 57.5%. The study concluded that the two virus subtypes, individually or simultaneously, were present in domestic waterfowl in regions of Basrah, and they were higher in ducks than in geese.
Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the swine industry, causing severe disease and resulting in substantial economic losses. Despite China’s implementation of a ...large-scale vaccine immunization strategy in recent years, various strains of PEDV, including classical attenuated vaccine strains, continue to emerge in immunized pig herds. Here, we established a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting a 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains, derived from classical strains. This assay effectively distinguishes between PEDV classical attenuated vaccine strains and wild-type strains, and we also explore the causes of this discriminatory target deficiency of this method through phylogenetic and recombination analysis. We found that these three classical attenuated vaccine strains exhibit closer phylogenetic relationships and higher sequence similarity with five cell-adapted strains. Recombination analysis revealed that although recombination is widespread in the PEDV genome, the 24-nucleotide deletion site remains stable without undergoing recombination and can be utilized as a target for identification. Further analysis revealed there are no enzyme cleavage sites near the 24-nucleotide site, suggesting that this deletion may have been lost during the process of culturing these viral strains in cells.The detection method we have established exhibits high specificity and sensitivity to PEDV, without cross-reactivity with other viruses causing diarrheal diseases. A total of 117 swine fecal samples were analyzed using this established one-step real-time reverse transcription PCR assay, indicating the presence of classical attenuated vaccine strains in pig herds in Gansu province, China. Additionally, the designed primer pairs and two probes can be placed in a single reaction tube to differentiate between these two types of strains, effectively reducing detection costs. These findings offer an efficient and cost-effective technological platform for clinical rapid identification testing of both wild-type and classical attenuated vaccine strains of PEDV, as well as for precise investigation of clinical data on natural infections and vaccine immunity in pig herds.
•A one-step real-time RT-PCR assay targeting a 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains, derived from classical strains.•This assay effectively distinguishes between PEDV classical attenuated vaccine strains and wild-type strains, while preliminarily exploring the potential reasons for the discriminatory target deficiency of this method through phylogenetic and recombination analysis.•Our method offers an efficient and cost-effective technological platform for precise investigation of clinical data on natural infections and vaccine immunity in pig herds.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease‐2019 (COVID‐19). ...Here, we examined the duration of clearance of SARS‐CoV‐2 in respiratory samples from 207 infected cases by real‐time reverse‐transcription polymerase chain reaction (RT‐PCR). A substantial proportion of COVID‐19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS‐CoV‐2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID‐19, irrespective of their Ct values, cleared SARS‐CoV‐2 RNA within 14 days of the initial detection. Longitudinal assessment of RT‐PCR test results in individuals requiring 15–30 days to clear SARS‐CoV‐2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow‐up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID‐19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS‐CoV‐2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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