Coronaviruses (Coronaviridae) of bats in the northern Caucasus and south of western Siberia.

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.

Prevalence of coronaviruses in European bison (Bison bonasus) in Poland.

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.

Dynamics of endemic human coronavirus and SARS-CoV-2 in a hospital of Madrid, Spain. Retrospective study from June 2020 to July 2023.

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.

A novel fast hybrid capture sequencing method for high-efficiency common human coronavirus whole-genome acquisition.

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.

Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

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.

Comparative Performance in the Detection of Four Coronavirus Genera from Human, Animal, and Environmental Specimens.

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.

Epidemiology of Human Seasonal Coronaviruses Among People With Mild and Severe Acute Respiratory Illness in Blantyre, Malawi, 2011-2017.

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.

Covid's cold cousins.

Four largely ignored coronaviruses circulate in humans without causing great harm and may portend the future for SARS-CoV-2.

Coronaviruses Are Abundant and Genetically Diverse in West and Central African Bats, including Viruses Closely Related to Human Coronaviruses.

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.

RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing.

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).

Surveillance of SARS-CoV-2 virus circulation using Acute Respiratory Infections sentinel system of Catalonia (PIDIRAC) during the 2019-2020 season: A retrospective observational study.

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.

Molecular detection of bat coronaviruses in three bat species in Indonesia.

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.

Antiviral Activity of Peptide-Based Assemblies.

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.

Prediction of pandemic risk for animal-origin coronavirus using a deep learning method.

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.

Description of the first isolates of guinea fowl corona and picornaviruses obtained from a case of guinea fowl fulminating enteritis.

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 HIGHLIGHTSFirst 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.

Traceable surveillance and genetic diversity analysis of coronaviruses in poultry from China in 2019.

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.

Epidemiology and Seasonality of Endemic Human Coronaviruses in South African and Zambian Children: A Case-Control Pneumonia Study.

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.

Human Coronaviruses Do Not Transfer Efficiently between Surfaces in the Absence of Organic Materials.

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.