Detection of Influenza D Antibodies in Dogs, Apulia Region, Italy, 2016 and 2023.

Dogs are known to be susceptible to influenza A viruses, although information on influenza D virus (IDV) is limited. We investigated the seroprevalence of IDV in 426 dogs in the Apulia region of Italy during 2016 and 2023. A total of 14 samples were positive for IDV antibodies, suggesting exposure to IDV in dogs.

ERDRP-0519 inhibits feline coronavirus in vitro.

BACKGROUND: Coronaviruses (CoVs) are major human and animal pathogens and antiviral drugs are pursued as a complementary strategy, chiefly if vaccines are not available. Feline infectious peritonitis (FIP) is a fatal systemic disease of felids caused by FIP virus (FIPV), a virulent pathotype of feline enteric coronavirus (FeCoV). Some antiviral drugs active on FIPV have been identified, but they are not available in veterinary medicine. ERDRP-0519 (ERDRP) is a non-nucleoside inhibitor, targeting viral RNA polymerase, effective against morbilliviruses in vitro and in vivo. RESULTS: The antiviral efficacy of ERDRP against a type II FIPV was evaluated in vitro in Crandell Reese Feline Kidney (CRFK) cells. ERDRP significantly inhibited replication of FIPV in a dose-dependent manner. Viral infectivity was decreased by up to 3.00 logarithms in cell cultures whilst viral load, estimated by quantification of nucleic acids, was reduced by nearly 3.11 logaritms. CONCLUSIONS: These findings confirm that ERDRP is highly effective against a CoV. Experiments will be necessary to assess whether ERDRP is suitable for treatment of FIPV in vivo.

In vitro virucidal activity of mouthwashes on SARS-CoV-2.

OBJECTIVES: The objective of the study was to evaluate the in vitro virucidal activity of commercial mouthwashes against SARS-CoV-2 and variants of concern. MATERIALS AND METHODS: Antiviral activity was assessed at different time intervals, based on common use of these products by titrating residual viral infectivity on Vero E6 cells. RESULTS: All the mouthwashes were effective to reduce the infectious titers of SARS-CoV-2 and its tested variants. Mouthwashes Listerine® Cool Mint milder taste and Listerine® Cavity Protection milder taste reduced the infectious viral titer by up to 3.9 log10 after 30 s, while mouthwash Cetilsan® Sugar Free was able to reduce the viral titer by 2.2-2.9 log10 at all tested time intervals. Mouthwash Curasept® ADS DNA Intensive treatment was less effective to decrease viral infectivity (0.7-2.2 log10 TCID50/ml at all tested time intervals). Interestingly, the Gamma variant appeared more resistant to treatment in vitro with the different mouthwashes. CONCLUSIONS: In this study, we were able to assess the ability of different mouthwashes to in vitro decrease the infectivity of SARS-CoV-2 and its variants, and we observed that Gamma variant of concern was more resistant to treatment with mouthwashes.

Virucidal activity in vitro of mouthwashes against a feline coronavirus type II.

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can occur through saliva and aerosol droplets deriving from the upper aerodigestive tract during coughing, sneezing, talking, and even during oral inspection or dental procedures. The aim of this study was to assess in vitro virucidal activity of commercial and experimental mouthwashes against a feline coronavirus (FCoV) strain. Commercial and experimental (commercial-based products with addition of either sodium dodecyl sulfate (SDS) or thymus vulgaris essential oil (TEO) at different concentrations) mouthwashes were placed in contact with FCoV for different time intervals, that is, 30 s (T30), 60 s (T60), and 180 s (T180); subsequently, the virus was titrated on Crandell Reese Feline Kidney cells. An SDS-based commercial mouthwash reduced the viral load by 5 log10 tissue culture infectious dose (TCID)50 /50 µl at T30 while a cetylpyridinium (CPC)-based commercial mouthwash was able to reduce the viral titer of 4.75 log10 at T60. Furthermore, five experimental mouthwashes supplemented with SDS reduced the viral titer by 4.75-5 log10 according to a dose- (up to 4 mM) and time-dependent fashion.

Identification of a Novel α-herpesvirus Associated with Ulcerative Stomatitis in Donkeys.

An outbreak of ulcerative stomatitis was observed in a donkey (Equus asinus) dairy herd. Similar lesions were also observed on the dams' udders and, sporadically, in genital areas. The lesions typically resolved in 1-3 weeks. An α-herpesvirus, Varicellovirus, genetically related to equid herpesvirus type 3, was identified.

Isolation and characterization of bovine alphaherpesvirus 2 strain from an outbreak of bovine herpetic mammillitis in a dairy farm.

BACKGROUND: Bovine alphaherpesvirus type 2 (BoHV-2) belongs to family Herpesviridae, subfamily Alphaherpesviridae and can cause two distinct, well-defined conditions: a generalized benign skin infection that somewhat mimics lumpy skin disease (LSD), referred to as Pseudo-Lumpy Skin Disease (PSLD) and a localized ulcerative mammillitis, referred to as Bovine Herpetic Mammillitis (BHM). BHM is a localized form of BoHV-2 infection that causes erosive-ulcerative self-limiting lesions on breast and nipples. BHM is chiefly a disease of lactating dairy cows and has been described sporadically in several countries. In this study we describe an outbreak of bovine herpetic mammillitis caused by BoHV-2 occurred in a dairy farm in Southern Italy. Clinical signs were observed in 26/59 lactating cows with the age ranging between 2 and 6 years. The affected animals were afebrile, showed lesions on the skin of nipples, breast and ventral surface of the abdomen, near the mammary veins and spontaneously recovered within 2 months. RESULTS: BoHV-2 DNA was detected in the crust samples by pan-herpes PCR and real-time quantitative PCR. The virus was isolated on bovine kidney cells and was characterised by deep sequencing technologies. The nucleotide identity to BoHV-2 of the strain ITA/2018/468 retrieved in this study ranged from 98.83 to 100%. Phylogenetic analyses based on three full-length gene (glycoprotein B, thymidine kinase and glycoprotein G) sequences confirmed the close relatedness of the strain ITA/2018/468 to BoHV-2 sequences. CONCLUSIONS: The report represents a significant outbreak of BHM in a dairy farm 50 years after the last description in Italy. However, outbreaks of PLSD have been described in Europe recently, indicating that the virus is present in European territories. Improving the diagnostic algorithms and enacting specific surveillance plans could be useful to understand better the epidemiological and pathogenetic patterns of BoHV-2 infection in livestock animals, and to develop, eventually, effective prophylaxis plans.

Novel Parvovirus Related to Primate Bufaviruses in Dogs.

A novel protoparvovirus species, related genetically to human bufaviruses, was identified in dogs with respiratory signs. The canine bufavirus was distantly related to the well-known canine protoparvovirus, canine parvovirus type 2, sharing low amino acid identities in the nonstructural protein 1 (40.6%) and in the capsid protein 1 (33.4%). By screening collections of fecal, nasal, and oropharyngeal samples obtained from juvenile dogs (<1 year of age), canine bufavirus DNA appeared as a common component of canine virome. The virus was common in the stool samples of dogs with or without enteric disease and in the nasal and oropharyngeal swab samples of dogs with respiratory signs. However, the virus was not detected in nasal and oropharyngeal swab samples from animals without clinical signs.

Detection and Full-Length Genome Characterization of Novel Canine Vesiviruses.

Vesiviruses have been detected in several animal species and as accidental contaminants of cells. We detected vesiviruses in asymptomatic kennel dogs (64.8%) and symptomatic (1.1%) and asymptomatic (3.5%) household dogs in Italy. The full-length genome of 1 strain, Bari/212/07/ITA, shared 89%-90% nt identity with vesiviruses previously detected in contaminated cells.

Assessing the virucidal activity of essential oils against feline calicivirus, a non-enveloped virus used as surrogate of norovirus.

Norovirus (NoV) causes serious gastrointestinal disease worldwide and is regarded as an important foodborne pathogen. Due the difficulties of in vitro cultivation for human NoV, alternative caliciviruses (i.e., feline calicivirus, FCV, or murine NoV) have long been used as surrogates for in vitro assessment of the efficacy of antivirals. Essential oils (EOs) are natural compounds that have displayed antimicrobial and antioxidant properties. We report in vitro the virucidal efficacy of four EOs, Melissa officinalis L. EO (MEO), Thymus vulgaris L. EO (TEO), Rosmarinus officinalis L. EO (REO), and Salvia officinalis L. EO (SEO) against FCV at different time contacts (10, 30 min, 1, 4 and 8 h). At the maximum non-cytotoxic concentration and at 10- and 100- fold concentrations over the cytotoxic threshold, the EOs did not decrease significantly FCV viral titers. However, MEO at 12,302.70 µg/mL exhibited a significant efficacy decreasing the viral titer by 0.75 log10 Tissue Culture Infectious Dose (TCID50)/50 µl after 10 min as compared to virus control. In this study, virucidal activity of four EOs against FCV, was investigated. A lack of virucidal efficacy of TEO, REO and SEO at different compound concentrations and time contacts against FCV was observed whilst MEO was able to significantly decrease FCV titer.

In Vitro Antiviral and Virucidal Activity of Ozone against Feline Calicivirus.

The Caliciviridae family includes several viral pathogens of humans and animals, including norovirus (NoV), genus Norovirus, and feline calicivirus (FCV), genus Vesivirus. Due to their resistance in the environment, NoV and FCV may give rise to nosocomial infections, and indirect transmission plays a major role in their diffusion in susceptible populations. A pillar of the control of viruses resistant to an environment is the adoption of prophylaR1.6ctic measures, including disinfection. Since NoVs are not cultivatable in common cell cultures, FCV has been largely used as a surrogate of NoV for the assessment of effective disinfectants. Ozone (O3), a molecule with strong oxidizing properties, has shown strong microbicidal activity on bacteria, fungi, protozoa, and viruses. In this study, the virucidal and antiviral activities of an O3/O2 gas mixture containing O3 were tested at different concentrations (20, 35, and 50 µg/mL) for distinct contact times against FCV. The O3/O2 gas mixture showed virucidal and antiviral activities against FCV in a dose- and contact time-dependent fashion. Ozonation could be considered as a valid strategy for the disinfection of environments at risk of contamination by FCV and NoV.

Mechanism of surfactant interactions with feline coronavirus: A physical chemistry perspective.

HYPOTHESIS: Surfactants are inexpensive chemicals with promising applications in virus inactivation, particularly for enveloped viruses. Yet, the detailed mechanisms by which surfactants deactivate coronaviruses remain underexplored. This study delves into the virucidal mechanisms of various surfactants on Feline Coronavirus (FCoV) and their potential applications against more pathogenic coronaviruses. EXPERIMENTS: By integrating virucidal activity assays with fluorescence spectroscopy, dynamic light scattering and laser Doppler electrophoresis, alongside liposome permeability experiments, we have analyzed the effects of non-ionic and ionic surfactants on viral activity. FINDINGS: The non-ionic surfactant octaethylene glycol monodecyl ether (C10EO8) inactivates the virus by disrupting the lipid envelope, whereas ionic surfactants like Sodium Dodecyl Sulfate and Cetylpyridinium Chloride predominantly affect the spike proteins, with their impact on the viral membrane being hampered by kinetic and thermodynamic constraints. FCoV served as a safe model for studying virucidal activity, offering a faster alternative to traditional virucidal assays. The study demonstrates that physicochemical techniques can expedite the screening of virucidal compounds, contributing to the design of effective disinfectant formulations. Our results not only highlight the critical role of surfactant-virus interactions but also contribute to strategic advancements in public health measures for future pandemic containment and the ongoing challenge of antimicrobial resistance.

West Nile Virus Infection in Occupational Settings-A Systematic Review.

BACKGROUND: West Nile virus (WNV) is an emerging mosquito-borne neurotropic virus, belonging to the Flaviviridae family and the Orthoflavivirus genus. The effective control of WNV requires a targeted preventive strategy that also needs the identification of the higher-risk populations. Hence, this study focused on a systematic literature review of WNV-acquired infection in work-related settings and the assessment of the exposure risks among different occupational categories. METHODS: A comprehensive search was conducted to identify studies until September 2023 in multiple databases such as PubMed/MEDLINE, SCOPUS and Web of Science, according to the PRISMA 2020 statement. Risk of bias of collected papers was assessed by the ROB tool of the National Toxicology Program's Office of Health Assessment and Translation handbook. RESULTS: A total of 21 studies were included in the systematic review, out of which seventeen were observational studies and four were case reports. Workers identified as at higher risk for WNV infection were military workers, veterinarians, agricultural workers, farmers, and laboratory workers with contact with infected fluids or aerosols. CONCLUSIONS: The identification of higher-risk workers could facilitate active surveillance by occupational physicians, which could improve our understanding of the epidemiology of WNV and, in addition, could help tailor appropriate preventive recommendations, reducing the overall burden of disease in high-risk areas.

Serological Evidence for Circulation of Influenza D Virus in the Ovine Population in Italy.

Influenza D virus (IDV) is a novel orthomyxovirus initially isolated from pigs exhibiting influenza-like disease in the USA. Since then, IDV has been detected worldwide in several host species, including livestock animals, whilst specific antibodies have been identified in humans, raising concerns about interspecies transmission and zoonotic risks. Few data regarding the seroprevalence of IDV in small ruminants have been available to date. In this study, we assessed the prevalence of antibodies against IDV in ovine serum samples in Sicily, Southern Italy. Six hundred serum samples, collected from dairy sheep herds located in Sicily in 2022, were tested by haemagglutination inhibition (HI) and virus neutralization (VN) assays using reference strains, D/660 and D/OK, representative of two distinct IDV lineages circulating in Italy. Out of 600 tested samples, 168 (28.0%) tested positive to either IDV strain D/660 or D/OK or to both by HI whilst 378 (63.0%) tested positive to either IDV strain D/660 or D/OK or to both by VN. Overall, our findings demonstrate that IDV circulates in ovine dairy herds in Sicily. Since IDV seems to have a broad host range and it has zoonotic potential, it is important to collect epidemiological information on susceptible species.

In Vitro Virucidal Activity of Different Essential Oils against Bovine Viral Diarrhea Virus Used as Surrogate of Human Hepatitis C Virus.

The hepatitis C virus (HCV) is a major hepatotropic virus that affects humans with increased risk of developing hepatocellular carcinoma. The bovine viral diarrhea virus (BVDV) causes abortion, calf mortality and poor reproductive performance in cattle. Due the difficulties of in vitro cultivation for HCV, BVDV has been used as surrogate for in vitro assessment of the efficacy of antivirals. Essential oils (EOs) display antiviral and virucidal activity on several viral pathogens. In this study, the virucidal activity of five EOs, Salvia officinalis L. EO (SEO), Melissa officinalis L. EO (MEO), Citrus lemon EO (LEO), Rosmarinus officinalis L. EO (REO) and Thymus vulgaris L. EO (TEO) against BVDV was evaluated in vitro at different concentrations for several time contacts. MEO and LEO were able to considerably inactivate BVDV with a time- and dose-dependent fashion. MEO and LEO at the highest concentrations decreased viral titer by 2.00 and 2.25 log10 TCID50/50 µL at 8 h contact time, respectively. SEO, REO and TEO displayed mild virucidal activity at the highest concentrations for 8 h contact times. In this study, the virucidal efficacies of MEO and LEO against BVDV were observed regardless of compound concentration and contact time. Further studies are needed to confirm the potential use of MEO and LEO as surface disinfectants.

Identification and characterization of a novel circovirus in Iberian lynx in Spain.

Circoviruses cause severe disease in pigs and birds. Canine circovirus has thus far only been associated with respiratory and gastrointestinal disorders and systemic disease in dogs. The Iberian lynx (Lynx pardinus) is one of the most endangered carnivores in Europe and the most endangered felid worldwide. Exploring the virome of these animals may be important in terms of virus discovery and assessing the interspecies-circulation of viruses from related carnivores. In this study, 162 spleen samples from Iberian lynx were screened for CRESS DNA viruses. Overall, 11 (6.8%) of 162 samples tested positive using a consensus PCR. Partial rep sequences were tightly related to each other (96.6-100%). Specific molecular protocols were designed on the partial rep sequences of the novel virus, Iberian lynx-associated circovirus-1 (ILCV-1). By screening a subset of 45 spleen samples, the infection rate of ILCV-1 in Iberian lynxes was 57.8% (26/45). ILCV-1 strains formed a separate cluster intermingled with bat, rodent, mongoose, and felid circoviruses. The genome of the novel virus displayed the highest nucleotide identity (64.3-65.3%) to mongoose circoviruses, thus representing a novel candidate circovirus species. The detection of these viruses in the spleen tissues could suggest systemic infection in the animal host. Overall, these findings suggest that this novel circovirus is common in the Iberian lynx. Further studies are warranted to assess the possible health implications of ILCV-1 in this endangered species.

Assessing Contact Time and Concentration of Thymus vulgaris Essential Oil on Antibacterial Efficacy In Vitro.

The overuse and misuse of antibiotics can pose the risk of spreading mutant strains that show antimicrobial resistance (AMR), with negative impacts on the management of bacterial infections and economic implications for healthcare systems. The research and development of natural antibacterial agents could be a priority in the next years to improve a number of effective antibacterial molecules and to reduce the AMR phenomenon and its development. The present study identified the most effective concentration and contact time of Thymus vulgaris L. essential oil (TEO) to obtain bactericidal effects in vitro against different Gram-positive and Gram-negative bacterial strains. Six clinically isolated (wild types) bacterial strains, (Citrobacter freundii, Enterococcus feciorum, Proteus mirabilis, Acinetobacter cioffi, Pseudomonas putrefaciens and Klebsiella pneumoniae) and two ATCCs (Staphylococcus aureus and Streptococcus mutans) were tested after 1 min, 3 min and 5 min of contact with TEO. The preliminary results on S. aureus after 24 h of incubation revealed a TEO concentration of 9.28 mg/mL (w/v) that completely inhibited bacteria growth, keeping cell viability. The total suppression of bacterial growth at all tested contact times was observed for all tested bacterial strains, and the results were confirmed after 48 h of incubation. Bacterial growth suppression was confirmed even with the presence of organic components. These preliminary results showed the in vitro antimicrobial efficacy of TEO against different Gram-positive and Gram-negative bacterial strains. Future studies are necessary to confirm the reproducibility of these results even on other strains and to define the exact molecular mechanisms of EOs in order to consider TEO as a valid alternative to classic antibiotic therapies and subsequently to reduce the occurrence of AMR.

In Vitro Activity of Ozone/Oxygen Gaseous Mixture against a Caprine Herpesvirus Type 1 Strain Isolated from a Goat with Vaginitis.

Alphaherpesviruses cause genital lesions and reproductive failure in both humans and animals. Their control is mainly based on prevention using hygienic prophylactic measures due to the absence of vaccines and limitations of antiviral drug therapy. Ozone is an oxidating gas showing a strong microbicidal activity on bacteria, fungi, viruses, and protozoa. The present study assessed the in vitro virucidal and antiviral activity of ozone against caprine herpesvirus type 1 (CpHV-1). The virucidal activity of a gaseous mixture containing O3 at 20 and 50 µg/mL was assessed against the virus at different contact times (30 s, 60 s, 90 s, 120 s, 180 s, and 300 s). Antiviral activity of a gaseous mixture containing O3 at 20 and 50 µg/mL was evaluated against the virus after 30 s and 60 s. Ozone displayed significant virucidal activity when used at all the tested concentrations whilst significant antiviral activity was observed using ozone at 50 µg/mL. The gaseous mixture, tested in the present study, showed virucidal and antiviral activity against CpHV-1 in a dose- and time contact-dependent fashion. Ozone therapy could be evaluated in vivo for the treatment of CpHV-1-induced genital lesions in goats using topical applications.

Analysis of oral microbiota in non-vital teeth and clinically intact external surface from patients with severe periodontitis using Nanopore sequencing: a case study.

Periodontal diseases include a wide range of pathological conditions, damaging the supporting structures of the teeth. Origin and propagation of periodontal disease is believed to be caused by dysbiosis of the commensal oral microbiota. The aim of this study was to evaluate the presence of bacteria in the pulp cavity of teeth with severe periodontal disease with clinically intact external surface. Periodontal (P) and endodontic (E) tissue samples of root canals from six intact teeth of three patients were sampled for analysis of microbial population using Nanopore technology. Streptococcus was the predominant genus in E samples. Porphyromonas (33.4%, p = 0.047), Tannerella (41.7%, p = 0.042), and Treponema (50.0%, p = 0.0064) were significantly more present in P than in E samples. Some samples (E6 and E1) exhibited a remarkable difference in terms of microbial composition, whilst Streptococcus was a common signature in samples E2 to E5, all which were obtained from the same patient. In conclusion, bacteria were identified on both the root surface and the root canal system, thus demonstrating the possibility of bacteria to spread directly from the periodontal pocket to the root canal system even in the absence of crown's loss of integrity.

Identification of Prototheca from the Cerebrospinal Fluid of a Cat with Neurological Signs.

Prototheca infections are rare in cats, and they are usually associated with cutaneous or subcutaneous infections by P. wickerhamii, with no evidence of neurological signs or systemic disease. In this study, we report the identification of prototheca in the cerebrospinal fluid (CSF) of a cat with neurological symptoms. Fourteen CSF samples were gathered from cats presented with neurological disease between 2012 and 2014. The inclusion criteria for the samples were an increase in CSF protein and cell number (pleocytosis), suggestive of an infectious inflammatory status of the central nervous system (CNS). Nine samples fulfilled the inclusion criteria (inflammatory samples), while five samples, used as control, did not (non-inflammatory samples). All the samples were screened molecularly for different pathogens associated with CNS disease in cats, including prototheca. Out of 14 CSF samples, only one inflammatory sample tested positive for prototheca. Upon sequence and phylogenetic analysis of the amplicon, the strain was characterized as P. bovis. This report is the first documented evidence of prototheca in the cerebrospinal fluid of a cat with neurological signs. Prototheca should be considered in the diagnostics procedures on the CNS of cats presented with infectious diseases.

Virucidal Activity of Lemon Essential Oil against Feline Calicivirus Used as Surrogate for Norovirus.

Norovirus (NoV) is regarded as a common cause of acute gastrointestinal illness worldwide in all age groups, with substantial morbidity across health care and community settings. The lack of in vitro cell culture systems for human NoV has prompted the use of cultivatable caliciviruses (such as feline calicivirus, FCV, or murine NoV) as surrogates for in vitro evaluation of antivirals. Essential oils (EOs) may represent a valid tool to counteract viral infections, particularly as food preservatives. In the present study, the virucidal efficacy of lemon EO (LEO) against FCV was assessed in vitro. The gas chromatography hyphenated with mass spectrometry (GC/MS) technique was used to reveal the chemical composition of LEO. The following small molecules were detected as major components of LEO: limonene (53%), ß-pinene (14.5%), γ-terpinene (5.9%), citral (3.8%), α-pinene (2.4%), and ß-thujene (1.94%). LEO at 302.0 µg/mL, exceeding the maximum non cytotoxic limit, significantly decreased viral titre of 0.75 log10 TCID50/50 µL after 8 h. Moreover, virucidal activity was tested using LEO at 3020.00 µg/mL, determining a reduction of viral titre as high as 1.25 log10 TCID50/50 µL after 8 h of time contact. These results open up perspectives for the development of alternative prophylaxis approaches for the control of NoV infection.