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An observational and retrospective study was carried out to analyse HCoV positivity from a multiplex PCR respiratory panel and RT-PCR for SARS-CoV-2 in respiratory samples from 1 June 2020 to 31 July 2023 at the Príncipe de Asturias University Hospital (HUPA) in Alcalá de Henares, Madrid, Spain. Out of 2802 respiratory panels, 1258 (44.8%) turned out positive. HCoV was detected in 114 (4%) cases (range 0-23; median 1.5; IQR 0-3.75) with positivity rates ranging from 0% to 14%. All four variants of HCoV circulated, and OC-43 was the most common in 62.3% of cases. After the onset of the pandemic, the HCoV season was delayed 22 weeks, with a peak positivity of 9% in the summer of 2021, showing an inverse relationship with the alpha and delta waves of SARS-CoV-2. In the two subsequent autumn-winter seasons, HCoV positivity increased (11-14%) with a reduction in the summer of 2022 and 2023 following the emergence of the omicron variant and the relaxation of social distancing measures. The seasonal spread pattern of endemic HCoV might be returning to normal in our region and likely in other temperate zones of the northern hemisphere after 3 years of the pandemic., (© 2024 The Author(s). APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

In 2020, Denmark buried approximately four million culled, farmed mink in mass graves treated with slaked lime due to widespread SARS-CoV-2 infections. After six months, environmental concerns prompted the exhumation of these cadavers. Our analysis encompassed visual inspections, soil pH measurements, and gas emission assessments of the grave environment. Additionally, we evaluated carcasses for decay status, cadaverine content, and the presence of various pathogens, including SARS-CoV-2 and mink coronavirus. Our findings revealed minimal microbial activity and limited carcass decomposition. Although viral RNA from SARS-CoV-2 and mink coronavirus, along with DNA from Aleutian mink disease virus, were detected, the absence of infectious SARS-CoV-2 in cell culture assays suggests slow natural degradation processes. This study provides critical insights for future considerations in managing mass burial scenarios during outbreaks of livestock-associated zoonotic pathogens., (© 2024. The Author(s).)

Background: The aim of this study was to characterize the epidemiology of human seasonal coronaviruses (HCoVs) in southern Malawi., Methods: We tested for HCoVs 229E, OC43, NL63, and HKU1 using real-time polymerase chain reaction (PCR) on upper respiratory specimens from asymptomatic controls and individuals of all ages recruited through severe acute respiratory illness (SARI) surveillance at Queen Elizabeth Central Hospital, Blantyre, and a prospective influenza-like illness (ILI) observational study between 2011 and 2017. We modeled the probability of having a positive PCR for each HCoV using negative binomial models, and calculated pathogen-attributable fractions (PAFs)., Results: Overall, 8.8% (539/6107) of specimens were positive for ≥1 HCoV. OC43 was the most frequently detected HCoV (3.1% [191/6107]). NL63 was more frequently detected in ILI patients (adjusted incidence rate ratio [aIRR], 9.60 [95% confidence interval {CI}, 3.25-28.30]), while 229E (aIRR, 8.99 [95% CI, 1.81-44.70]) was more frequent in SARI patients than asymptomatic controls. In adults, 229E and OC43 were associated with SARI (PAF, 86.5% and 89.4%, respectively), while NL63 was associated with ILI (PAF, 85.1%). The prevalence of HCoVs was similar between children with SARI and controls. All HCoVs had bimodal peaks but distinct seasonality., Conclusions: OC43 was the most prevalent HCoV in acute respiratory illness of all ages. Individual HCoVs had distinct seasonality that differed from temperate settings., Competing Interests: Potential conflicts of interest. J. M. R. has received funding from UK Research and Innovation (UKRI) (MR/V038613/1). A. H. was supported by a Wellcome Trust Clinical PhD Fellowship (097464) and receives funding from UKRI, the Medical Research Council, British Society for Antimicrobial Chemotherapy, Wellcome Trust, and the Medical Research Foundation, unrelated to this work. S. A. was supported by a Wellcome Trust Clinical PhD Fellowship (099962). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Introduction: Bats are natural reservoirs of coronaviruses ( Coronaviridae ), which have caused three outbreaks of human disease SARS, MERS and COVID-19 or SARS-2 over the past decade. The purpose of the work is to study the diversity of coronaviruses among bats inhabiting the foothills and mountainous areas of the Republics of Dagestan, Altai and the Kemerovo region., Materials and Methods: Samples of bat oral swabs and feces were tested for the presence of coronavirus RNA by reverse transcription-polymerase chain reaction (RT-PCR)., Results: It has been shown that the greater horseshoe bats ( Rhinolophus ferrumequinum ), inhabiting the Republic of Dagestan, are carriers of two different coronaviruses. One of the two coronaviruses is a member of the Sarbecovius subgenus of the Betacoronavirus genus, which includes the causative agents of SARS and COVID-19. The second coronavirus is assigned to the Decacovirus subgenus of the Alphacoronavirus genus and is most similar to viruses identified among Rhinolophus spp. from European and Middle Eastern countries. In the Altai Republic and Kemerovo region, coronaviruses belonging to the genus Alphacoronavirus , subgenus Pedacovirus , were found in the smooth-nosed bats: Ikonnikov`s bat ( Myotis ikonnikovi ) and the eastern bat ( Myotis petax ). The virus from the Altai Republic from M. ikonnikovi is close to viruses from Japan and Korea, as well as viruses from Myotis spp. from European countries. The virus from the Kemerovo region from M. petax groups with coronaviruses from Myotis spp. from Asian countries and is significantly different from coronaviruses previously discovered in the same natural host.

Coronaviruses have been confirmed to infect a variety of species, but only one case of associated winter dysentery of European bison has been described. The study aimed to analyze the prevalence, and define the impact on the species conservation, the source of coronavirus infection, and the role of the European bison in the transmission of the pathogen in Poland. Molecular and serological screening was performed on 409 European bison from 6 free-ranging and 14 captive herds over the period of 6 years (2017-2023). Presence of coronavirus was confirmed in one nasal swab by pancoronavirus RT-PCR and in 3 nasal swab samples by bovine coronavirus (BCoV) specific real time RT-PCR. The detected virus showed high (> 98%) homology in both RdRp and Spike genes to BCoV strains characterised recently in Polish cattle and strains isolated from wild cervids in Italy. Antibodies specific to BCoV were found in 6.4% of tested samples, all originating from free-ranging animals. Seroprevalence was higher in adult animals over 5 years of age (p = 0.0015) and in females (p = 0.09). Our results suggest that European bison play only a limited role as reservoirs of bovine-like coronaviruses. Although the most probable source of infections in the European bison population in Poland is cattle, other wild ruminants could also be involved. In addition, the zoonotic potential of bovine coronaviruses is quite low., (© 2024. The Author(s).)

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/μL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi., Competing Interests: Declaration of competing interest Hechao Zhu, Geng Wang, Teng Yu, Weichao Zhang and Lumei Ni are employees of Guangxi Yangxiang Co., LTD. The remaining authors declared that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Rapid and accurate sequencing of the entire viral genome, coupled with continuous monitoring of genetic changes, is crucial for understanding the epidemiology of coronaviruses. We designed a novel method called micro target hybrid capture system (MT-Capture) to enable whole-genome sequencing in a timely manner. The novel design of probes used in target binding exhibits a unique and synergistic "hand-in-hand" conjugation effect. The entire hybrid capture process is within 2.5 hours, overcoming the time-consuming and complex operation characteristics of the traditional liquid-phase hybrid capture (T-Capture) system. By designing specific probes for these coronaviruses, MT-Capture effectively enriched isolated strains and 112 clinical samples of coronaviruses with cycle threshold values below 37. Compared to multiplex PCR sequencing, it does not require frequent primer updates and has higher compatibility. MT-Capture is highly sensitive and capable of tracking variants.IMPORTANCEMT-Capture is meticulously designed to enable the efficient acquisition of the target genome of the common human coronavirus. Coronavirus is a kind of virus that people are generally susceptible to and is epidemic and infectious, and it is the virus with the longest genome among known RNA viruses. Therefore, common human coronavirus samples are selected to evaluate the accuracy and sensitivity of MT-Capture. This method utilizes innovative probe designs optimized through probe conjugation techniques, greatly shortening the time and simplifying the handwork compared with traditional hybridization capture processes. Our results demonstrate that MT-Capture surpasses multiplex PCR in terms of sensitivity, exhibiting a thousandfold increase. Moreover, MT-Capture excels in the identification of mutation sites. This method not only is used to target the coronaviruses but also may be used to diagnose other diseases, including various infectious diseases, genetic diseases, or tumors., Competing Interests: The authors declare no conflict of interest.

Emerging coronaviruses (CoVs) are understood to cause critical human and domestic animal diseases; the spillover from wildlife reservoirs can result in mild and severe respiratory illness in humans and domestic animals and can spread more readily in these naïve hosts. A low-cost CoV molecular method that can detect a variety of CoVs from humans, animals, and environmental specimens is an initial step to ensure the early identification of known and new viruses. We examine a collection of 50 human, 46 wastewater, 28 bat, and 17 avian archived specimens using 3 published pan-CoV PCR assays called Q-, W-, and X-CoV PCR, to compare the performance of each assay against four CoV genera. X-CoV PCR can detect all four CoV genera, but Q- and W-CoV PCR failed to detect δ-CoV. In total, 21 (42.0%), 9 (18.0%), and 21 (42.0%) of 50 human specimens and 30 (65.22%), 6 (13.04%), and 27 (58.70%) of 46 wastewater specimens were detected using Q-, W-, and X-CoV PCR assays, respectively. The X-CoV PCR assay has a comparable sensitivity to Q-CoV PCR in bat CoV detection. Combining Q- and X-CoV PCR assays can increase sensitivity and avoid false negative results in the early detection of novel CoVs., Competing Interests: The authors declare no conflicts of interest.

Bats are at the origin of human coronaviruses, either directly or via an intermediate host. We tested swabs from 4597 bats (897 from the Democratic Republic of Congo (DRC), 2191 from Cameroon and 1509 from Guinea) with a broadly reactive PCR in the RdRp region. Coronaviruses were detected in 903 (19.6%) bats and in all species, with more than 25 individuals tested. The highest prevalence was observed in Eidolon helvum (239/733; 39.9%) and Rhinolophus sp. (306/899; 34.1%), followed by Hipposideros sp. (61/291; 20.9%). Frugivorous bats were predominantly infected with beta coronaviruses from the Nobecovirus subgenus (93.8%), in which at least 6 species/genus-specific subclades were observed. In contrast, insectivorous bats were infected with beta-coronaviruses from different subgenera (Nobecovirus (8.5%), Hibecovirus (32.8%), Merbecovirus (0.5%) and Sarbecovirus (57.6%)) and with a high diversity of alpha-coronaviruses. Overall, our study shows a high prevalence and genetic diversity of coronaviruses in bats and illustrates that Rhinolophus bats in Africa are infected at high levels with the Sarbecovirus subgenus, to which SARS-CoV-2 belongs. It is important to characterize in more detail the different coronavirus lineages from bats for their potential to infect human cells, their evolution and to study frequency and modes of contact between humans and bats in Africa.

Background: In the context of COVID-19 pandemic in Catalonia (Spain), the present study analyses respiratory samples collected by the primary care network using Acute Respiratory Infections Sentinel Surveillance System (PIDIRAC) during the 2019-2020 season to complement the pandemic surveillance system in place to detect SARS-CoV-2. The aim of the study is to describe whether SARS-CoV-2 was circulating before the first confirmed case was detected in Catalonia, on February 25th, 2020., Methods: The study sample was made up of all samples collected by the PIDIRAC primary care network as part of the Influenza and Acute Respiratory Infections (ARI) surveillance system activities. The study on respiratory virus included coronavirus using multiple RT-PCR assays. All positive samples for human coronavirus were subsequently typed for HKU1, OC43, NL63, 229E. Every respiratory sample was frozen at-80°C and retrospectively studied for SARS-CoV-2 detection. A descriptive study was performed, analysing significant differences among variables related to SARS-CoV- 2 cases comparing with rest of coronaviruses cases through a bivariate study with Chi-squared test and statistical significance at 95%., Results: Between October 2019 and April 2020, 878 respiratory samples from patients with acute respiratory infection or influenza syndrome obtained by PIDIRAC were analysed. 51.9% tested positive for influenza virus, 48.1% for other respiratory viruses. SARS-CoV-2 was present in 6 samples. The first positive SARS-CoV-2 case had symptom onset on 2 March 2020. These 6 cases were 3 men and 3 women, aged between 25 and 50 years old. 67% had risk factors, none had previous travel history nor presented viral coinfection. All of them recovered favourably., Conclusion: Sentinel Surveillance PIDIRAC enhances global epidemiological surveillance by allowing confirmation of viral circulation and describes the epidemiology of generalized community respiratory viruses' transmission in Catalonia. The system can provide an alert signal when identification of a virus is not achieved in order to take adequate preparedness measures., Competing Interests: The author declares that neither they nor any of the co-authors have a conflict of interest regarding the publication of this manuscript.

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands)., Competing Interests: The authors have declared that no competing interests exist.

Mammal-associated coronaviruses have a long evolutionary history across global bat populations, which makes them prone to be the most likely ancestral origins of coronavirus-associated epidemics and pandemics globally. Limited coronavirus research has occurred at the junction of Europe and Asia, thereby investigations in Georgia are critical to complete the coronavirus diversity map in the region. We conducted a cross-sectional coronavirus survey in bat populations at eight locations of Georgia, from July to October of 2014. We tested 188 anal swab samples, remains of previous pathogen discovery studies, for the presence of coronaviruses using end-point pan-coronavirus RT-PCR assays. Samples positive for a 440 bp amplicon were Sanger sequenced to infer coronavirus subgenus or species through phylogenetic reconstructions. Overall, we found a 24.5% positive rate, with 10.1% for Alphacoronavirus and 14.4% for Betacoronavirus . Albeit R. euryale , R. ferrumequinum , M. blythii and M. emarginatus were found infected with both CoV genera, we could not rule out CoV co-infection due to limitation of the sequencing method used and sample availability. Based on phylogenetic inferences and genetic distances at nucleotide and amino acid levels, we found one putative new subgenus and three new species of Alphacoronavirus , and two new species of Betacoronavirus .

Guinea fowl fulminating enteritis has been reported in France since the 1970s. In 2014, a coronavirus was identified and appeared as a possible viral pathogen involved in the disease. In the present study, intestinal content from a guinea fowl involved in a new case of the disease in 2017 was analysed by deep sequencing, revealing the presence of a guinea fowl coronavirus (GfCoV) and a picornavirus (GfPic). Serial passage assays into the intra-amniotic cavity of 13-day-old specific pathogen-free chicken eggs and 20-day-old conventional guinea fowl eggs were attempted. In chicken eggs, isolation assays failed, but in guinea fowl eggs, both viruses were successfully obtained. Furthermore, two GfCoV and two GfPic isolates were obtained from the same bird but from different sections of its intestines. This shows that using eggs of the same species, in which the virus has been detected, can be the key for successful isolation. The consensus sequence of the full-length genomes of both GfCoV isolates was highly similar, and correlated to those previously described in terms of genome organization, ORF length and phylogenetic clustering. According to full-length genome analysis and the structure of the Internal Ribosome Entry Site, both GfPic isolates belong to the Anativirus genus and specifically the species Anativirus B . The availability of the first isolates of GfCoV and GfPic will now provide a means of assessing their pathogenicity in guinea fowl in controlled experimental conditions and to assess whether they are primary viral pathogens of the disease "guinea fowl fulminating enteritis". RESEARCH HIGHLIGHTS First isolation of guinea fowl coronaviruses and picornaviruses.Eggs homologous to the infected species are key for isolation.Isolates available to precisely evaluate the virus roles in fulminating enteritis.First full-length genome sequences of guinea fowl picornaviruses.

Coronavirus disease 2019 (COVID-19), caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China, and rapidly spread throughout the world. This newly emerging pathogen is highly transmittable and can cause fatal disease. More than 35 million cases have been confirmed, with a fatality rate of about 2.9% to October 9, 2020. However, the original and intermediate hosts of SARS-CoV-2 remain unknown. Here, 3160 poultry samples collected from 14 provinces of China between September and December 2019 were tested for SARS-CoV-2 infection. All the samples were SARS-CoV-2 negative, but 593 avian coronaviruses were detected, including 485 avian infectious bronchitis viruses, 72 duck coronaviruses, and 36 pigeon coronaviruses, with positivity rates of 15.35%, 2.28%, and 1.14%, respectively. Our surveillance demonstrates the diversity of avian coronaviruses in China, with higher prevalence rates in some regions. Furthermore, the possibility that SARS-CoV-2 originated from a known avian-origin coronavirus can be preliminarily ruled out. More surveillance of and research into avian coronaviruses are required to better understand the diversity, distribution, cross-species transmission, and clinical significance of these viruses., (Copyright © 2021. Published by Elsevier B.V.)

Bats are an important reservoir of several zoonotic diseases. However, the circulation of bat coronaviruses (BatCoV) in live animal markets in Indonesia has not been reported. Genetic characterization of BatCoV was performed by sequencing partial RdRp genes. Real-time polymerase chain reaction based on nucleocapsid protein (N) gene and Enzyme-linked immunosorbent assay against the N protein were conducted to detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA and antibody, respectively. We identified the presence of BatCoV on Cynopterus brachyotis , Macroglossus minimus , and Rousettus amplexicaudatus . The results showed that the BatCoV included in this study are from an unclassified coronavirus group. Notably, SARS-CoV-2 viral RNA and antibodies were not detected in the sampled bats., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Korean Society of Veterinary Science.)

Recirculating air purification technologies are employed as potential means of reducing exposure to aerosol particles and airborne viruses. Toward improved testing of recirculating air purification units, we developed and applied a medium-scale single-pass wind tunnel test to examine the size-dependent collection of particles and the collection and inactivation of viable bovine coronavirus (BCoV, a betacoronavirus), porcine respiratory coronavirus (PRCV, an alphacoronavirus), and influenza A virus (IAV), by a commercial air purification unit. The tested unit, the Molekule Air Mini, incorporates a MERV 16 filter as well as a photoelectrochemical oxidating layer. It was found to have a collection efficiency above 95.8% for all tested particle diameters and flow rates, with collection efficiencies above 99% for supermicrometer particles with the minimum collection efficiency for particles smaller than 100 nm. For all three tested viruses, the physical tracer-based log reduction was near 2.0 (99% removal). Conversely, the viable virus log reductions were found to be near 4.0 for IAV, 3.0 for BCoV, and 2.5 for PRCV, suggesting additional inactivation in a virus family- and genus-specific manner. In total, this work describes a suite of test methods which can be used to rigorously evaluate the efficacy of recirculating air purification technologies., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

To investigate the presence of respiratory viruses in the middle ear cavity of the individuals with a healthy middle ear and the children with otitis media with effusion (OME). A total of 72 middle ear samples were collected from 25 children with OME (Group 1) and 47 individuals with no middle ear disease (Group 2). Multiplex real-time polymerase chain reaction was used to investigate the presence of 20 different respiratory viruses. Virus results were compared with bacteriomes of the same populations. At least one respiratory virus was detected in 56% of the patients in Group 1 and 12.8% of the individuals in Group 2. The viral co-infection rate for Group 1 and 2 was 8% and 2.1%, respectively. In Group 1, adenovirus was the most frequently detected virus with a rate of 24%, either alone (16%) or concurrent with other viruses (8%), followed by influenza B (12%), rhinovirus, and bocavirus (8%) each. Parainfluenza 4, coronavirus OC43, and RSV A/B were detected in 4% of the sample each. In Group 2, rhinovirus was detected in two samples (4.3%) followed by adenovirus, coronavirus OC43, coronavirus E299, and coronavirus NL63 with a rate of 2.1% each. The detection rate of respiratory viruses was significantly higher in children aged 6 to 11 years. There was no positive association between virus and bacteria found in the middle ear cavity. The current study has provided comprehensive data indicating the presence of diverse respiratory viruses in the healthy middle ear cavity. Our results also suggest that respiratory viruses might have a contribution to OME pathogenesis., (© 2021 Wiley Periodicals LLC.)

Background: Coronaviruses can be isolated from bats, civets, pangolins, birds and other wild animals. As an animal-origin pathogen, coronavirus can cross species barrier and cause pandemic in humans. In this study, a deep learning model for early prediction of pandemic risk was proposed based on the sequences of viral genomes., Methods: A total of 3257 genomes were downloaded from the Coronavirus Genome Resource Library. We present a deep learning model of cross-species coronavirus infection that combines a bidirectional gated recurrent unit network with a one-dimensional convolution. The genome sequence of animal-origin coronavirus was directly input to extract features and predict pandemic risk. The best performances were explored with the use of pre-trained DNA vector and attention mechanism. The area under the receiver operating characteristic curve (AUROC) and the area under precision-recall curve (AUPR) were used to evaluate the predictive models., Results: The six specific models achieved good performances for the corresponding virus groups (1 for AUROC and 1 for AUPR). The general model with pre-training vector and attention mechanism provided excellent predictions for all virus groups (1 for AUROC and 1 for AUPR) while those without pre-training vector or attention mechanism had obviously reduction of performance (about 5-25%). Re-training experiments showed that the general model has good capabilities of transfer learning (average for six groups: 0.968 for AUROC and 0.942 for AUPR) and should give reasonable prediction for potential pathogen of next pandemic. The artificial negative data with the replacement of the coding region of the spike protein were also predicted correctly (100% accuracy). With the application of the Python programming language, an easy-to-use tool was created to implements our predictor., Conclusions: Robust deep learning model with pre-training vector and attention mechanism mastered the features from the whole genomes of animal-origin coronaviruses and could predict the risk of cross-species infection for early warning of next pandemic., (© 2021. The Author(s).)

The COVID-19 pandemic highlighted the importance of developing surfaces and coatings with antiviral activity. Here, we present, for the first time, peptide-based assemblies that can kill viruses. The minimal inhibitory concentration (MIC) of the assemblies is in the range tens of micrograms per milliliter. This value is 2 orders of magnitude smaller than the MIC of metal nanoparticles. When applied on a surface, by drop casting, the peptide spherical assemblies adhere to the surface and form an antiviral coating against both RNA- and DNA-based viruses including coronavirus. Our results show that the coating reduced the number of T4 bacteriophages (DNA-based virus) by 3 log, compared with an untreated surface and 6 log, when compared with a stock solution. Importantly, we showed that this coating completely inactivated canine coronavirus (RNA-based virus). This peptide-based coating can be useful wherever sterile surfaces are needed to reduce the risk of viral transmission.

Despite the discovery of animal coronaviruses related to SARS-CoV-2, the evolutionary origins of this virus are elusive. We describe a meta-transcriptomic study of 411 bat samples collected from a small geographical region in Yunnan province, China, between May 2019 and November 2020. We identified 24 full-length coronavirus genomes, including four novel SARS-CoV-2-related and three SARS-CoV-related viruses. Rhinolophus pusillus virus RpYN06 was the closest relative of SARS-CoV-2 in most of the genome, although it possessed a more divergent spike gene. The other three SARS-CoV-2-related coronaviruses carried a genetically distinct spike gene that could weakly bind to the hACE2 receptor in vitro. Ecological modeling predicted the co-existence of up to 23 Rhinolophus bat species, with the largest contiguous hotspots extending from South Laos and Vietnam to southern China. Our study highlights the remarkable diversity of bat coronaviruses at the local scale, including close relatives of both SARS-CoV-2 and SARS-CoV., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Respiratory viral infections (RVIs) affect children year-round, with seasonal-specific patterns. Pediatric oncology patients are uniquely vulnerable to infection, but whether this predisposes them to different patterns of RVIs than healthy children is unknown. There is also limited data on the impact of RVIs on cancer patients. We conducted a retrospective study of children ages 1-21 with cancer presenting to the clinic and emergency department (ED) and a randomly selected subset of patients without cancer presenting to the ED who had positive nasopharyngeal viral polymerase chain reactions at our institution from 2014 to 2019. Sixty-seven cancer patients (206 RVI episodes) and 225 pediatric non-cancer patients (237 RVI episodes) were included. Human rhino/enterovirus (HRE) was the most common infection in both groups in the spring, summer, and fall. In the winter, the most common RVI was influenza in cancer patients verses respiratory syncytial virus in non-cancer patients. On age-adjusted analysis, the likelihood of detecting coronavirus in the winter, HRE in the spring and fall, and parainfluenza in the summer was significantly greater in cancer patients (OR = 2.60, 2.52, 5.73, 3.59 respectively). Among cancer RVI episodes, 50% received parenteral antibiotics, 22% were severely neutropenic, 22% had chemotherapy delays for a median of six days, 16% were hospitalized, and 6% received intravenous immunoglobulin. We conclude that there are differences in the seasonal patterns of RVIs between children with and without cancer. RVIs also cause significant morbidity in children with cancer.

Endemic human coronaviruses (HCoV) are capable of causing a range of diseases from the common cold to pneumonia. We evaluated the epidemiology and seasonality of endemic HCoVs in children hospitalized with clinical pneumonia and among community controls living in countries with a high HIV burden, namely South Africa and Zambia, between August 2011 to October 2013. Nasopharyngeal/oropharyngeal swabs were collected from all cases and controls and tested for endemic HCoV species and 12 other respiratory viruses using a multiplex real-time PCR assay. We found that the likelihood of detecting endemic HCoV species was higher among asymptomatic controls than cases (11% vs. 7.2%; 95% CI: 1.2-2.0). This was however only observed among children > 6 months and was mainly driven by the Betacoronavirus endemic species (HCoV-OC43 and -HKU1). Endemic HCoV species were detected through the year; however, in Zambia, the endemic Betacoronavirus species tended to peak during the winter months (May-August). There was no association between HIV status and endemic HCoV detection.

Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 10 4 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.

Human coronaviruses, including SARS-CoV-2, are known to spread mainly via close contact and respiratory droplets. However, other potential means of transmission may be present. Fomite-mediated transmission occurs when viruses are deposited onto a surface and then transfer to a subsequent individual. Surfaces can become contaminated directly from respiratory droplets or from a contaminated hand. Due to mask mandates in many countries around the world, the former is less likely. Hands can become contaminated if respiratory droplets are deposited on them (i.e., coughing or sneezing) or through contact with fecal material where human coronaviruses (HCoVs) can be shed. The focus of this paper is on whether human coronaviruses can transfer efficiently from contaminated hands to food or food contact surfaces. The surfaces chosen were: stainless steel, plastic, cucumber and apple. Transfer was first tested with cellular maintenance media and three viruses: two human coronaviruses, 229E and OC43, and murine norovirus-1, as a surrogate for human norovirus. There was no transfer for either of the human coronaviruses to any of the surfaces. Murine norovirus-1 did transfer to stainless steel, cucumber and apple, with transfer efficiencies of 9.19%, 5.95% and 0.329%, respectively. Human coronavirus OC43 transfer was then tested in the presence of fecal material, and transfer was observed for stainless steel (0.52%), cucumber (19.82%) and apple (15.51%) but not plastic. This study indicates that human coronaviruses do not transfer effectively from contaminated hands to contact surfaces without the presence of fecal material.

The SARS-CoV-2 coronavirus is the focus of attention as it has caused more than three million human deaths globally. This and other coronaviruses, such as MERS-CoV, have been suggested to be related to coronaviruses that are hosted in bats. This work shows, through a bibliographic review, the frequency of detection of coronavirus in bats species of the Americas. The presence of coronavirus in bats has been examined in 25 investigations in 11 countries of the Americas between 2007 and 2020. Coronaviruses have been explored in 9371 individuals from 160 species of bats, and 187 coronavirus sequences have been deposited in GenBank distributed in 43 species of bats. While 91% of the coronaviruses sequences identified infect a single species of bat, the remainder show a change of host, dominating the intragenera change. So far, only Mex-CoV-6 is related to MERS-CoV, a coronavirus pathogenic for humans, so further coronavirus research effort in yet unexplored bat species is warranted.

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 alpha- and 6 betacoronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance., Competing Interests: Metabiota (https://metabiota.com), Labyrinth Global Health (https://www.labyrinthgh.com), and Mosaic (https://mosaic.cm) are private contractors who receive funds from international donor organizations to conduct technical assistance and research. Metabiota employs or employed CK, FM, PMK, INL, FNK, JAL, PM, MM, UT, AG, BSS, DJM, JAA, CM, DOJ, KS, NDW, EMR, and CEL, Mosaic employs ML, and Labyrinth Global Health employs KS, MM and CEL. There are no patents, products in development or marketed products associated with this research to declare. The association with commercial entities that some of the authors have does not alter our adherence to PLOS ONE policies on sharing data and materials.

The pandemic caused by SARS-CoV-2 has had a significant impact on the whole world. In a theory of the origin of SARS-CoV-2, pangolins are considered as a potential intermediate host. To assemble the genome of suspicious coronavirus (CoV) found in pangolins, SARS-CoV-2 was used as a reference in most of the previous studies, implicitly assuming the pangolin CoV and SARS-CoV-2 are the closest neighbors in evolution. However, this assumption may not be true. We investigated how the choice of reference genome affected the resulting CoV genome assembly. We explored various representative CoVs as the reference genome, and found significant differences in the resulting assemblies. The assembly obtained using RaTG13 as a reference showed better statistics in total length, N50, and pairwise distance reconstruction (PDR) scores than the assembly guided by SARS-CoV-2, indicating that RaTG13 may be a better reference. Therefore, RaTG13 should also be considered as a reference for assembling suspicious CoV found in pangolins and other potential intermediate hosts.

Outbreaks of severe virus infections with the potential to cause global pandemics are increasingly concerning. One type of those commonly emerging and re-emerging pathogens are coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2). Wild animals are hosts of different coronaviruses with the potential risk of cross-species transmission. However, little is known about the reservoir and host of coronaviruses in wild animals in Qinghai Province, where has the greatest biodiversity among the world's high-altitude regions. Here, from the next-generation sequencing data, we obtained a known beta-coronavirus (beta-CoV) genome and a novel delta-coronavirus (delta-CoV) genome from faecal samples of 29 marmots, 50 rats and 25 birds in Yushu Tibetan Autonomous Prefecture, Qinghai Province, China in July 2019. According to the phylogenetic analysis, the beta-CoV shared high nucleotide identity with Coronavirus HKU24. Although the novel delta-CoV (MtCoV) was closely related to Sparrow deltacoronavirus ISU42824, the protein spike of the novel delta-CoV showed highest amino acid identity to Sparrow coronavirus HKU17 (73.1%). Interestingly, our results identified a novel host (Montifringilla taczanowskii) for the novel delta-CoV and the potential cross-species transmission. The most recent common ancestor (tMRCA) of MtCoVs along with other closest members of the species of Coronavirus HKU15 was estimated to be 289 years ago. Thus, this study increases our understanding of the genetic diversity of beta-CoVs and delta-CoVs, and also provides a new perspective of the coronavirus hosts.

Data about the diagnostic efficiency of bilateral bronchoalveolar lavage (BAL) samples and endotracheal aspirates (EA) testing for common viral respiratory infections are scarce. We analyzed data from 167 cases, where bilateral BAL samples were tested, and from 101 cases, where BAL samples and EA were tested. Multiplex polymerase chain reaction (PCR) was performed with the fast track diagnostics viral respiratory panel, producing data on the adenovirus, coronavirus, enterovirus, human metapneumovirus, bocavirus, influenza virus, parainfluenza virus, rhinovirus, and respiratory syncytial virus status of patients with respiratory disease symptoms. In the bilateral BAL cohort, 46 (27.5%) cases were positive for at least one of the viruses mentioned above in both samples. Discrepant results (virus not detected on one side) were seen in six (3.6%) cases. In the BAL versus EA cohort, 12 (11.9%) cases were positive in both materials, discrepant results (only one material being positive) were observed in 11 (10.9%) cases, with seven (63.6%) BAL samples, and four (36.4%) EA being positive. Bilateral sampling does not significantly improve the diagnostic efficiency of BAL for the detection of common respiratory viral pathogens via PCR. The diagnostic quality of EA and BAL samples for the detection of common viral respiratory pathogens is comparable., (© 2020 Wiley Periodicals LLC.)

Rapid, sensitive, and precise multiplexed assays for serological analysis during candidate COVID-19 vaccine development would streamline clinical trials. The VaxArray Coronavirus (CoV) SeroAssay quantifies IgG antibody binding to 9 pandemic, potentially pandemic, and endemic human CoV spike antigens in 2 h with automated results analysis. IgG antibodies in serum bind to the CoV spike protein capture antigens printed in a microarray format and are labeled with a fluorescent anti-species IgG secondary label. The assay demonstrated excellent lower limits of quantification ranging from 0.3 to 2.0 ng/mL and linear dynamic ranges of 76 to 911-fold. Average precision of 11 % CV and accuracy (% recovery) of 92.5 % over all capture antigens were achieved over 216 replicates representing 3 days and 3 microarray lots. Clinical performance on 263 human serum samples (132 SARS-CoV-2 negatives and 131 positives based on donor-matched RT-PCR and/or date of collection) produced 98.5 % PPA and 100 % NPA., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: SARS-CoV-2 infection can present with a broad clinical differential that includes many other respiratory viruses; therefore, accurate tests are crucial to distinguish true COVID-19 cases from pathogens that do not require urgent public health interventions. Co-circulation of other respiratory viruses is largely unknown during the COVID-19 pandemic but would inform strategies to rapidly and accurately test patients with respiratory symptoms., Methods: This study retrospectively examined 298,415 respiratory specimens collected from symptomatic patients for SARS-CoV-2 testing in the three months since COVID-19 was initially documented in the province of Alberta, Canada (March-May, 2020). By focusing on 52,285 specimens that were also tested with the Luminex Respiratory Pathogen Panel for 17 other pathogens, this study examines the prevalence of 18 potentially co-circulating pathogens and their relative rates in prior years versus since COVID-19 emerged, including four endemic coronaviruses., Results: SARS-CoV-2 was identified in 2.2% of all specimens. Parallel broad multiplex testing detected additional pathogens in only 3.4% of these SARS-CoV-2-positive specimens: significantly less than in SARS-CoV-2-negative specimens (p < 0.0001), suggesting very low rates of SARS-CoV-2 co-infection. Furthermore, the overall co-infection rate was significantly lower among specimens with SARS-CoV-2 detected (p < 0.0001). Finally, less than 0.005% of all specimens tested positive for both SARS-CoV-2 and any of the four endemic coronaviruses tested, strongly suggesting neither co-infection nor cross-reactivity between these coronaviruses., Conclusions: Broad respiratory pathogen testing rarely detected additional pathogens in SARS-CoV-2-positive specimens. While helpful to understand co-circulation of respiratory viruses causing similar symptoms as COVID-19, ultimately these broad tests were resource-intensive and inflexible in a time when clinical laboratories face unprecedented demand for respiratory virus testing, with further increases expected during influenza season. A transition from broad, multiplex tests toward streamlined diagnostic algorithms targeting respiratory pathogens of public health concern could simultaneously reduce the overall burden on clinical laboratories while prioritizing testing of pathogens of public health importance. This is particularly valuable with ongoing strains on testing resources, exacerbated during influenza seasons.

Background: A novel coronavirus (SARS-CoV-2) emerging has put global public health institutes on high alert. Little is known about the epidemiology and clinical characteristics of human coronaviruses infections in relation to infections with other respiratory viruses., Methods: From February 2017 to December 2019, 3660 respiratory samples submitted to Zhejiang Children Hospital with acute respiratory symptoms were tested for four human coronaviruses RNA by a novel two-tube multiplex reverse transcription polymerase chain reaction assays. Samples were also screened for the occurrence of SARS-CoV-2 by reverse transcription-PCR analysis., Results: Coronavirus RNAs were detected in 144 (3.93%) specimens: HCoV-HKU1 in 38 specimens, HCoV-NL63 in 62 specimens, HCoV-OC43 in 38 specimens and HCoV-229E in 8 specimens. Genomes for SARS-CoV-2 were absent in all specimens by RT-PCR analysis during the study period. The majority of HCoV infections occurred during fall months. No significant differences in gender, sample type, year were seen across species. 37.5 to 52.6% of coronaviruses detected were in specimens testing positive for other respiratory viruses. Phylogenic analysis identified that Zhejiang coronaviruses belong to multiple lineages of the coronaviruses circulating in other countries and areas., Conclusion: Common HCoVs may have annual peaks of circulation in fall months in the Zhejiang province, China. Genetic relatedness to the coronaviruses in other regions suggests further surveillance on human coronaviruses in clinical samples are clearly needed to understand their patterns of activity and role in the emergence of novel coronaviruses.

PCR Single-Strand Conformation Polymorphism is a method used to identify and detect mutations and is now well known for its many applications on living beings. This paper will discuss the experimental details, limitations and sensitivity of the PCR Single-Strand Conformation Polymorphism method in relation to all existing literature available to us until today. Genomic DNA extraction, PCR amplification and Single-Strand Conformation Polymorphism conditions (concentration of polyacrylamide slab gel electrophoresis, dissociation treatment of double- stranded DNA) and comparison with PCR Restriction Fragment Length Polymorphism are presented. Since its discovery in 1989, there have been many variations, innovations, and modifications of the method, which makes it very easy, safe, fast and for this reason widely applied in clinical diagnostic, forensic medicine, biochemical, veterinary, microbiological, food and environmental laboratories. One of the possible applications of the method is the diagnosis and identification of mutations in new strains of coronaviruses, because science needs more tools to tackle the problem of this pandemic. The PCR Single-Strand Conformation Polymorphism method can be applied in many cases provided that control samples are available and the required conditions of the method are achieved.

Coronavirus (CoV) spillover events from wildlife reservoirs can result in mild to severe human respiratory illness. These spillover events underlie the importance of detecting known and novel CoVs circulating in reservoir host species and determining CoV prevalence and distribution, allowing improved prediction of spillover events or where a human-reservoir interface should be closely monitored. To increase the likelihood of detecting all circulating genera and strains, we have modified primers published by Watanabe et al. in 2010 to generate a semi-nested pan-CoV PCR assay. Representatives from the four coronavirus genera (α-CoVs, β-CoVs, γ-CoVs and δ-CoVs) were tested and all of the in-house CoVs were detected using this assay. After comparing both assays, we found that the updated assay reliably detected viruses in all genera of CoVs with high sensitivity, whereas the sensitivity of the original assay was lower. Our updated PCR assay is an important tool to detect, monitor and track CoVs to enhance viral surveillance in reservoir hosts.

The current global fight against coronavirus disease (COVID-19) to flatten the transmission curve is put forth by the World Health Organization (WHO) as there is no immediate diagnosis or cure for COVID-19 so far. In order to stop the spread, researchers worldwide are working around the clock aiming to develop reliable tools for early diagnosis of severe acute respiratory syndrome (SARS-CoV-2) understanding the infection path and mechanisms. Currently, nucleic acid-based molecular diagnosis (real-time reverse transcription polymerase chain reaction (RT-PCR) test) is considered the gold standard for early diagnosis of SARS-CoV-2. Antibody-based serology detection is ineffective for the purpose of early diagnosis, but a potential tool for serosurveys, providing people with immune certificates for clearance from COVID-19 infection. Meanwhile, there are various blooming methods developed these days. In this review, we summarise different types of coronavirus discovered which can be transmitted between human beings. Methods used for diagnosis of the discovered human coronavirus (SARS, MERS, COVID-19) including nucleic acid detection, gene sequencing, antibody detection, antigen detection, and clinical diagnosis are presented. Their merits, demerits and prospects are discussed which can help the researchers to develop new generation of advanced diagnostic tools for accurate and effective control of human coronavirus transmission in the communities and hospitals.

Competing Interests: The authors declare no competing interest.

We performed viral metagenomics analysis of Japanese quail affected with enteritis to elucidate the viral etiology. Metagenomics generated 21,066,442 sequence reads via high-throughput sequencing, with a mean length of 136 nt. Enrichment in viral sequences suggested that at least three viruses were present in quail samples. Coronavirus and picornavirus were identified and are known as pathogens causing quail enteritis that match the observed morphology. Abundant reads of coronavirus from quail samples yielded four fragment sequences exhibiting six genomes of avian coronavirus. Sequence analysis showed that this quail coronavirus was related to turkey coronavirus and chicken infectious bronchitis virus. Quail picornavirus 8177 bp in size was identified and was similar to the QPV1/HUN/01 virus detected in quails without clinical symptoms in Hungary with 84.6% nucleotide and 94.6% amino acid identity. Our results are useful for understanding the genetic diversity of quail viruses. Further studies must be performed to determine whether quail coronavirus and quail picornavirus are pathogens of the digestive tract of quails.

Background: Management and control of coronavirus disease 2019 (COVID-19) relies on reliable diagnostic testing., Objectives: To evaluate the diagnostic test accuracy (DTA) of nucleic acid amplification tests (NAATs) for the diagnosis of coronavirus infections., Data Sources: PubMed, Web of Science, the Cochrane Library, Embase, Open Grey and conference proceeding until May 2019. PubMed and medRxiv were updated for COVID-19 on 31st August 2020., Study Eligibility: Studies were eligible if they reported on agreement rates between different NAATs using clinical samples., Participants: Symptomatic patients with suspected upper or lower respiratory tract coronavirus infection., Methods: The new NAAT was defined as the index test and the existing NAAT as reference standard. Data were extracted independently in duplicate. Risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. Confidence regions (CRs) surrounding summary sensitivity/specificity pooled by bivariate meta-analysis are reported. Heterogeneity was assessed using meta-regression., Results: Fifty-one studies were included, 22 of which included 10 181 persons before COVID-19 and 29 including 8742 persons diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The overall summary sensitivity was 89.1% (95%CR 84.0-92.7%) and specificity 98.9% (95%CR 98.0-99.4%). Nearly all the studies evaluated different PCRs as both index and reference standards. Real-time RT PCR assays resulted in significantly higher sensitivity than other tests. Reference standards at high risk of bias possibly exaggerated specificity. The pooled sensitivity and specificity of studies evaluating SARS-COV-2 were 90.4% (95%CR 83.7-94.5%) and 98.1% (95%CR 95.9-99.2), respectively. SARS-COV-2 studies using samples from the lower respiratory tract, real-time RT-PCR, and tests targeting the N or S gene or more than one gene showed higher sensitivity, and assays based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), especially when targeting only the RNA-dependent RNA polymerase (RdRp) gene, showed significantly lower sensitivity compared to other studies., Conclusions: Pooling all studies to date shows that on average 10% of patients with coronavirus infections might be missed with PCR tests. Variables affecting sensitivity and specificity can be used for test selection and development., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Pigs (Sus scrofa) may be important surveillance targets for risk assessment and risk-based control planning against emerging zoonoses. Pigs have high contact rates with humans and other animals, transmit similar pathogens as humans including CoVs, and serve as reservoirs and intermediate hosts for notable human pandemics. Wild and domestic pigs both interface with humans and each other but have unique ecologies that demand different surveillance strategies. Three fundamental questions shape any surveillance program: where, when, and how can surveillance be conducted to optimize the surveillance objective? Using theory of mechanisms of zoonotic spillover and data on risk factors, we propose a framework for determining where surveillance might begin initially to maximize a detection in each host species at their interface. We illustrate the utility of the framework using data from the United States. We then discuss variables to consider in refining when and how to conduct surveillance. Recent advances in accounting for opportunistic sampling designs and in translating serology samples into infection times provide promising directions for extracting spatio-temporal estimates of disease risk from typical surveillance data. Such robust estimates of population-level disease risk allow surveillance plans to be updated in space and time based on new information (adaptive surveillance) thus optimizing allocation of surveillance resources to maximize the quality of risk assessment insight., (Published by Elsevier B.V.)

Four endemic human coronaviruses (HCoVs) are commonly associated with acute respiratory infection in humans. B cell responses to these "common cold" viruses remain incompletely understood. Here we report a comprehensive analysis of CoV-specific antibody repertoires in 231 children and 1168 adults using phage immunoprecipitation sequencing. Seroprevalence of antibodies against endemic HCoVs ranged between approximately 4% and 27% depending on the species and cohort. We identified at least 136 novel linear B cell epitopes. Antibody repertoires against endemic HCoVs were qualitatively different between children and adults in that anti-HCoV IgG specificities more frequently found among children targeted functionally important and structurally conserved regions of the spike, nucleocapsid, and matrix proteins. Moreover, antibody specificities targeting the highly conserved fusion peptide region and S2' cleavage site of the spike protein were broadly cross-reactive with peptides of epidemic human and nonhuman coronaviruses. In contrast, an acidic tandem repeat in the N-terminal region of the Nsp3 subdomain of the HCoV-HKU1 polyprotein was the predominant target of antibody responses in adult donors. Our findings shed light on the dominant species-specific and pan-CoV target sites of human antibody responses to coronavirus infection, thereby providing important insights for the development of prophylactic or therapeutic monoclonal antibodies and vaccine design.

Background: Although the mechanisms of adaptive immunity to pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are still unknown, the immune response to the widespread endemic coronaviruses HKU1, 229E, NL63, and OC43 provide a useful reference for understanding repeat infection risk., Methods: Here we used data from proactive sampling carried out in New York City from fall 2016 to spring 2018. We combined weekly nasal swab collection with self-reports of respiratory symptoms from 191 participants to investigate the profile of recurring infections with endemic coronaviruses., Results: During the study, 12 individuals tested positive multiple times for the same coronavirus. We found no significant difference between the probability of testing positive at least once and the probability of a recurrence for the betacoronaviruses HKU1 and OC43 at 34 weeks after enrollment/first infection. We also found no significant association between repeat infections and symptom severity, but found strong association between symptom severity and belonging to the same family., Conclusions: This study provides evidence that reinfections with the same endemic coronavirus are not atypical in a time window shorter than 1 year and that the genetic basis of innate immune response may be a greater determinant of infection severity than immune memory acquired after a previous infection., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

A rapid, sensitive and simple point-of-care (POC) nucleic acid diagnostic test is needed to prevent spread of infectious diseases. Paper-based toehold reaction, a recently emerged colorimetric POC nucleic acid diagnostic test, has been widely used for pathogen detection and microbiome profiling. Here, we introduce an amplification method called reverse transcription loop-mediated amplification (RT-LAMP) prior to the toehold reaction and modify it to enable more sensitive and faster colorimetric detection of RNA viruses. We show that incorporating the modified RT-LAMP to the toehold reaction detects as few as 120 copies of coronavirus RNA in 70 min. Cross-reactivity test against other coronaviruses indicates this toehold reaction with the modified RT-LAMP is highly specific to the target RNA. Overall, the paper-based toehold switch sensors with the modified RT-LAMP allow fast, sensitive, specific and colorimetric coronavirus detection.

Gastrointestinal disorders caused by enteric viruses are frequently reported in dogs worldwide, with significant mortality rates in unvaccinated individuals. This study reports the identification and molecular characterization of Canine parvovirus (CPV-2), Canine coronavirus (CcoV), Canine astrovirus (AstV), and Canine calicivirus (CcaV) in a panel of dogs showing severe enteric clinical signs sampled in a typical Mediterranean environment (Sardinia, Italy). At least one of these viral species was detected in 92.3% samples. CPV-2 was the most frequently detected virus (87.2%), followed by AsTv (20.5%), CCoV-IIa (18%), and CCoV-I (10.3%). CCoV-IIb and CaCV were not detected in any sample. Single infection was detected in 24 samples (66.7%), mainly related to CPV-2 (91.7%). Coinfections were present in 33.3% samples with constant detection of CPV-2. Canine coronavirus was present only in coinfected animals. The VP2 sequence analysis of CPV-2 positive samples confirmed the presence of all variants, with CPV-2b most frequently detected. Phylogeny based on the CcoV-IIa spike protein (S) gene allowed to identify 2 different clades among Sardinian isolates but failed to distinguish enteric from pantropic viruses. Study on presence and prevalence of enteroviruses in dogs increase our knowledge about the circulation of these pathogens in the Mediterranean area and highlight the need for dedicated routine vaccine prophylaxis. Molecular analyses of enteric viruses are fundamental to avoid failure of vaccines caused by frequent mutations observed in these enteroviruses.

The search for therapeutic drugs that can neutralize the effects of COVID-2019 (SARS-CoV-2) infection is the main focus of current research. The coronavirus main protease (M pro ) is an attractive target for anti-coronavirus drug design. Further, α-ketoamide is proved to be very effective as a reversible covalent-inhibitor against cysteine proteases. Herein, we report on the non-covalent to the covalent adduct formation mechanism of α-ketoamide-based inhibitor with the enzyme active site amino acids by QM/SQM model (QM = quantum mechanical, SQM = semi-empirical QM). To uncover the mechanism, we focused on two approaches: a concerted and a stepwise fashion. The concerted pathway proceeds via deprotonation of the thiol of cysteine (here, Cys 145 SγH) and simultaneous reversible nucleophilic attack of sulfur onto the α-ketoamide warhead. In this work, we propose three plausible concerted pathways. On the contrary, in a traditional two-stage pathway, the first step is proton transfer from Cys 145 SγH to His 41 Nδ forming an ion pair, and consecutively, in the second step, the thiolate ion attacks the α-keto group to form a thiohemiketal. In this reaction, we find that the stability of the tetrahedral intermediate oxyanion/hydroxyl group plays an important role. Moreover, as the α-keto group has two faces Si or Re for the nucleophilic attack, we considered both possibilities of attack leading to S- and R-thiohemiketal. We computed the structural, electronic, and energetic parameters of all stationary points including transition states via ONIOM and pure DFT method. Additionally, to characterize covalent, weak noncovalent interaction (NCI) and hydrogen-bonds, we applied NCI-reduced density gradient (NCI-RDG) methods along with Bader's Quantum Theory of Atoms-in-Molecules (QTAIM) and natural bonding orbital (NBO) analysis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Modelling the spread of coronavirus globally while learning trends at global and country levels remains crucial for tackling the pandemic. We introduce a novel variational-LSTM Autoencoder model to predict the spread of coronavirus for each country across the globe. This deep Spatio-temporal model does not only rely on historical data of the virus spread but also includes factors related to urban characteristics represented in locational and demographic data (such as population density, urban population, and fertility rate), an index that represents the governmental measures and response amid toward mitigating the outbreak (includes 13 measures such as: 1) school closing, 2) workplace closing, 3) cancelling public events, 4) close public transport, 5) public information campaigns, 6) restrictions on internal movements, 7) international travel controls, 8) fiscal measures, 9) monetary measures, 10) emergency investment in health care, 11) investment in vaccines, 12) virus testing framework, and 13) contact tracing). In addition, the introduced method learns to generate a graph to adjust the spatial dependences among different countries while forecasting the spread. We trained two models for short and long-term forecasts. The first one is trained to output one step in future with three previous timestamps of all features across the globe, whereas the second model is trained to output 10 steps in future. Overall, the trained models show high validation for forecasting the spread for each country for short and long-term forecasts, which makes the introduce method a useful tool to assist decision and policymaking for the different corners of the globe., Competing Interests: The authors have declared that no competing interests exist.