Molecular Detection of Feline Coronavirus Based on Recombinase Polymerase Amplification Assay.

Feline coronavirus (FCoV) is endemic in cat populations worldwide. Persistently, subclinically infected cats play a significant role in spreading the infection. Testing fecal samples of cats may facilitate efforts to decrease the viral burden within a population. Real-time RT-PCR is highly sensitive and specific for the detection of FCoV but must be performed in a fully equipped laboratory. A simple and accurate assay is needed to identify FCoV at the point-of-need. The aim of this study was to develop a rapid FCoV detection assay based on isothermal amplification technology, i.e., reverse transcription-recombinase polymerase amplification (RT-RPA). Primers were designed to target the highly conserved 3' untranslated region of the 7b gene. Running on a constant temperature of 42 °C, reverse transcription as well as DNA amplification and detection was achieved in a maximum of 15 min. A probit analysis revealed a detection limit of 58.5 RNA copies/reaction. For cross-detection, nucleic acids from 19 viruses were tested. Both RT-RPA and real-time RT-PCR showed cross-detection with canine coronavirus and transmissible gastroenteritis virus, but not with other pathogens. To evaluate clinical performance, RNA was extracted from 39 fecal samples from cats. All samples were tested simultaneously with real-time RT-PCR resulting in a RT-RPA sensitivity and specificity of 90.9% and 100%, respectively. RT-RPA can be considered a promising simple method for rapid detection of FCoV.

2022 AAFP/EveryCat Feline Infectious Peritonitis Diagnosis Guidelines.

CLINICAL IMPORTANCE: Feline infectious peritonitis (FIP) is one of the most important infectious diseases and causes of death in cats; young cats less than 2 years of age are especially vulnerable. FIP is caused by a feline coronavirus (FCoV). It has been estimated that around 0.3% to 1.4% of feline deaths at veterinary institutions are caused by FIP. SCOPE: This document has been developed by a Task Force of experts in feline clinical medicine as the 2022 AAFP/EveryCat Feline Infectious Peritonitis Diagnosis Guidelines to provide veterinarians with essential information to aid their ability to recognize cats presenting with FIP. TESTING AND INTERPRETATION: Nearly every small animal veterinary practitioner will see cases. FIP can be challenging to diagnose owing to the lack of pathognomonic clinical signs or laboratory changes, especially when no effusion is present. A good understanding of each diagnostic test's sensitivity, specificity, predictive value, likelihood ratio and diagnostic accuracy is important when building a case for FIP. Before proceeding with any diagnostic test or commercial laboratory profile, the clinician should be able to answer the questions of 'why this test?' and 'what do the results mean?' Ultimately, the approach to diagnosing FIP must be tailored to the specific presentation of the individual cat. RELEVANCE: Given that the disease is fatal when untreated, the ability to obtain a correct diagnosis is critical. The clinician must consider the individual patient's history, signalment and comprehensive physical examination findings when selecting diagnostic tests and sample types in order to build the index of suspicion 'brick by brick'. Research has demonstrated efficacy of new antivirals in FIP treatment, but these products are not legally available in many countries at this time. The Task Force encourages veterinarians to review the literature and stay informed on clinical trials and new drug approvals.

Secondary zoonotic dog-to-human transmission of SARS-CoV-2 suggested by timeline but refuted by viral genome sequencing.

PURPOSE: The risk of secondary zoonotic transmission of SARS-CoV-2 from pet animals remains unclear. Here, we report on a 44 year old Caucasian male presenting to our clinic with COVID-19 pneumonia, who reported that his dog displayed respiratory signs shortly prior to his infection. The dog tested real-time-PCR (RT-PCR) positive for SARS-CoV-2 RNA and the timeline of events suggested a transmission from the dog to the patient. METHODS: RT-PCR and serological assays were used to confirm SARS-CoV-2 infection in the nasopharyngeal tract in the dog and the patient. We performed SARS-CoV-2-targeted amplicon-based next generation sequencing of respiratory samples from the dog and patient for sequence comparisons. RESULTS: SARS-CoV-2 infection of the dog was confirmed by three independent PCR-positive pharyngeal swabs and subsequent seroconversion. Sequence analysis identified two separate SARS-CoV-2 lineages in the canine and the patient's respiratory samples. The timeline strongly suggested dog-to-human transmission, yet due to the genetic distance of the canine and the patient's samples paired-transmission was highly unlikely. CONCLUSION: The results of this case support current knowledge about the low risk of secondary zoonotic dog-to-human transmissions of SARS-CoV-2 and emphasizes the strength of genomic sequencing in deciphering viral transmission chains.

Correlation of Feline Coronavirus Shedding in Feces with Coronavirus Antibody Titer.

BACKGROUND: Feline coronavirus (FCoV) infection is ubiquitous in multi-cat households. Responsible for the continuous presence are cats that are chronically shedding a high load of FCoV. The aim of the study was to determine a possible correlation between FCoV antibody titer and frequency and load of fecal FCoV shedding in cats from catteries. METHODS: Four fecal samples from each of 82 cats originating from 19 German catteries were examined for FCoV viral loads by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). Additionally, antibody titers were determined by an immunofluorescence assay. RESULTS: Cats with antibodies were more likely to be FCoV shedders than non-shedders, and there was a weak positive correlation between antibody titer and mean fecal virus load (Spearman r = 0.2984; p = 0.0072). Antibody titers were significantly higher if cats shed FCoV more frequently throughout the study period (p = 0.0063). When analyzing only FCoV shedders, cats that were RT-qPCR-positive in all four samples had significantly higher antibody titers (p = 0.0014) and significantly higher mean fecal virus loads (p = 0.0475) than cats that were RT-qPCR-positive in only one, two, or three samples. CONCLUSIONS: The cats' antibody titers correlate with the likelihood and frequency of FCoV shedding and fecal virus load. Chronic shedders have higher antibody titers and shed more virus. This knowledge is important for the management of FCoV infections in multi-cat environments, but the results indicate that antibody measurement cannot replace fecal RT-qPCR.

Clinical Follow-Up and Postmortem Findings in a Cat That Was Cured of Feline Infectious Peritonitis with an Oral Antiviral Drug Containing GS-441524.

This is the first report on a clinical follow-up and postmortem examination of a cat that had been cured of feline infectious peritonitis (FIP) with ocular manifestation by successful treatment with an oral multicomponent drug containing GS-441524. The cat was 6 months old when clinical signs (recurrent fever, lethargy, lack of appetite, and fulminant anterior uveitis) appeared. FIP was diagnosed by ocular tissue immunohistochemistry after enucleation of the affected eye. The cat was a participant in a FIP treatment study, which was published recently. However, 240 days after leaving the clinic healthy, and 164 days after the end of the 84 days of treatment, the cured cat died in a road traffic accident. Upon full postmortem examination, including histopathology and immunohistochemistry, there were no residual FIP lesions observed apart from a generalized lymphadenopathy due to massive lymphoid hyperplasia. Neither feline coronavirus (FCoV) RNA nor FCoV antigen were identified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry, respectively, in any tissues or body fluids, including feces. These results prove that oral treatment with GS-441524 leads to the cure of FIP-associated changes and the elimination of FCoV from all tissues.

Detection of Feline Coronavirus Variants in Cats without Feline Infectious Peritonitis.

(1) Background: This study aimed to detect feline coronavirus (FCoV) and characterize spike (S) gene mutation profiles in cats suffering from diseases other than feline infectious peritonitis (FIP) using commercial real-time reverse transcription polymerase chain reaction (RT-qPCR) and reevaluating results by sequencing. (2) Methods: In 87 cats in which FIP was excluded by histopathology and immunohistochemistry, FCoV 7b gene and S gene mutation RT-qPCR was performed prospectively on incisional biopsies and fine-needle aspirates of different organs, body fluids, and feces. Samples positive for S gene mutations or mixed FCoV underwent sequencing. (3) Results: In 21/87 cats, FCoV RNA was detectable. S gene mutations were detected by commercial RT-qPCR (and a diagnostic algorithm that was used at the time of sample submission) in at least one sample in 14/21 cats (66.7%), with only mutated FCoV in 2/21, only mixed in 1/21, and different results in 11/21 cats; in the remaining 7/21 cats, RNA load was too low to differentiate. However, sequencing of 8 tissue samples and 8 fecal samples of 9 cats did not confirm mutated FCoV in any of the FCoV RNA-positive cats without FIP. (4) Conclusions: Sequencing results did not confirm results of the commercial S gene mutation RT-qPCR.

Fecal Feline Coronavirus RNA Shedding and Spike Gene Mutations in Cats with Feline Infectious Peritonitis Treated with GS-441524.

As previously demonstrated by our research group, the oral multicomponent drug Xraphconn® containing GS-441524 was effective at curing otherwise fatal feline infectious peritonitis (FIP) in 18 feline coronavirus (FCoV)-infected cats. The aims of the current study were to investigate, using samples from the same animals as in the previous study, (1) the effect of treatment on fecal viral RNA shedding; (2) the presence of spike gene mutations in different body compartments of these cats; and (3) viral RNA shedding, presence of spike gene mutations, and anti-FCoV antibody titers in samples of 12 companion cats cohabitating with the treated cats. Eleven of the eighteen treated FIP cats (61%) were shedding FCoV RNA in feces within the first three days after treatment initiation, but all of them tested negative by day 6. In one of these cats, fecal shedding reoccurred on day 83. Two cats initially negative in feces were transiently positive 1-4 weeks into the study. The remaining five cats never shed FCoV. Viral RNA loads in feces decreased with time comparable with those in blood and effusion. Specific spike gene mutations linked to systemic FCoV spread were consistently found in blood and effusion from treated FIP cats, but not in feces from treated or companion cats. A new mutation that led to a not yet described amino acid change was identified, indicating that further mutations may be involved in the development of FIP. Eight of the twelve companion cats shed FCoV in feces. All but one of the twelve companion cats had anti-FCoV antibodies. Oral treatment with GS-441524 effectively decreased viral RNA loads in feces, blood, and effusion in cats with FIP. Nonetheless, re-shedding can most likely occur if cats are re-exposed to FCoV by their companion cats.

Vaccination of Immunocompromised Cats.

Immunocompromise is a common condition in cats, especially due to widespread infections with immunosuppressive viruses, such as feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV), but also due to chronic non-infectious diseases, such as tumours, diabetes mellitus, and chronic kidney disease, as well as treatment with immunosuppressive drugs, such as glucocorticoids, cyclosporins, or tumour chemotherapy. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from eleven European countries, discusses the current knowledge and rationale for vaccination of immunocompromised cats. So far, there are few data available on vaccination of immunocompromised cats, and sometimes studies produce controversial results. Thus, this guideline summarizes the available scientific studies and fills in the gaps with expert opinion, where scientific studies are missing. Ultimately, this review aims to help veterinarians with their decision-making in how best to vaccinate immunocompromised cats.

Parents' and Childcare Workers' Perspectives Toward SARS-CoV-2 Test and Surveillance Protocols in Pre-school Children Day Care Centers: A Qualitative Study Within the German Wü-KiTa-CoV Project.

Background: Feasibility of surveillance through continuous SARS-CoV-2 testing in pre-school children and childcare workers (CCWs) to prevent closure of day care centers (DCCs) was proven in the Wü-KiTa-CoV study. The purpose of this study was to describe the factors that facilitate or hinder the implementation of continuous SARS-CoV-2 testing from the perspective of parents and CCWs involved in the study. Methods: A total of 148 semi-structured telephone interviews, repeated before and after the implementation of the surveillance protocols, were conducted with parents and CCWs belonging to the DCCs involved in Wü-KiTa-CoV and analyzed using qualitative content analysis. Results: Five main topical categories that influences implementation of surveillance protocols for SARS-CoV-2 in DCCs emerged: Generating valuable knowledge, Impact on daily life, Communication and information, Children's wellbeing and the Sense of security. Smooth integration in daily routines, quickly delivered test results, and efficient communication and information between the study team and the participants were identified as factors that had a positive impact on implementation. To ensure children's wellbeing, the introduction of non-invasive testing procedures such as saliva testing, parental involvement to motivate, and prepare children for the procedure, the creation of a child-friendly environment for testing, and use of child-friendly explanations were considered critical. The surveillance was found to increase the sense of security during the pandemic. Conversely, reliability of tests in the surveillance protocols, low participation rates, non-transparent communication, the need to travel to testing sites, fear of quarantine in case of positive test results, concerns about higher workloads, the fear of unpleasant feelings for children, their young age, and changing test teams were considered as hindering factors. Conclusion: This qualitative study of parents of children in day care and DCC staff under surveillance through continuous testing for SARS-CoV-2 in nine German DCCs identified several factors that facilitate or hinder its implementation. These should be considered when planning screening interventions to prevent the spread of SARS-CoV-2 or other infectious diseases in pre-school children DCCs.

Role of Feline Coronavirus as Contributor to Diarrhea in Cats from Breeding Catteries.

(1) Background: Feline coronavirus infection (FCoV) is common in multi-cat environments. A role of FCoV in causing diarrhea is often assumed, but has not been proven. The aim of this study was to evaluate an association of FCoV infection with diarrhea in multi-cat environments. (2) Methods: The study included 234 cats from 37 catteries. Fecal samples were analyzed for FCoV RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Potential co-infections were determined by applying a qPCR panel on different potential enteropathogens and fecal flotation. A fecal scoring system was used to categorize feces as diarrheic or non-diarrheic. (3) Results: Of the 234 cats included, 23 had diarrhea. The prevalence of FCoV infection was 87.0% in cats with and 58.8% in cats without diarrhea. FCoV infection was significantly associated with diarrhea (Odds Ratio (OR) 5.01; p = 0.008). In addition, presence of Clostridium perfringens α toxin (OR 6.93; p = 0.032) and feline panleukopenia virus (OR 13.74; p = 0.004) were associated with an increased risk of diarrhea. There was no correlation between FCoV load and fecal score. FCoV-positive cats with co-infections were not more likely to have diarrhea than FCoV-positive cats without co-infections (p = 0.455). (4) Conclusions: FCoV infection is common in cats from catteries and can be associated with diarrhea.

Very Low Incidence of SARS-CoV-2, Influenza and RSV but High Incidence of Rhino-, Adeno- and Endemic Coronaviruses in Children With Acute Respiratory Infection in Primary Care Pediatric Practices During the Second and Third Wave of the SARS-CoV-2 Pandemic.

Respiratory viruses were detected by multiplex-polymerase chain reaction from oropharyngeal swabs in 114/168 (67.9%) children with acute respiratory infection presenting to 5 pediatric practices in Germany between November 2020 and April 2021. In contrast to rhino- (48.8%), adeno- (14.3%) and endemic coronaviruses (14.9%), SARS-CoV-2 and influenza virus were detected only once; respiratory syncytial virus was not detected. This demonstrates differing impacts of pandemic infection control measures on the spread of respiratory viruses.

Feasibility of SARS-CoV-2 Surveillance Testing Among Children and Childcare Workers at German Day Care Centers: A Nonrandomized Controlled Trial.

Importance: Closure of day care centers has been implemented globally to contain the COVID-19 pandemic but has negative effects on children's health and psychosocial well-being. Objective: To investigate the feasibility of surveillance among children and childcare workers and to model the efficacy of surveillance on viral spread prevention. Design, Setting, and Participants: This nonrandomized controlled trial was conducted at 9 day care centers in Wuerzburg, Germany, from October 2020 to March 2021. Participants included children attending day care, childcare workers, and household members. Participating day care centers were assigned to different surveillance modules in a nonrandomized feasibility study. A mathematical model for SARS-CoV-2 spread in day care centers was developed to identify optimal surveillance. Interventions: Modules 1, 2, and 3 involved continuous surveillance of asymptomatic children and childcare workers by SARS-CoV-2 polymerase chain reaction testing of either midturbinate nasal swabs twice weekly (module 1) or once weekly (module 2) or self-sampled saliva samples twice weekly (module 3). Module 4 involved symptom-based, on-demand testing of children, childcare workers, and their household members by oropharyngeal swabs. All participants underwent SARS-CoV-2 antibody status testing before and after the sampling period. Questionnaires on attitudes and perception of the pandemic were administered in weeks 1, 6, and 12. Mathematical modeling was used to estimate SARS-CoV-2 spread in day care centers. Main Outcomes and Measures: The primary outcomes were acceptance of the respective surveillance protocols (feasibility study) and the estimated number of secondary infections (mathematical modeling). Results: Of 954 eligible individuals (772 children and 182 childcare workers), 592 (62%), including 442 children (median [IQR] age, 3 [2-4] years; 214 [48.6%] female) and 150 childcare workers (median [IQR] age, 29 [25-44] years; 129 [90.8%] female) participated in the surveillance. In total, 4755 tests for SARS-CoV-2 detected 2 infections (1 childcare worker and 1 adult household member). Acceptance for continuous surveillance was highest for biweekly saliva testing (150 of 221 eligible individuals [67.9%; 95% CI, 61.5%-73.7%]) compared with biweekly (51 of 117 individuals [43.6%; 95% CI, 35.0%-52.6%]) and weekly (44 of 128 individuals [34.4%; 95% CI, 26.7%-43.0%]) midturbinate swabbing (P < .001). Dropout rates were higher for midturbinate swabbing (biweekly, 11 of 62 participants [18%]; once weekly, 11 of 55 participants [20%]) than for saliva testing (6 of 156 participants [4%]). Mathematical modeling based on study and literature data identified biweekly testing of at least 50% of children and childcare workers as minimal requirements to limit secondary infections. Conclusions and Relevance: In this nonrandomized controlled trial, surveillance for SARS-CoV-2 in 9 German day care centers was feasible and well accepted. Mathematical modeling estimated that testing can minimize the spread of SARS-CoV-2 in day care centers. These findings enable setup of surveillance programs to maintain institutional childcare. Trial Registration: German Registry for Clinical Trials Identifier: DRKS00023721.

SARS-CoV-2 Infection in Dogs and Cats from Southern Germany and Northern Italy during the First Wave of the COVID-19 Pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people globally since its first detection in late 2019. Besides humans, cats and, to some extent, dogs were shown to be susceptible to SARS-CoV-2, highlighting the need for surveillance in a One Health context. Seven veterinary clinics from regions with high incidences of coronavirus disease (COVID-19) were recruited during the early pandemic (March to July 2020) for the screening of patients. A total of 2257 oropharyngeal and nasal swab specimen from 877 dogs and 260 cats (including 18 animals from COVID-19-affected households and 92 animals with signs of respiratory disease) were analyzed for the presence of SARS-CoV-2 RNA using reverse transcriptase real-time polymerase chain reaction (RT-qPCR) targeting the viral envelope (E) and RNA dependent RNA polymerase (RdRp) genes. One oropharyngeal swab from an Italian cat, living in a COVID-19-affected household in Piedmont, tested positive in RT-qPCR (1/260; 0.38%, 95% CI: 0.01-2.1%), and SARS-CoV-2 infection of the animal was serologically confirmed six months later. One oropharyngeal swab from a dog was potentially positive (1/877; 0.1%, 95% CI: 0.002-0.63%), but the result was not confirmed in a reference laboratory. Analyses of convenience sera from 118 animals identified one dog (1/94; 1.1%; 95% CI: 0.02-5.7%) from Lombardy, but no cats (0/24), as positive for anti-SARS-CoV-2 receptor binding domain (RBD) antibodies and neutralizing activity. These findings support the hypothesis that the prevalence of SARS-CoV-2 infection in pet cat and dog populations, and hence, the risk of zoonotic transmission to veterinary staff, was low during the first wave of the pandemic, even in hotspot areas.

One Health: EAACI Position Paper on coronaviruses at the human-animal interface, with a specific focus on comparative and zoonotic aspects of SARS-CoV-2.

The latest outbreak of a coronavirus disease in 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), evolved into a worldwide pandemic with massive effects on health, quality of life, and economy. Given the short period of time since the outbreak, there are several knowledge gaps on the comparative and zoonotic aspects of this new virus. Within the One Health concept, the current EAACI position paper dwells into the current knowledge on SARS-CoV-2's receptors, symptoms, transmission routes for human and animals living in close vicinity to each other, usefulness of animal models to study this disease and management options to avoid intra- and interspecies transmission. Similar pandemics might appear unexpectedly and more frequently in the near future due to climate change, consumption of exotic foods and drinks, globe-trotter travel possibilities, the growing world population, the decreasing production space, declining room for wildlife and free-ranging animals, and the changed lifestyle including living very close to animals. Therefore, both the society and the health authorities need to be aware and well prepared for similar future situations, and research needs to focus on prevention and fast development of treatment options (medications, vaccines).

Advances in Feline Viruses and Viral Diseases.

Viral diseases play a very important role in feline medicine, and research on feline viruses and viral diseases is a well-established field that helps to safeguard the health of domestic cats and non-domestic felids, many of which are endangered [...].

Anthropogenic Infection of Cats during the 2020 COVID-19 Pandemic.

COVID-19 is a severe acute respiratory syndrome (SARS) caused by a new coronavirus (CoV), SARS-CoV-2, which is closely related to SARS-CoV that jumped the animal-human species barrier and caused a disease outbreak in 2003. SARS-CoV-2 is a betacoronavirus that was first described in 2019, unrelated to the commonly occurring feline coronavirus (FCoV) that is an alphacoronavirus associated with feline infectious peritonitis (FIP). SARS-CoV-2 is highly contagious and has spread globally within a few months, resulting in the current pandemic. Felids have been shown to be susceptible to SARS-CoV-2 infection. Particularly in the Western world, many people live in very close contact with their pet cats, and natural infections of cats in COVID-19-positive households have been described in several countries. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from 11 European Countries, discusses the current status of SARS-CoV infections in cats. The review examines the host range of SARS-CoV-2 and human-to-animal transmissions, including infections in domestic and non-domestic felids, as well as mink-to-human/-cat transmission. It summarises current data on SARS-CoV-2 prevalence in domestic cats and the results of experimental infections of cats and provides expert opinions on the clinical relevance and prevention of SARS-CoV-2 infection in cats.