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.

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.

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.

Circoviridae Survey in Captive Non-Human Primates, Italy.

Circoviruses (CVs) and cycloviruses (CyVs), members of the family Circoviridae, have been identified only occasionally in non-human primates (NHPs). In this study, we investigated the presence and genetic features of these viruses in 48 NHPs housed in the Bioparco-Rome Zoological Garden (Italy) and in the Anima Natura Wild Sanctuary Semproniano (Grosseto, Italy), testing fecal, saliva, and serum samples with a broadly reactive consensus nested PCR able of amplifying a partial region of the replicase (Rep) gene of members of the family Circoviridae. Viral DNA was detected in a total of 10 samples, including a saliva swab and 9 fecal samples collected, respectively from five Japanese macaques (Macaca fuscata) and four mandrills (Mandrillus sphinx), with an overall prevalence of 18.7% (9/48). On genome sequencing, five strains revealed the highest nucleotide identity (98.3-98.6%) to a CyV strain (RI196/ITA) detected in the intestinal content of a Maltese wall lizard (Podarcis filfolensis) in Italy. Although the origin of the Italian NHP strains, genetically distant from previously detected NHP CyVs, is uncertain, our results also highlight that the virome of captive animals is modulated by the different dietary and environmental sources of exposure.

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.

Identification of a novel intra-genotype reassortant G1P[8] rotavirus in Italy, 2021.

OBJECTIVES: Rotaviruses G1P[8] are epidemiologically relevant and are targeted by vaccines. The introduction of vaccines has altered rotavirus epidemiology. Hospital-based surveillance conducted in Sicily, Italy, showed a progressive decline in rotavirus prevalence since 2014, along with an increasing vaccine coverage (63.8% in 2020), and a marked decrease in circulation of G1P[8] strains. Surprisingly in 2021, G1P[8] viruses accounted for 90.5% (19/21) of rotavirus infections. This study aimed to understand if the increased activity of G1P[8]'s was related to virus-related peculiarities. DESIGN: In 2021, 266 patients <15 years of age were hospitalized with acute gastroenteritis (AGE) and included in rotavirus surveillance. Viral proteins (VP7 and VP4) genotyping and sequence data were generated from all rotavirus-positive samples. The genetic makeup of G1P[8] rotaviruses was investigated by full-genome sequencing. RESULTS: Peculiar G1P[8] rotaviruses, with VP7 and VP4 belonging to novel sub-lineages, circulated in 2021, accounting for 76.2% (16/21) of all rotavirus infections. On full-genome analysis, the novel G1P[8] variant displayed an intra-genotype (Wa-like) reassortant constellation, involving G12 and G1 strains, into a unique arrangement never observed before. The novel G1P[8] variant showed peculiar amino acid substitutions in 8-1 and 8-3 epitopes of the VP4 with respect to the Rotarix strain. CONCLUSIONS: Prompt identification of virus variants circulating in the human population is pivotal to understanding epidemiological trends and assessing vaccine efficacy.

Detection of vesivirus in minks (Neovison vison), Italy 2021.

Vesiviruses are important animal pathogens with a broad host range, and they have also been involved in accidental contamination of cells used for the production of drugs for rare and life-threatening human diseases. A vesivirus (family Caliciviridae) was detected in minks (Neovison vison) with respiratory and neurological signs, during syndromic surveillance for SARS-CoV-2 conducted in Italy. The complete genome (8,397 nucleotides in length) of the vesivirus strain ITA/2021/mink/TE (OR130287) was obtained by combining NGS approach with 5' and 3' RACE protocols. The virus was seemingly more related (95.9-97.2% nt identity in the partial RNA-dependent RNA polymerase) to American vesivirus isolates 9/1980/US, 12/1980/US, and 20/1980/US dating back to the early 1980s than to recent mink strains. These results highlight the importance of gathering information on the virome of animals.

Molecular Surveillance for Bocaparvoviruses and Bufaviruses in the European Hedgehog (Erinaceus europaeus).

The presence of bocaparvoviruses (BoVs) and bufaviruses (BuVs) in the European hedgehog (Erinaceus europaeus) was investigated by screening duodenal and liver samples collected from 183 carcasses, delivered to wildlife rescue centers located in northwestern Italy. BoV DNA was detected in 15 animals (8.2%), with prevalences of 7.1% (13/183) and 2.7% (5/183) in intestine and liver samples, respectively. Upon the sequence analyses of the NS1 gene, two highly divergent BoVs (65.5-67.8% nt identities) were identified. Fourteen strains showed the highest identity (98.3-99.4% nt) to the hedgehog BoV strains recently detected in China in Amur hedgehogs (Erinaceus amurensis), whilst four strains were genetically related (98.9-99.4% nt identities) to the porcine BoVs identified in pigs and classified in the species Bocaparvovirus ungulate 4, which included related viruses also found in rats, minks, shrews, and mice. BuV DNA was detected in the duodenal samples of two hedgehogs, with a prevalence rate of 1.1%. The nearly full-length genome of two BuV strains, Hedgehog/331DU-2022/ITA and Hedgehog/1278DU/2019/ITA, was reconstructed. Upon phylogenetic analysis based on the NS and VP aa sequences, the Italian hedgehog BuVs tightly clustered with the BuVs recently identified in the Chinese Amur hedgehogs, within a potential novel candidate species of the genus Protoparvovirus.

Metagenomic Identification of Novel Eukaryotic Viruses with Small DNA Genomes in Pheasants.

A panel of intestinal samples collected from common pheasants (Phasianus colchicus) between 2008 and 2017 was used for metagenomic investigation using an unbiased enrichment protocol and different bioinformatic pipelines. The number of sequence reads in the metagenomic analysis ranged from 1,419,265 to 17,507,704 with a viral sequence read rate ranging from 0.01% to 59%. When considering the sequence reads of eukaryotic viruses, RNA and DNA viruses were identified in the samples, including but not limited to coronaviruses, reoviruses, parvoviruses, and CRESS DNA viruses (i.e., circular Rep-encoding single-stranded DNA viruses). Partial or nearly complete genome sequences were reconstructed of at least three different parvoviruses (dependoparvovirus, aveparvovirus and chaphamaparvovirus), as well as gyroviruses and diverse CRESS DNA viruses. Generating information of virus diversity will serve as a basis for developing specific diagnostic tools and for structured epidemiological investigations, useful to assess the impact of these novel viruses on animal health.

Assessing the Prevalence of Astroviruses in Water Environments: A Systematic Review and Meta-analysis.

Astroviruses (AstVs) are a major cause of gastroenteritis, especially in children. They can be transmitted through various pathways, including environmental contamination via water matrices. This study aimed to investigate the prevalence of AstV in different types of water, such as untreated and treated wastewater, surface water (e.g., rivers, lakes, and seawater), groundwater, drinking water, and other water matrices (e.g., irrigation water, gray water, reservoir water, floodwater, and pig slaughterhouse effluents). The meta-analysis included 80 articles, and the overall prevalence of AstV in water matrices was 36.6% [95% confidence interval (CI) of 29.6-44.0]. The highest prevalence was found in untreated wastewater at 56.8% (95% CI of 41.5-71.5), followed by treated wastewater at 48.5% (95% CI of 30.6-66.5), surface water at 28.6% (95% CI of 21.1-36.7), other matrices at 9.8% (95% CI of 0.7-25.3), drinking water at 3.3% (95% CI of 0.2-8.7), and groundwater at 0.5% (95% CI of 0.0-3.4). The most frequent AstVs detected in water environments were human AstVs, but canine and feline AstVs were also detected. Our findings highlight the importance of water as a potential route for AstV transmission, even in high-income countries. Effective water surveillance and treatment measures are necessary to minimize AstV environmental circulation and human infection through water.

Hepadnavirus Infection in a Cat with Chronic Liver Disease: A Multi-Disciplinary Diagnostic Approach.

A 3-year-old female stray, shorthair cat, with clinical signs and serum chemistry markers indicative of hepatic disease, was diagnosed with domestic cat hepadnavirus (DCH) infection. Coupling molecular and serological data, the infection was seemingly contextualized into a chronic phase, since IgM anti-core antibodies, a marker of early-stage Hepatitis B Virus (HBV) infection, were not detected. However, the cat possessed IgG anti-core, a common indicator of chronic HBV infection in human patients and did not show seroconversion to the anti-DCH surface antigen, considered protective during HBV infection and associated with long-term protective immunity. On genome sequencing, the DCH strain showed 98.3% nucleotide identity to strains previously identified in Italy.

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.

Biological Specimen Banking as a Time Capsule to Explore the Temporal Dynamics of Norovirus Epidemiology.

Norovirus is recognised as a major cause of epidemic and sporadic acute gastroenteritis (AGE) in all age groups. Information on the genetic diversity of the noroviruses circulating in the 1980s and 1990s, before the development and adoption of dedicated molecular assays, is limited compared with the last decades. Between 1986 and 2020, uninterrupted viral surveillance was conducted in symptomatic children hospitalized with AGE in Palermo, Italy, providing a unique time capsule for exploring the epidemiological and evolutionary dynamics of enteric viruses. A total of 8433 stool samples were tested using real-time RT-PCR. All samples were stored at -20 or -80 °C until processing. In this 35-year long time span, noroviruses of genogroup II (GII) were detected in 15.6% of AGE requiring hospitalization, whilst GI noroviruses were detected in 1.4% of AGE. Overall, the predominant norovirus capsid (Cap) genotype was GII.4 (60.8%), followed by GII.3 (13.3%) and GII.2 (12.4%). Temporal replacement of the GII.4 Cap variants associated with different polymerase (Pol) types were observed over the study period. The chronology of emergence and circulation of the different GII.4 variants were consistent with data available in the literature. Also, for GII.3 and GII.2 NoVs, the circulation of different lineages/strains, differing in either the Cap or Pol genes or in both, was observed. This long-term study revealed the ability of noroviruses to continuously and rapidly modify their genomic makeup and highlights the importance of surveillance activities in vaccine design.

A Snapshot on the Genomic Epidemiology of Turkey Reovirus Infections, Hungary.

Reovirus infections in turkeys are associated with arthritis and lameness. Viral genome sequence data are scarce, which makes an accurate description of the viral evolution and epidemiology difficult. In this study, we isolated and characterized turkey reoviruses from Hungary. The isolates were identified in 2016; these isolates were compared with earlier Hungarian turkey reovirus strains and turkey reoviruses isolated in the 2010s in the United States. Gene-wise sequence and phylogenetic analyses identified the cell-receptor binding protein and the main neutralization antigen, σC, to be the most conserved. The most genetically diverse gene was another surface antigen coding gene, µB. This gene was shown to undergo frequent reassortment among chicken and turkey origin reoviruses. Additional reassortment events were found primarily within members of the homologous turkey reovirus clade. Our data showed evidence for low variability among strains isolated from independent outbreaks, a finding that suggests a common source of turkey reoviruses in Hungarian turkey flocks. Given that commercial vaccines are not available, identification of the source of these founder virus strains would permit a more efficient prevention of disease outbreaks before young birds are settled to fattening facilities.

Surveillance for rat hepatitis E in wastewater networks, Italy.

IMPORTANCE: Hepatitis E virus (HEV) infection constitutes a significant health problem worldwide. In recent years, in addition to the zoonotic HEV3 and HEV4, emerging highly divergent hepevirus of rat origin (rat HEV [RHEV]) has been associated with human acute and chronic hepatitis. As environmental surveillance can be a complementary tool to explore emerging viruses of human and rodent origin, we investigated the epidemiology and the genetic variability of RHEV targeting 14 wastewater treatment plants in an Italian geographic area considered a hot spot for HEV infection in humans. Our results revealed that RHEV is a significant component of the wastewater microbiota with viral RNA detected in 43.9% of the specimens tested, adding further evidence to the need to investigate more in depth the real burden of RHEV infections in humans.

Evidence of Circulation and Phylogenetic Analysis of Hepatitis E Virus (HEV) in Wild Boar in South-East Italy.

Hepatitis E virus (HEV) is an important cause of acute viral hepatitis in humans worldwide. The food-borne transmission of HEV appears to be a major route in Europe through the consumption of pork and wild boar meat. HEV epidemiology in wild boars has been investigated mainly in Northern and Central Italian regions, whilst information from Southern Italy is limited. We investigated the occurrence of HEV in wild boar in the Apulia and Basilicata regions (Southern Italy). Thirteen (10.4%) out of one hundred and twenty-five wild boar samples tested positive for HEV using a quantitative reverse transcription PCR. HEV prevalence was 12% in Apulia and 9.3% in Basilicata. Seven samples were genotyped, and different subtypes (c, f, m) of genotype 3 were identified. The complete genome of a 3m strain was determined, and the virus showed the highest nucleotide identity to a human HEV strain identified in France in 2017. These findings demonstrate the substantial circulation of HEV in the wild boar population in Italian Southern regions. Gathering information on the HEV strains circulating in different geographical areas is useful for tracking the origin of HEV outbreaks and assessing the epidemiological role of wild boar as a potential virus reservoir for domestic pigs.

An Outbreak of Limping Syndrome Associated with Feline Calicivirus.

Feline calicivirus (FCV) is a common viral pathogen found in domestic cats. FCV is highly contagious and demonstrates a high genetic variability. Upper respiratory tract disease, oral ulcerations, salivation, and gingivitis-stomatitis have been regarded as typical clinical signs of FCV infection. Ulcerative dermatitis, abortion, severe pneumonia, enteritis, chronic stomatitis, and virulent systemic disease have been reported more sporadically. Limping syndrome has been also described either in naturally or experimentally FCV-infected cats. In this study, we monitored a small outbreak of FCV infection in two household cats, in which limping disease was monitored with a 12-day lag time. The complete genome sequence was determined for the viruses isolated from the oropharyngeal and rectal swabs of the two animals, mapping up to 39 synonymous nucleotide mutations. The four isolates were sensitive to low pH conditions and trypsin treatment, a pattern usually associated with viruses isolated from the upper respiratory tract. Overall, the asynchronous pattern of infections and the results of genome sequencing suggest that a virus of respiratory origin was transmitted between the animals and that the FCV strain was able to retain the limping disease pathotype during the transmission chain, as previously observed in experimental studies with FCV strains associated with lameness.

Novel parvovirus in an outbreak of fatal enteritis in European hedgehogs (Erinaceus europaeus), Italy, 2022.

Starting from June 2022, increased mortality associated with enteric signs was reported in European hedgehogs (Erinaceus europaeus) recovered at a regional wildlife rescue center, in Apulia, Italy. Cases of enteric disease were observed until the end of the breeding season, despite increased biosafety measures. A novel parvovirus was identified using metaviromic, and parvovirus-like particles were observed in the stools on electron microscopy observation. The virus was detected in the fecal samples of all the animals tested (n = 9) and in the internal organs (liver, spleen, and kidney) of three out of nine animals using a specific quantitative assay. In the full-length genome, the parvovirus was closely related (90.4% nt) to a chaphamaparvovirus identified in an Amur hedgehog (Erinaceus amurensis) in Asia and to chaphamaparvoviruses (≤ 70% nt) detected in bats and rodents. Since chaphamaparvoviruses are considered as pathogen in rodents, it will be important to investigate the pathogenic role, if any, of these parvoviruses in hedgehogs. IMPORTANCE European hedgehogs (Erinaceus europaeus) are common in Europe. This species has been shown to harbor occasionally zoonotic pathogens, including bacteria, fungi, and viruses. Exploring the virome of wildlife animals is important for animal conservation and also to assess zoonotic risks. Our metaviromic investigation identified a novel parvovirus from an outbreak of enteritis in European hedgehogs housed in a wildlife rescue center, extending the spectrum of potential viral pathogens in this species.

Identification of new astroviruses in synanthropic squamates.

Astroviruses have been identified in a wide variety of animal species and are associated with gastro-intestinal disease in humans. Pathologies due to extra-intestinal localization are known in different hosts. We report the detection of astroviruses in synanthropic squamate reptile species (Podercis siculus and Tarentola mauritanica). Fecal samples were collected from 100 squamates from urban and peri-urban areas of three regions in South Italy and tested for the presence of astroviruses using a broadly reactive (pan-astrovirus) RT-PCR protocol targeting the RNA-dependent RNA polymerase. Astrovirus RNA was detected in 11% of the samples and for six strains a 3 kb-long fragment at the 3' end of the genome was sequenced, obtaining information on the complete capsid-encoding ORF2 sequence. Viral RNA was also detected in the brain of one of the positive animals. The sequences generated from the astrovirus strains shared low nucleotide identities in the ORF2 (< 43.7%) with other known reptilian astrovirus sequences, hinting to the massive genetic diversity of members of this viral family. Based on the partial RdRp gene of the sequenced strains, however, we observed species-specific patterns, regardless of the geographic origin of the animals, and we also identified a possible inter-species transmission event between geckoes and lizards.

Investigating the genetic diversity of CRESS DNA viruses in cats identifies a novel feline circovirus and unveils exposure of cats to canine circovirus.

Circular replication-associated protein (Rep)-encoding single stranded (CRESS) DNA viruses include Circoviruses which have been found in several animal species and in human specimens. Circoviruses are associated with severe disease in pigs and birds and with respiratory and gastrointestinal disorders and systemic disease in dogs. In cats there are only a few anecdotical studies reporting CRESS DNA viruses. In this study, a total of 530 samples (361 sera, 131 stools, and 38 respiratory swabs) from cats, were screened for the presence of CRESS DNA viruses. Overall, 48 (9.0%) of 530 samples tested positive using a pan-Rep PCR. A total of 30 Rep sequences were obtained. Ten sequences of fecal origin were tightly related to each other (82.4-100% nt identity) and more distantly related to mongoose circoviruses (68.3 to 77.2% nt identity). At genome level these circoviruses displayed the highest nt identity (74.3-78.7%) to mongoose circoviruses thus representing a novel circovirus species. Circoviruses from different animal hosts (n = 12) and from humans (n = 8) were also identified. However, six Rep sequences were obtained from serum samples, including canine circoviruses, a human cyclovirus and human and fish-associated CRESS DNA viruses. The presence of these viruses in the sera would imply, to various extent, virus replication in the animal host, able to sustain viremia. Overall, these findings indicate a wide genetic diversity of CRESS DNA viruses in cats and warrant further investigations.