viral isolation
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2022 ◽  
Author(s):  
Anupriya Aggarwal ◽  
Alberto Stella ◽  
Anouschka Akerman ◽  
Gregory Walker ◽  
Vanessa Milogiannakis ◽  
...  

Abstract From late 2020 the world observed the rapid emergence of many distinct SARS-CoV-2 variants. At the same time, pandemic responses coalesced into significant global vaccine roll-out that have now significantly lowered Covid-19 hospital and mortality rates in the developed world. Over this period, we developed a rapid platform (R-20) for viral isolation and characterisation using primary remnant diagnostic swabs. This combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterisation of all major SARS-CoV-2 variants (all variants of concern and 6 variants of interest) globally with a 4-month period. This platform facilitated viral variant isolation and enabled rapid resolution of variant phenotype by allowing determining end point viral titers from primary nasopharyngeal swabs and through ranking of evasion of neutralising antibodies. In late 2021, when the Delta variant was dominating, Omicron rapidly emerged. Using this platform, we isolated and tested the first cases of this variant within Australia. In this setting we observed Omicron to diverge from other variants at two levels: Firstly, it ranks at the mots evasive to neutralisation antibodies compared to all VOCs and major VUIs. Secondly, it no longer engages TMPRSS2 during the late stages of fusion.


Author(s):  
Juan More-Bayona ◽  
Mercy Ramírez ◽  
Ben Hause ◽  
Eric Nelson ◽  
Hermelinda Rivera

Porcine Deltacoronavirus is a newly emergent enteric pathogen affecting swine farms worldwide. It has been detected in several countries in Europe, Asia and North America; yet, it has not been reported in South America. In November 2019, an enteric disease outbreak in a pig farm located in San Martin, Peru; was reported along with submission of three intestinal samples from pigs who succumbed to the disease. Samples were processed for molecular detection by qRT-PCR, viral isolation and further sequencing analysis. A taqman-based RT-PCR was performed to differentiate among the most relevant swine enteric coronaviruses described to date. All samples were positive to Porcine Deltacoronavirus with a cycle threshold (Ct) value between 9-14, revealing a high viral load, while testing negative to Porcine Epidemic diarrhea and Transmissible Gastroenteritis viruses. Following detection, viral isolation was performed using PK-15 and Vero cell lines. After 5 days of inoculation, no cytopathic effect was observed. A second blind passage allowed the observation of cytopathic effect on PK-15 cells, while it remained absent in Vero cells. A fluorescence test using an anti-N monoclonal antibody confirmed viral replication. One sample was processed for whole genome sequencing (NGS). In short, raw reads were imported into CLC genomics and assembled de novo. Out of 479k reads generated from the sample, 436k assembled into a 25501 bp contig which was 99.5% identical to a reference Porcine Deltacoronavirus strain from US within the North American phylogroup. Yet, there are relevant differences at the nucleotide and amino acid levels compared to previously described Porcine Deltacoronavirus strains. Altogether, our findings represent the first report of Porcine Deltacoronavirus in South America, its genomic characterization, which provides information of its evolutionary origin. Thus, this study offers new insights into the molecular epidemiology of Porcine Deltacoronavirus infections in the swine industry.


Author(s):  
Emiko Igarashi ◽  
Hideki Tani ◽  
Kosuke Tamura ◽  
Masae Itamochi ◽  
Takahisa Shimada ◽  
...  

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1430
Author(s):  
Vladimir Stevanovic ◽  
Irena Tabain ◽  
Tatjana Vilibic-Cavlek ◽  
Maja Mauric Maljkovic ◽  
Iva Benvin ◽  
...  

Over a year into the COVID-19 pandemic, there is growing evidence that SARS-CoV-2 infections among dogs are more common than previously thought. In this study, the prevalence of SARS-CoV-2 antibodies was investigated in two dog populations. The first group was comprised of 1069 dogs admitted to the Veterinary Teaching Hospital for any given reason. The second group included dogs that shared households with confirmed COVID-19 cases in humans. This study group numbered 78 dogs. In COVID-19 infected households, 43.9% tested ELISA positive, and neutralising antibodies were detected in 25.64% of dogs. Those data are comparable with the secondary attack rate in the human population. With 14.69% of dogs in the general population testing ELISA positive, there was a surge of SARS-CoV-2 infections within the dog population amid the second wave of the pandemic. Noticeably seroprevalence of SARS-CoV-2 in the dog and the human population did not differ at the end of the study period. Male sex, breed and age were identified as significant risk factors. This study gives strong evidence that while acute dog infections are mostly asymptomatic, they can pose a significant risk to dog health. Due to the retrospective nature of this study, samples for viral isolation and PCR were unavailable. Still, seropositive dogs had a 1.97 times greater risk for developing central nervous symptoms.


Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1379
Author(s):  
Sandra Barroso-Arévalo ◽  
Belén Rivera ◽  
Lucas Domínguez ◽  
José M. Sánchez-Vizcaíno

Natural SARS-CoV-2 infection in pets has been widely documented during the last year. Although the majority of reports suggested that dogs’ susceptibility to the infection is low, little is known about viral pathogenicity and transmissibility in the case of variants of concern, such as B.1.1.7 in this species. Here, as part of a large-scale study on SARS-CoV-2 prevalence in pets in Spain, we have detected the B.1.1.7 variant of concern (VOC) in a dog whose owners were infected with SARS-CoV-2. The animal did not present any symptoms, but viral loads were high in the nasal and rectal swabs. In addition, viral isolation was possible from both swabs, demonstrating that the dog was shedding infectious virus. Seroconversion occurred 23 days after the first sampling. This study documents the first detection of B.1.1.7 VOC in a dog in Spain and emphasizes the importance of performing active surveillance and genomic investigation on infected animals.


2021 ◽  
Vol 26 (14) ◽  
Author(s):  
Mina Park ◽  
Colleen Pawliuk ◽  
Tribesty Nguyen ◽  
Amanda Griffitt ◽  
Linda Dix-Cooper ◽  
...  

Introduction Standard testing for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is based on RT-PCR tests, but detection of viral genetic material alone does not indicate ongoing infectious potential. The ability to isolate whole virus represents a better proxy for infectivity. Aim The objective of this study was to gain an understanding of the current literature and compare the reported periods of positive SARS-CoV-2 detection from studies that conducted RT-PCR testing in addition to experiments isolating whole virus. Methods Using a rapid review approach, studies reporting empirical data on the duration of positive RT-PCR results and/or successful viral isolation following SARS-CoV-2 infection in humans were identified through searches of peer-reviewed and pre-print health sciences literature. Articles were screened for relevance, then data were extracted, analysed, and synthesised. Results Of the 160 studies included for qualitative analysis, 84% (n = 135) investigated duration of positive RT-PCR tests only, 5% (n = 8) investigated duration of successful viral isolations, while 11% (n = 17) included measurements on both. There was significant heterogeneity in reported data. There was a prolonged time to viral clearance when deduced from RT-PCR tests compared with viral isolations (median: 26 vs 9 days). Discussion Findings from this review support a minimum 10-day period of isolation but certain cases where virus was isolated after 10 days were identified. Given the extended time to viral clearance from RT-PCR tests, future research should ensure standard reporting of RT-PCR protocols and results to help inform testing policies aimed at clearance from isolation.


2021 ◽  
Vol 37 (1) ◽  
Author(s):  
Meenesh Juvekar ◽  
Baisali Sarkar

Abstract Background Coronavirus disease 2019 was first identified in Wuhan, the capital of China’s Hubei province, in December 2019. India has witnessed a massive surge of coronavirus cases. Main text This study details the measures to be taken by the clinicians involved in doing otorhinolaryngology and head neck surgery in light of the recent coronavirus disease 2019 pandemic. All COVID-positive patients should be admitted in a separate COVID ward, and patients should be screened for COVID-19 before admission. Only emergent ENT surgeries should be done in an operating room having a negative pressure environment with high-frequency air changes, and all staff must wear personal protective equipment. The anesthetist intubates the patient while the surgical team waits outside the operation theater post-intubation for 21 min. For otology surgery, double draping of the microscope should be done; for rhinology surgery, concept of negative-pressure otolaryngology viral isolation drape (NOVID) system should be used. Smoke evacuation system is set up inside the tent to evacuate any smoke produced during the surgery. Tracheostomy should be done at least after 10 days of mechanical ventilation with cuffed, non-fenestrated tracheal tube inserted through the tracheal window, and a separate closed suction system is used for suctioning. After the surgery is completed, disposal of PPE kit needs to be done according to local guidelines. After completion of the surgery, the full anesthesia unit should be disinfected for 2 h with 12 % hydrogen peroxide. Chlorine-containing disinfectant (2000 mg/L) is used to clean the floor of the operation theater and clean all the reusable medical equipment. Ultra-low volume 20 to 30 mL/m of 3% hydrogen peroxide is used to fumigate the OT for 2 h. Conclusions COVID-19 is a newly discovered infectious disease. Measures need to be taken to prevent transmission and attain a plateau and decline in the disease. Otorhinolaryngologists and head neck surgeons are at high risk of this infection. This review summarizes the protocol for otorhinolaryngologists and head neck surgeons caring for patients in this current scenario. Protocols need to be strictly followed to prevent the spread of this disease.


2021 ◽  
Vol 8 (1) ◽  
pp. e000830
Author(s):  
Souichi Yamada ◽  
Shuetsu Fukushi ◽  
Hitomi Kinoshita ◽  
Makoto Ohnishi ◽  
Tadaki Suzuki ◽  
...  

BackgroundAn outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19.MethodsWe developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan.ResultsThe SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus.ConclusionIn combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.


2021 ◽  
Vol 2 (2) ◽  
pp. 212-215
Author(s):  
S. A. Bida ◽  
F. K. Ramsey ◽  
C. O. Njoku ◽  
E. U. Eze ◽  
F. I. Eid

SHEEP pox in two northern states of Nigeria is reported. The disease produced nodules which became popular, vesicular and ulcerated on the skin. Lesions also developed in the respiratory, urogenital and alimentary organs The importance of the disease in animal production and its control are discussed. Viral isolation was not done but the disease was experimentally reproduced. Diagnosis was based on clinical history, experimental transmission and morphological examination


PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0239916
Author(s):  
Juscilânia Furtado Araújo ◽  
Alice Andrioli ◽  
Raymundo Rizaldo Pinheiro ◽  
Lucia Helena Sider ◽  
Ana Lídia Madeira de Sousa ◽  
...  

This study aimed to evaluate by means of Nested Polymerase Chain Reaction (nPCR), co-cultivation and sequencing, with genetic comparison between strains (mother/newborn), the occurrence of vertical transmission of Small Ruminant Lentiviruses (SRLV) from naturally occurring nannies infected for their offspring. For the detection of SRLV seropositive progenitors, blood was collected from 42 nannies in the final third of gestation in tubes with and without anticoagulant. The diagnostic tests used were Western Blot (WB) and nPCR. During the period of birth, the same blood collection procedure was performed on 73 newborns at zero hours of birth, with the same diagnostic tests. Seventeen blood samples from seven-day-old kids, proven positive for SRLV by nPCR, chosen at random, were subjected to coculture in goat synovial membrane (GSM) cells for 105 days. The pro-viral DNA extracted from the cell supernatant from the coculture was subjected to nPCR. For DNA sequencing from the nPCR products, nine positive samples were chosen at random, four nannies with their respective offspring, also positive. Each sample was performed in triplicate, thus generating 27 nPCR products of which only 19 were suitable for analysis. Among the 42 pregnant goats, in 50% (21/42) pro-viral DNA was detected by nPCR, while in the WB, only 7.14% (3/42) presented antibodies against SRLV. Regarding neonates, of the 73 kids, 34 (46.57%) were positive for the virus, using the nPCR technique, while in the serological test (WB), three positive animals (4.10%) were observed. The coculture of the 17 samples with a positive result in the nPCR was confirmed in viral isolation by amplification of the SRLV pro-viral DNA. When aligned, the pro-viral DNA sequences (nannies and their respective offspring) presented homology in relation to the standard strain CAEV Co. It was concluded that the transmission of SRLV through intrauterine route was potentially the source of infection in the newborn goats.


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