Efficacy of three EPA-registered antimicrobials and steam against two human norovirus surrogates on nylon carpets with two backing types.

Carpet cleaning guidelines currently do not include the use of an antimicrobial, except after a bodily fluid event. To address this gap, we compared the efficacy of three antimicrobials-two hydrogen peroxide-based (H2O2) products (A and B) and one chlorine-based product (C)-and a steam treatment against two norovirus surrogates, specifically feline calicivirus (FCV) and Tulane virus (TuV). These tests were performed on nylon carpets with either water-permeable or waterproof backing types. The effect of repeated antimicrobial use on carpet properties was also evaluated. For a carpet with water-permeable backing, products A, B, and C achieved a 0.8, 3.1, and 0.9 log10 PFU/coupon reduction of FCV and 0.3, 2.5, and 0.4 log10 TCID50/coupon reduction of TuV, respectively, following a 30 min contact time. For carpet with waterproof backing, only product B achieved a 5.0 log10 PFU/coupon reduction of FCV and >3.0 log10 TCID50/coupon reduction of TuV, whereas products A and C achieved a 2.4 and 1.6 log10 PFU/coupon reduction of FCV and a 1.2 and 1.2 log10 TCID50/coupon reduction of TuV, respectively. Steam treatment achieved a ≥ 5.2 log10 PFU/coupon reduction of FCV and a > 3.2 log10 TCID50/coupon reduction of TuV in 15 seconds on the carpet with both backing types. The repeated use of products A and B decreased the tensile strength of the carpet backing, while use of product B resulted in cracks on carpet fibers. Overall, steam treatment for 15 seconds was efficacious on both carpet types, but only product B achieved efficacy after a 30-minute exposure on the carpet with waterproof backing.IMPORTANCECarpets are common in long-term care facilities, despite its potential as a vehicle for transmission of agents associated with healthcare-associated infections, including human norovirus (NoV). Presently, our understanding of carpet disinfection is limited; hence, there are no commercial antimicrobials against norovirus available for use on carpets. Our findings showed that steam treatment, which minimally affected the properties of carpet fibers and backing, was more efficacious against human norovirus surrogates on carpets compared to the three chemical antimicrobials tested. Additionally, the two surrogates were more sensitive to chemical antimicrobials on the carpet with waterproof backing compared to carpets with water-permeable backing. These findings can inform development of antimicrobials for use on carpets contaminated with human norovirus.

VP2 mediates the release of the feline calicivirus RNA genome by puncturing the endosome membrane of infected cells.

Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release.IMPORTANCEResearch on the biology and pathogenicity of certain caliciviruses, such as Norovirus and Sapovirus, is hindered by the lack of easy-to-use cell culture system. Feline calicivirus (FCV), which grows effectively in cell lines, is used as a substitute. At present, there is limited understanding of the genome release mechanism in caliciviruses. Our findings suggest that FCV uses VP2 to pierce the endosome membrane for genome release and provide new insights into the calicivirus gRNA release mechanism.

Identification and genomic characterization of feline calicivirus from a leopard cat (Prionailurus bengalensis) in Taiwan.

The leopard cat (Prionailurus bengalensis) is an endangered wildlife that is protected under Taiwan's regulations. The body of a road-killed leopard cat was found to contain sequences of feline calicivirus (FCV), designated W109-1443. Analysis of the complete genomic sequence revealed that it shared approximately 81% similarity with a Chinese strain of FCV found in a domestic cat. Phylogenetic analysis of the VP1 gene indicated that the W109-1443 isolate belonged to genogroup II. Recombination analysis revealed that the W109-1443 isolate may have resulted from recombination between two FCV strains. Given the potential impact of FCV on the health and survival of wild felids, further investigation is necessary to assess its pathogenicity in the leopard cat population.

Ultrasensitive and visual detection of Feline herpesvirus type-1 and Feline calicivirus using one-tube dRPA-Cas12a/Cas13a assay.

BACKGROUND: Feline herpesvirus type 1 (FHV) and Feline calicivirus (FCV) are the primary co-infecting pathogens that cause upper respiratory tract disease in cats. However, there are currently no visual detection assays available for on-site testing. Here, we develop an ultrasensitive and visual detection method based on dual recombinase polymerase amplification (dRPA) reaction and the hybrid Cas12a/Cas13a trans-cleavage activities in a one-tube reaction system, referred to as one-tube dRPA-Cas12a/Cas13a assay. RESULTS: The recombinant plasmid DNAs, crRNAs, and RPA oligonucleotides targeting the FCV ORF1 gene and FHV-1 TK gene were meticulously prepared. Subsequently, dual RPA reactions were performed followed by screening of essential reaction components for hybrid CRISPR-Cas12a (targeting the FHV-1 TK gene) and CRISPR-Cas13a (targeting the FCV ORF1 gene) trans-cleavage reaction. As a result, we successfully established an ultra-sensitive and visually detectable method for simultaneous detection of FCV and FHV-1 nucleic acids using dRPA and CRISPR/Cas-powered technology in one-tube reaction system. Visual readouts were displayed using either a fluorescence detector (Fluor-based assay) or lateral flow dipsticks (LDF-based assay). As expected, this optimized assay exhibited high specificity towards only FHV-1 and FCV without cross-reactivity with other feline pathogens while achieving accurate detection for both targets with limit of detection at 2.4 × 10- 1 copies/µL for the FHV-1 TK gene and 5.5 copies/µL for the FCV ORF1 gene, respectively. Furthermore, field detection was conducted using the dRPA-Cas12a/Cas13a assay and the reference real-time PCR methods for 56 clinical samples collected from cats with URTD. Comparatively, the results of Fluor-based assay were in exceptional concordance with the reference real-time PCR methods, resulting in high sensitivity (100% for both FHV-1 and FCV), specificity (100% for both FHV-1 and FCV), as well as consistency (Kappa values were 1.00 for FHV-1 and FCV). However, several discordant results for FHV-1 detection were observed by LDF-based assay, which suggests its prudent use and interpretaion for clinical detection. In spite of this, incorporating dRPA-Cas12a/Cas13a assay and visual readouts will facilitate rapid and accurate detection of FHV-1 and FCV in resource-limited settings. CONCLUSIONS: The one-tube dRPA-Cas12a/Cas13a assay enables simultaneously ultrasensitive and visual detection of FHV-1 and FCV with user-friendly modality, providing unparalleled convenience for FHV-1 and FCV co-infection surveillance and decision-making of URTD management.

The CDE region of feline Calicivirus VP1 protein is a potential candidate subunit vaccine.

BACKGROUND: Feline calicivirus (FCV) infection causes severe upper respiratory disease in cats, but there are no effective vaccines available for preventing FCV infection. Subunit vaccines have the advantages of safety, low cost and excellent immunogenicity, but no FCV subunit vaccine is currently available. The CDE protein is the dominant neutralizing epitope region of the main antigenic structural protein of FCV, VP1. Therefore, this study evaluated the effectiveness of the CDE region as a truncated FCV VP1 protein in preventing FCV infection to provide a strategy for developing potential FCV subunit vaccines. RESULTS: Through the prediction of FCV VP1 epitopes, we found that the E region is the dominant neutralizing epitope region. By analysing the spatial structure of VP1 protein, 13 amino acid sites in the CD and E regions were found to form hydrogen bonding interactions. The results show the presence of these interaction forces supports the E region, helping improve the stability and expression level of the soluble E protein. Therefore, we selected the CDE protein as the immunogen for the immunization of felines. After immunization with the CDE protein, we found significant stimulation of IgG, IgA and neutralizing antibody production in serum and swab samples, and the cytokine TNF-α levels and the numbers of CD4+ T lymphocytes were increased. Moreover, a viral challenge trial indicated that the protection generated by the CDE subunit vaccine significantly reduced the incidence of disease in animals. CONCLUSIONS: For the first time, we studied the efficacy of the CDE protein, which is the dominant neutralizing epitope region of the FCV VP1 protein, in preventing FCV infection. We revealed that the CDE protein can significantly activate humoral, mucosal and cellular immunity, and the resulting protective effect can significantly reduce the incidence of animal disease. The CDE region of the FCV capsid is easy to produce and has high stability and excellent immunogenicity, which makes it a candidate for low-cost vaccines.

Molecular epidemiology and phylogenetic analysis of feline calicivirus in Kunshan, China.

Feline calicivirus (FCV) is a highly contagious virus in cats, which typically causes respiratory tract and oral infections. Despite vaccination against FCV being a regular practice in China, new FCV cases still occur. Antigenic diversity of FCV hinders the effective control by vaccination. This is first report which aims to investigate the molecular epidemiology and molecular characteristics of FCV in Kunshan, China. The nasopharyngeal swabs were collected from cats showing variable clinical signs from different animal clinics in Kunshan from 2022 to 2023. Preliminary detection and sequencing of the FCV capsid gene were performed to study genetic diversity and evolutionary characteristics. FCV-RNA was identified in 52 (26%) of the samples using RT-PCR. A significant association was found between FCV-positive detection rate, age, gender, vaccination status and living environment, while a non-significant association was found with breed of cats. Nucleotide analysis revealed two genotypes, GI and GII. GII predominated in Kunshan, with diverse strains and amino acid variations potentially affecting vaccination efficacy and FCV detection. Notably, analysis pinpointed certain strains' association with FCV-virulent systemic disease pathotypes. This investigation sheds light on FCV dynamics, which may aid in developing better prevention strategies and future vaccine designs against circulating FCV genotypes.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Genetic and pathogenicity analysis for the two FCV strains isolated from Eastern China.

In this study, the diversity and regularity of two new feline calicivirus (FCV) isolates, QD-7 and QD-164, were investigated. The genomes of these new strains were compared with 39 strains from the NCBI database including isolates from China, United States, Germany, South Korea, the United Kingdom and Japan. The nucleotide sequence identities ranged from 75-88%, indicating a high degree of variability. These variations were not related to distributions of the virus by time of isolation and geographical location. Cats that were experimentally infected with the new isolate QD-164 showed typical clinical symptoms of sneezing, fever and conjunctivitis and all recovered within 30 days. In contrast, QD-7 infections were asymptomatic and the virus was cleared within 16 days. These results indicate that QD-7 and QD-164 were naturally attenuated strains. NNS mutations characteristic of highly virulent strains at positions 441-443 were absent in QD-7 while QD-164 possessed an N at position 442. This indicated that mutations in regions 441-443 may be linked to disease severity.

Broad-Spectrum, Potent, and Durable Ceria Nanoparticles Inactivate RNA Virus Infectivity by Targeting Virion Surfaces and Disrupting Virus-Receptor Interactions.

There is intense interest in developing long-lasting, potent, and broad-spectrum antiviral disinfectants. Ceria nanoparticles (CNPs) can undergo surface redox reactions (Ce3+ ↔ Ce4+) to generate ROS without requiring an external driving force. Here, we tested the mechanism behind our prior finding of potent inactivation of enveloped and non-enveloped RNA viruses by silver-modified CNPs, AgCNP1 and AgCNP2. Treatment of human respiratory viruses, coronavirus OC43 and parainfluenza virus type 5 (PIV5) with AgCNP1 and 2, respectively, prevented virus interactions with host cell receptors and resulted in virion aggregation. Rhinovirus 14 (RV14) mutants were selected to be resistant to inactivation by AgCNP2. Sequence analysis of the resistant virus genomes predicted two amino acid changes in surface-located residues D91V and F177L within capsid protein VP1. Consistent with the regenerative properties of CNPs, surface-applied AgCNP1 and 2 inactivated a wide range of structurally diverse viruses, including enveloped (OC43, SARS-CoV-2, and PIV5) and non-enveloped RNA viruses (RV14 and feline calicivirus; FCV). Remarkably, a single application of AgCNP1 and 2 potently inactivated up to four sequential rounds of virus challenge. Our results show broad-spectrum and long-lasting anti-viral activity of AgCNP nanoparticles, due to targeting of viral surface proteins to disrupt interactions with cellular receptors.

Genetic Diversity and Evolution of Viruses Infecting Felis catus: A Global Perspective.

Cats harbor many important viral pathogens, and the knowledge of their diversity has been greatly expanded thanks to increasingly popular molecular sequencing techniques. While the diversity is mostly described in numerous regionally defined studies, there lacks a global overview of the diversity for the majority of cat viruses, and therefore our understanding of the evolution and epidemiology of these viruses was generally inadequate. In this study, we analyzed 12,377 genetic sequences from 25 cat virus species and conducted comprehensive phylodynamic analyses. It revealed, for the first time, the global diversity for all cat viruses known to date, taking into account highly virulent strains and vaccine strains. From there, we further characterized and compared the geographic expansion patterns, temporal dynamics and recombination frequencies of these viruses. While respiratory pathogens such as feline calicivirus showed some degree of geographical panmixes, the other viral species are more geographically defined. Furthermore, recombination rates were much higher in feline parvovirus, feline coronavirus, feline calicivirus and feline foamy virus than the other feline virus species. Collectively, our findings deepen the understanding of the evolutionary and epidemiological features of cat viruses, which in turn provide important insight into the prevention and control of cat pathogens.

Prevalence and risk factors for common respiratory pathogens within a cohort of pet cats in the UK.

OBJECTIVES: Feline herpesvirus (FHV), feline calicivirus (FCV) and Chlamydia felis are common causes of upper respiratory tract disease (URTD) in cats. Their prevalence in the UK pet cat population has not been reported and little is known regarding the risk factors for their oral carriage. METHODS: Total nucleic acid was extracted from owner-collected buccal swabs (n=600) from cats enrolled in a self-selected longitudinal cohort study. Duplex quantitative PCRs for the detection of FHV and C. felis genomic DNA and reverse-transcriptase quantitative PCRs for the detection of FCV genomic RNA were performed. Duplicates, swabs with insufficient host DNA/RNA, and cats with missing data were excluded. Selected epidemiological data were interrogated using univariable and multi-variable logistic regression modelling to identify risk factors. RESULTS: Data from 430 cats were included in the final statistical model. Of these, 2.1% (n=9/430; 95% CI 1.0% to 3.9%) were positive for FHV, 13.3% (n=57/430; 95% CI 10.2% to 16.8%) positive for FCV and 1.2% (n=5/430; 95% CI 0.4% to 2.7%) positive for C. felis. FCV co-infection was present in five (44%) FHV-positive cats and three (60%) C. felis-positive cats. FCV carriage was more frequent in purebred cats (odds ratio 2.48; 95% CI 1.37 to 4.49) and in cats with current or historical clinical signs compatible with URTD (odds ratio 2.98; 95% CI 1.22 to 7.27). CLINICAL SIGNIFICANCE: FCV was the most frequently encountered URTD pathogen in this sample of cats; this should be noted for disinfectant choice. In cats suspected of having FHV or C. felis infection, assessment for co-infection with FCV is recommended.

Development and application of a dual ERA method for the detection of Feline Calicivirus and Feline Herpesvirus Type I.

Feline calicivirus (FCV) and feline herpesvirus type I (FHV-1) are the most common viral pathogens responsible for cat respiratory diseases, and coinfection with these two pathogens is often found. In veterinary clinics, the main diagnostic methods for FCV and FHV-1 are test strips and polymerase chain reaction (PCR). However, the sensitivity of test strips are not sufficient, and PCR is time-consuming. Therefore, developing a rapid and high-performance clinical diagnostic test is imperative for the prevention and treatment of these diseases. Enzymatic recombinase amplification (ERA) is an automated isothermal nucleic acid amplification technique that maintains a constant temperature, and is both rapid and highly accurate. In this study, a dual ERA method was developed using the Exo probe for a differential detection of FCV and FHV-1. This dual ERA method demonstrated high performance with the detection limit of 101 copies for both viruses, and no cross-reactions with feline parvovirus virus and F81 cells. To test the utility of the method for clinical applications, 50 nasopharyngeal swabs from cats with respiratory symptoms were collected and tested. The positive rates of FCV and FHV-1 were 40% (20/50, 95% confidence interval [CI], 26.4 to 54.8%) and 14% (7/50, 95% CI, 5.8 to 26.7%), respectively. The rate of coinfection with FCV and FHV-1 was 10% (5/50, 95% CI, 3.3 to 21.8%). These results were in agreement with those found using quantitative real-time PCR. Therefore, this dual ERA method is a novel and efficient clinical diagnostic tool for FCV and FHV-1 detection.

Feline Calicivirus P39 Inhibits Innate Immune Responses by Autophagic Degradation of Retinoic Acid Inducible Gene I.

Feline calicivirus (FCV) is a feline pathogen that can cause severe upper respiratory tract disease in cats, thus posing a major threat to their health. The exact pathogenic mechanism of FCV is still unclear, although it has been identified as having the ability to induce immune depression. In this study, we discovered that FCV infection triggers autophagy and that its non-structural proteins, P30, P32, and P39, are responsible for initiating this process. Additionally, we observed that altering autophagy levels via chemical modulation resulted in different influences on FCV replication. Moreover, our findings indicate that autophagy can modify the innate immunity induced by FCV infection, with increased autophagy further suppressing FCV-induced RIG-I signal transduction. This research provides insights into the mechanism of FCV replication and has the potential to aid in the development of autophagy-targeted drugs to inhibit or prevent FCV infection.

POSTVACCINAL VIRAL DISEASE PRESENTATION IN TWO LITTERS OF CHEETAHS (ACINONYX JUBATUS).

Protective antibody titers against core vaccines have not been standardized for cheetahs (Acinonyx jubatus) under human care. Vaccine-induced disease has been suspected after administration of modified live virus vaccine (MLVV), but it has not been confirmed as the causative agent. MLVV and killed virus vaccines (KVV) elicit humoral response in cheetahs; however, the use of both vaccines for initial immunization in cheetah cubs <6 months old within the same population has not been reported. The current case series describes viral disease presentation in two cheetah litters after using both vaccines and presents results for serum neutralization titers against feline calicivirus (FCV) and feline herpesvirus-1 (FHV-1) and hemagglutination inhibition titers against feline panleukopenia virus (FPV). For Litter 1, MLVV was administered at 6 and 9 wk old. On week 11, one male developed ocular, oral, and dermal lesions. Viral isolation recovered FCV. Because of suspected vaccine-induced FCV, KVV was administered on weeks 13 and 16. Litter 2 was vaccinated with KVV via the same vaccination schedule. Fifty-three days after the last booster, two cubs presented with ocular, respiratory, and oral clinical signs; both were PCR positive for FHV-1. Serology reported a better anamnestic response and protective titers against FCV and FPV with the protocol used with Litter 1. In Litter 2, FCV and FHV-1 titer measurement failed in three of four cubs, limiting comparison of titers between litters. In spite of limited measurements, absence of a statistical evaluation, and presence of infection, serology showed a better humoral response when MLVV was used.

Antiviral Effects of Quillaja saponaria Extracts Against Human Noroviral Surrogates.

Aqueous extracts of Quillaja saponaria Molina are US FDA approved as food additives in beverages with known antiviral activity. Due to lack of commercially available vaccines against human noroviruses (HNoVs), alternate methods to prevent their spread and the subsequent emergence of variant strains are being researched. Furthermore, HNoVs are not yet culturable at high enough titers to determine inactivation, therefore surrogates continue to be used. This research analyzed the effect of aqueous Quillaja saponaria extracts (QE) against HNoV surrogates, Tulane virus (TV), murine norovirus (MNV-1), and feline calicivirus (FCV-F9) at room temperature (RT) and 37 °C. Viruses (~ 5 log PFU/mL) were individually treated with 1:1 or 1:5 (v/v) diluted QE (pH ~ 3.75), malic acid control (pH 3.0) or phosphate-buffered saline (pH 7.2, as control) at 37 °C or RT for up to 6 h. Individual treatments were replicated three times using duplicate plaque assays for each treatment. FCV-F9 at ~ 5 log PFU/mL was not detectable after 15 min by 1:1 QE at 37 °C and RT. At RT, 1:5 QE lowered FCV-F9 titers by 2.05, 2.14 and 2.74 log PFU/mL after 0.5 h, 1 h and 2 h, respectively. MNV-1 showed marginal reduction of < 1 log PFU/mL after 15 min with 1:1 or 1:5 QE at 37 °C without any significant reduction at RT, while TV titers decreased by 2.2 log PFU/mL after 30 min and were undetectable after 3 h at 37 °C. Longer incubation with higher QE concentrations may be required for improved antiviral activity against MNV-1 and TV.

Using a gradient boosted model for case ascertainment from free-text veterinary records.

Case ascertainment for prevalence and incidence studies from veterinary clinical data poses a major challenge because medical notes are not consistently structured or complete. Using natural language processing (NLP) and machine learning, this study aimed to obtain accurate case recognition for feline upper respiratory tract infections (primarily caused by viruses such as feline herpes virus (FHV-1) and feline calici virus (FCV), and bacteria such as Chlamydophila felis, Mycoplasma felis and Bordetella bronchiseptica using retrospective electronic veterinary records from the Royal Society for Prevention of Cruelty to Animals, Queensland (RSPCA Qld). Data cleaning and NLP on eight years of free-text veterinary records from RSPCA Queensland was carried out to derive text-based predictors. The NLP steps included sorting records by length of stay, vectorising, tokenising and spell checking against a bespoke veterinary database. A gradient boosted model (GBM) was trained to predict the probability of each animal having a diagnosis of upper respiratory infection. A manually annotated dataset was used for training the algorithm to learn dominant patterns between predictors (frequencies of n-grams) and responses (manual binary case classification). The GBM's performance was tested against an out of sample validation dataset, and model agnostics were used to interrogate the model's learning process. The GBM used patient-level frequencies of 1250 unique n-grams as predictor variables and was able to predict the probability of cases in the validation dataset with an accuracy of 0.95 (95% CI 0.92, 0.97) and F1 score of 0.96. Predictors that exerted the highest influence on the model included frequencies of "doxycycline", "flu", "sneezing", "doxybrom" and "ocular". The trained GBM was deployed on the full dataset spanning eight years, comprising 60,258 clinical entries. The prevalence in the full dataset was predicted to be 23.59%, which is in line with domain expertise from practicing veterinarians at the shelter. Case ascertainment is a crucial step for further epidemiological study of cat flu. Ultimately, this tool can be extended to other clinical procedures, conditions, and diseases such as intensive care treatment due to snake bites and tick paralysis, physical injuries such as orthopaedic fractures or chest injuries and labour-intensive infectious diseases like parvovirus, canine cough, and ringworm, all of which require prolonged quarantine and care.

A cDNA-based reverse genetics system for feline calicivirus identifies the 3' untranslated region as an essential element for viral replication.

Virulent systemic feline calicivirus (VS-FCV) is a newly emerging FCV variant that is associated with a severe acute multisystem disease in cats that is characterized by jaundice, oedema, and high mortality (approximately 70%). VS-FCV has spread throughout the world, but there are no effective vaccines or therapeutic options to combat infection. VS-FCV may therefore pose a serious threat to the health of felines. The genomic characteristics and functions of VS-FCV are still poorly understood, and the reason for its increased pathogenicity is unknown. Reverse genetics systems are powerful tools for studying the molecular biology of RNA viruses, but a reverse genetics system for VS-FCV has not yet been reported. In this study, we developed a plasmid-based reverse genetics system for VS-FCV in which infectious progeny virus is produced in plasmid-transfected CRFK cells. Using this system, we found that the 3' untranslated region (UTR) and poly(A) tail are important for maintaining the infection and replication capacity of VS-FCV and that shortening of the poly(A) tail to less than 28 bases eliminated the ability to rescue infectious progeny virus. Whether these observations are unique to VS-FCV or represent more-general features of FCV remains to be determined. In conclusion, we successfully established a rapid and efficient VS-FCV reverse genetics system, which provides a good platform for future research on the gene functions and pathogenesis of VS-FCV. The effects of the deletion of 3' UTR and poly(A) tail on VS-FCV infectivity and replication also provided new information about the pathogenesis of VS-FCV.

A Retrospective Study of Viral Molecular Prevalences in Cats in Southern Italy (Campania Region).

From 2019 to 2021, a retrospective molecular study was conducted in the Campania region (southern Italy) to determine the prevalence of viral diseases in domestic cats. A total of 328 dead animals were analyzed by Real-Time PCR for the presence of feline panleukopenia virus (FPV), feline leukemia virus (FeLV), feline enteric coronavirus (FCoV), rotavirus (RVA), feline herpesvirus type 1 (FHV-1), and feline calicivirus (FCV). The possible presence of SARS-CoV-2 was also investigated by Real-Time PCR. The cats included in this study were specifically sourced and referred by local veterinarians and local authorities to the Zooprofilactic Experimental Institute of Southern Italy (IZSM) for pathological evaluation. The samples consisted of owners, catteries, and stray cats. Results revealed: 73.5% positive cats for FPV (189/257), 23.6% for FeLV (21/89), 21.5% for FCoV (56/266), 11.4% for RVA (16/140), 9.05% for FeHV-1 (21/232), and 7.04 for FCV (15/213). In contrast, SARS-CoV-2 was never detected. FPV was more prevalent in winter (p = 0.0027). FCoV FHV-1, FCV, and RVA predominated in autumn, whereas FeLV predominated in summer. As expected, viral infections were found more frequently in outdoor and shelter cats than in indoor ones, although no statistical association was found between animal lifestyle and viral presence. The study showed a high prevalence of FPV, FeLV, and FCoV and a moderate prevalence of RVA, FHV-1, and FCV. Moreover, the prevalence of these pathogens varied among the cat populations investigated.

Molecular Characterization and Phylogenetic Analysis of Feline Calicivirus Isolated in Guangdong Province, China from 2018 to 2022.

Feline calicivirus (FCV) is a common feline infectious pathogen that mainly causes upper respiratory tract disease. To investigate the prevalence of FCV in Guangdong Province in China, a total of 152 nasal and throat swabs from cats suspected of FCV infection were collected in veterinary clinics or shelters from 2018 to 2022. The positive detection rate of FCV was 28.9% (44/152) by RT-PCR. In addition, twenty FCV isolates were successfully isolated and purified. Eleven out of twenty isolates were selected for further phylogenetic analyses based on the capsid protein VP1; our results revealed that seven isolates were in genogroup I, and four were in genogroup II. Notably, according to the whole genome phylogenetic tree, FCV-SCAU-11 was in the same branch as Korean isolates, and recombination analysis revealed that the FCV-SCAU-11 isolate showed potential recombinant events between the FCV-SH isolate and FCV-GXNN03-20 isolate. Furthermore, the virus replication kinetics indicated that FCV-SCAU-10, with clinically severe symptoms in patient cats, performed a more efficient replication in vitro. In conclusion, this study revealed the genetic diversity of FCVs in Guangdong Province, providing a reference for novel vaccine candidate strains and the development of effective strategies for preventing FCV infection in cats.