One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

The Luciferase Immunoprecipitation System (LIPS) Targeting the Spike Protein of SARS-CoV-2 Is More Accurate than Nucleoprotein-Based LIPS and ELISAs for Mink Serology

Since anthropo-zoonotic outbreaks of SARS-CoV-2 have been reported in mink farms, it is important to monitor the seroprevalence within this population. To investigate the accuracy of nucleo (N) or spike (S) protein-based assays to detect anti-SARS-CoV-2 antibodies in animal serum, we compared four assays, two commercial N-based enzyme-linked immunosorbent assays (ELISA) validated for animal sera and two luciferase immunoprecipitation systems (LIPS-N and LIPS-S), to the reference standard plaque reduction neutralisation test (PRNT). Samples included in this study were derived from a naturally infected mink population. For the first time in this study, serum samples of mink were collected over a 307-day period, at different time points, thus providing an overview of performances of four different rapid serological tests over time. The assays were compared by performing a correlation analysis using R2, Spearman's rank-order correlation coefficient, and Fleiss' and Cohen's kappa for analysis of agreement to PRNT, and an UpSet chart was created to visualize the number of shared positive samples between assays. Cohen's kappa test on categorical data showed an excellent agreement between PRNT and LIPS-S, while agreements between PRNT and N-based methods decreased from fair for LIPS-N to poor agreements for the ELISA kits. In addition, LIPS-S revealed the highest number of true-positive SARS-CoV-2 samples compared to N-based methods. Despite an excellent agreement between LIPS-S and PRNT, a weak correlation was detectable between PRNT titres and relative light units. This study shows that the LIPS-S assay can be used for serological surveillance within a naturally exposed mink population, while N-based serological assays are less accurate providing a higher number of false-negative results, especially at a later stage of infection, thus indicating that N antibodies are less persistent in naturally exposed mink. Our findings provide crucial information for veterinarians and competent authorities involved in surveillance and outbreak investigation in wild and farmed minks. [ FROM AUTHOR] Copyright of Transboundary & Emerging Diseases is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Discovery of a coronavirus in the Eurasian badger (Meles meles) belonging to a putative new genus.

In the aftermath of COVID-19, coronaviruses gained renewed attention by the scientific community. The study reports the identification and genetic characterization of a novel coronavirus in the European badger (Meles meles) obtained in the framework of passive surveillance implemented in Italian wildlife in response to the pandemic. Positive samples were characterized using next generation sequencing as well as genetic and phylogenetic analyses, aiming for taxonomic placement under ICTV guidelines of the viruses contained in each sample. Results obtained for six conserved domains within the polyprotein showed that the virus clustered as outgroup and shared <46% amino acid identity with other coronaviruses, supporting the assumption that it belongs to a new putative genus Epsiloncoronavirus. This finding highlights that mammals still hide diverse coronaviruses whose zoonotic and epizootic potential remains unknown.

Host Response of Syrian Hamster to SARS-CoV-2 Infection including Differences with Humans and between Sexes.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the importance of having proper tools and models to study the pathophysiology of emerging infectious diseases to test therapeutic protocols, assess changes in viral phenotypes, and evaluate the effects of viral evolution. This study provided a comprehensive characterization of the Syrian hamster (Mesocricetus auratus) as an animal model for SARS-CoV-2 infection using different approaches (description of clinical signs, viral load, receptor profiling, and host immune response) and targeting four different organs (lungs, intestine, brain, and PBMCs). Our data showed that both male and female hamsters were susceptible to the infection and developed a disease similar to the one observed in patients with COVID-19 that included moderate to severe pulmonary lesions, inflammation, and recruitment of the immune system in the lungs and at the systemic level. However, all animals recovered within 14 days without developing the severe pathology seen in humans, and none of them died. We found faint evidence for intestinal and neurological tropism associated with the absence of lesions and a minimal host response in intestines and brains, which highlighted another crucial difference with the multiorgan impairment of severe COVID-19. When comparing male and female hamsters, we observed that males sustained higher viral RNA shedding and replication in the lungs, suffered from more severe symptoms and histopathological lesions, and triggered higher pulmonary inflammation. Overall, these data confirmed the Syrian hamster as a suitable model for mild to moderate COVID-19 and reflected sex-related differences in the response against the virus observed in humans.

SARS-CoV-2 intra-host evolution during prolonged infection in an immunocompromised patient.

OBJECTIVES: Intra-host SARS-CoV-2 evolution during chronic infection in immunocompromised hosts has been suggested as being the possible trigger of the emergence of new variants. METHODS: Using a deep sequencing approach, we investigated the SARS-CoV-2 intra-host genetic evolution in a patient with HIV over a period of 109 days. RESULTS: Sequencing of nasopharyngeal swabs at three time points demonstrated dynamic changes in the viral population, with the emergence of 26 amino acid mutations and two deletions, 57% of them in the Spike protein. Such a combination of mutations has never been observed in other SARS-CoV-2 lineages detected so far. CONCLUSION: Our data confirm that persistent infection in certain immunocompromised individuals for a long time may favor the dangerous emergence of new SARS-CoV-2 variants with immune evasion properties.

Active Surveillance for Highly Pathogenic Avian Influenza Viruses in Wintering Waterbirds in Northeast Italy, 2020-2021.

The increasing involvement of wild waterfowl in H5 Highly Pathogenic Avian Influenza Virus (HPAIV) circulation continues to pose a threat to animal and public health worldwide. In winter 2020-2021, two field surveillance activities were carried out on a weekly basis, through virological and serological analyses, in 823 hunted and 521 trapped migratory aquatic birds in northeast Italy. Sixty Eurasian teals were recaptured several times, which allowed us to follow the progression of the HPAI H5 infection in naturally infected wild waterfowl. Oropharyngeal, cloacal, and feather swabs (OS, CS and FS) were collected from each duck and tested by real time rRT-PCR Type A influenza. The identified viruses were characterized and pathotyped by sequencing. Several viruses belonging to three different HPAI H5 subtypes were detected: H5N8, H5N5, and H5N1. High prevalence of infection with HPAI H5 clade 2.3.4.4b during November-December 2020 (up to 27.1%) was observed in captured Eurasian teals, while infection rates in hunted dabbling ducks, mainly Eurasian wigeons, showed the highest prevalence of infection in November 2020 (8.9%) and January 2021 (10.2%). All HPAI positive birds were also clinically healthy when recaptured weeks apart. The OS and FS showed the highest detection efficiency of HPAIV. Our results highlight that HPAI passive surveillance should be complemented by a targeted active surveillance to more efficiently detect novel HPAI viruses.

Impact of Full Vaccination with mRNA BNT162b2 on SARS-CoV-2 Infection: Genomic and Subgenomic Viral RNAs Detection in Nasopharyngeal Swab and Saliva of Health Care Workers.

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

Prevalence of SARS-CoV-2 RNA on inanimate surfaces: a systematic review and meta-analysis.

Coronavirus disease (COVID-19) is a respiratory disease affecting many people and able to be transmitted through direct and perhaps indirect contact. Direct contact transmission, mediated by aerosols or droplets, is widely demonstrated, whereas indirect transmission is only supported by collateral evidence such as virus persistence on inanimate surfaces and data from other similar viruses. The present systematic review aims to estimate SARS-CoV-2 prevalence on inanimate surfaces, identifying risk levels according to surface characteristics. Data were obtained from studies in published papers collected from two databases (PubMed and Embase) with the last search on 1 September 2020. Included studies had to be papers in English, had to deal with coronavirus and had to consider inanimate surfaces in real settings. Studies were coded according to our assessment of the risk that the investigated surfaces could be contaminated by SARS-CoV-2. A meta-analysis and a metaregression were carried out to quantify virus RNA prevalence and to identify important factors driving differences among studies. Thirty-nine out of forty retrieved paper reported studies carried out in healthcare settings on the prevalence of virus RNA, five studies carry out also analyses through cell culture and six tested the viability of isolated viruses. Overall prevalences of SARS-CoV-2 RNA on high-, medium- and low-risk surfaces were 0.22 (CI95 [0.152-0.296]), 0.04 (CI95 [0.007-0.090]), and 0.00 (CI95 [0.00-0.019]), respectively. The duration surfaces were exposed to virus sources (patients) was the main factor explaining differences in prevalence.

Mild SARS-CoV-2 Infections and Neutralizing Antibody Titers.

BACKGROUND: Recent evidence suggests that neutralizing antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 may persist over time; however, knowledge regarding pediatric subjects is limited. METHODS: A single-center, prospective observational study was conducted on 57 family clusters of coronavirus disease 2019, including children of neonatal and pediatric age attending the University Hospital of Padua (Italy). For each patient, blood samples were collected for both the quantification of nAbs through a plaque reduction neutralizing test and the detection of antinucleocapsid-spike protein immunoglobulin G and/or immunoglobulin M. RESULTS: We analyzed 283 blood samples collected from 152 confirmed coronavirus disease 2019 cases (82 parents and 70 children or older siblings of median age of 8 years, interquartile range: 4-13), presenting asymptomatic or with mildly symptomatic disease. Despite the decrease of immunoglobulin G over time, nAbs were found to persist up to 7 to 8 months in children, whereas adults recorded a modest declining trend. Interestingly, children aged <6 years, and, in particular, those aged <3 years, developed higher long-lasting levels of nAbs compared with older siblings and/or adults. CONCLUSIONS: Mild and asymptomatic severe acute respiratory syndrome coronavirus 2 infections in family clusters elicited higher nAbs among children.

SARS-Cov-2 Natural Infection in a Symptomatic Cat: Diagnostic, Clinical and Medical Management in a One Health Vision.

Despite the reported increase in SARS-CoV-2-infected pets, the description of the clinical features from natural infection and the medical follow up in symptomatic pets is still not sufficiently documented. This study reports the case of an indoor cat that displayed respiratory signs and a gastrointestinal syndrome, following the COVID-19 diagnosis of his owners. Thoracic radiographies were suggestive of bronchial pneumonia, while blood tests were indicative of a mild inflammatory process. Nasal and oropharyngeal swabs tested positive through RT-qPCR assays targeting SARS-CoV-2 genes 14 days after his owners tested positive for the virus. Nasal swabs persisted to be RT-qPCR positive after 31 days. Serology confirmed the presence of antibodies through ELISA, electrochemiluminescence analysis and plaque reduction neutralization test, recording a high antibody titre after 31 days. The cat improved after medical treatment and clinically recovered. This study suggests that exposure to SARS-CoV-2 could lead to a natural infection with bronchial pneumonia in cats along with a possible prolonged persistence of SARS-CoV-2 RNA in the upper airways, albeit at a low level. The cat developed neutralizing antibodies, reaching a high titre after 31 days. Further descriptions of SARS-CoV-2 naturally infected pets, their medical management and diagnostic findings would be useful to enhance knowledge about COVID-19 in susceptible animals.

The first familial cluster of the B.1.1.7 variant of SARS-CoV-2 in the northeast of Italy.

PURPOSE: We report on the first identified cluster of the B.1.1.7 variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in the northeast of Italy. METHODS: The cluster was recognized in January 2021 with an epidemiological started from the hospitalization of a 68-year-old man suffering from coronavirus disease 2019 (COVID-19) related pneumonia and we surprisingly found three families involved in the same cluster. RESULTS: We retrospectively rebuilt the pathway of infection and performed a virological analysis. CONCLUSION: This allow us to make clear the very high attack rate and the great infective capacity of this B.1.1.7 variant of SARS-CoV-2.

A systematic review of human coronaviruses survival on environmental surfaces.

The current pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led people to implement preventive measures, including surface disinfection and use of alcohol-based hand gel, in order to avoid viral transmission via fomites. However, the role of surface transmission is still debated. The present systematic review aims to summarize all the evidence on surface survival of coronaviruses infecting humans. The analysis of 18 studies showed the longest coronavirus survival time is 28 days at room temperature (RT) on different surfaces: polymer banknotes, vinyl, steel, glass, and paper banknotes. Concerning SARS-CoV-2 human infection from contaminated surfaces, dangerous viral load on surfaces for up to 21 days was determined on polymer banknotes, steel, glass and paper banknotes. For viruses other than SARS-CoV-2, the longest period of survival was 14 days, recorded on glass. Environmental conditions can affect virus survival, and indeed, low temperatures and low humidity support prolonged survival of viruses on contaminated surfaces independently of surface type. Furthermore, it has been shown that exposure to sunlight significantly reduces the risk of surface transmission. Although studies are increasingly investigating the topic of coronavirus survival, it is difficult to compare them, given the methodology differences. For this reason, it is advisable to define a reference working protocol for virus survival trials, but, as an immediate measure, there is also a need for further investigations of coronavirus survival on surfaces.