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Severe clinical diseases associated to αCoronavirus (αCoV) infections were recently demonstrated for the first time in humans and a closely related but distinct canine CoV (CCoV) variant was identified in the nasopharyngeal swabs of children with pneumonia hospitalized in Malaysia, in 2017-2018. The complete genome sequence analysis demonstrated that the isolated strain, CCoV-HuPn-2018, was a novel canine-feline-like recombinant virus with a unique nucleoprotein. The occurrence of three human epidemics/pandemic caused by CoVs in the recent years and the detection of CCoV-HuPn-2018, raises questions about the ability of these viruses to overcome species barriers from their reservoirs jumping to humans. Interestingly, in this perspective, it is interesting to consider the report concerning new CCoV strains with a potential dual recombinant origin through partial S-gene exchange with porcine transmissible gastroenteritis virus (TGEV) identified in pups died with acute gastroenteritis in 2009. The significance of the ability of CCoVs to evolve is still unclear, but several questions arisen on the biology of these viruses, focusing important epidemiological outcomes in the field, in terms of both virus evolution and prophylaxis. The new CCoV-Hupn-2018 should lead researchers to pay more attention to the mechanisms of recombination among CoVs, rather than to the onset of variants as a result of mutations, suggesting a continuous monitoring of these viruses and in particular of SARS-CoV-2.

Feline infectious peritonitis (FIP) is a fatal systemic disease of felids caused by a Coronavirus (CoV) (FIPV). In spite of its clinical relevance and impact on feline health, currently the therapeutic possibilities for treatment of FIP in cats are limited. The emergence of the pandemic Severe Respiratory Syndrome (SARS) coronavirus (CoV) type 2 (SARS-CoV-2), etiological agent of the 2019 Coronavirus Disease (COVID-19), able to infect a broad spectrum of animal species including cats, triggered the interest for the development of novel molecules with antiviral activity for treatment of CoV infections in humans and animals. Essential oils (EOs) have raised significant attention for their antiviral properties integrating and, in some cases, replacing conventional drugs. Thymus vulgaris EO (TEO) has been previously shown to be effective against several RNA viruses including CoVs. In the present study the antiviral efficacy of TEO against FIPV was evaluated in vitro. TEO at 27 μg/ml was able to inhibit virus replication with a significant reduction of 2 log10 TCID50/50 μl. Moreover, virucidal activity was tested using TEO at 27 and 270 μg/ml, over the cytotoxic threshold, determining a reduction of viral titre as high as 3.25 log10 TCID50/50 μl up to 1 h of time contact. These results open several perspectives in terms of future applications and therapeutic possibilities for coronaviruses considering that FIPV infection in cats could be a potential model for the study of antivirals against CoVs.

Feline coronavirus (FCoV) exists as two different genotypes, FCoV type I and II, each including two biotypes, feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), the latter being a virulent variant originating from the former virus. Recently, two amino acid substitutions, M1058L and S1060A, within the spike protein have been associated to the FECV/FIPV virulence change. In this study, we have analysed the frequency of detection of such mutations in FIPV and FECV strains circulating in Italian cats and obtained information about their evolutionary relationships with reference isolates. A total of 40 FCoV strains, including 19 strains from effusions or tissue samples of FIP cats and 21 strains from faecal samples of non-FIP cats, were analysed. Mutation M1058L was detected in 16/18 FCoV-I and 1/1 FCoV-II strains associated with FIP, while change S1060A was presented by two FIPV strains. By phylogenetic analysis, FCoV sequences clustered according to the genotype but not according to the biotype, with FECV/FIPV strains recovered from the same animal being closely related. Further studies are needed to better define the genetic signatures associated with the FECV/FIPV virulence shift.

Caprine alphaherpesvirus 1 (CpHV-1) induces genital lesions in its natural host similar to those caused by Human alphaherpesvirus 2 (HHV-2), commonly named herpes simplex virus 2 (HSV-2) in human patients. CpHV-1 infection in goats could represent a useful homologous animal model for the study of HSV-2 infection, chiefly for the assessment of antiviral drugs in in vivo studies. PHA767491 is a potent inhibitor of HSV-1 and HSV-2, being able to limit replication of HHVs both in vitro and in the mouse model. In the present study the antiviral efficacy of PHA767491 against CpHV-1 was evaluated in vitro in MDBK cells. PHA767491 inhibited significantly CpHV-1 replication in a dose-dependent fashion by up to 2.50 log10 TCID50/50 μl and was able to decrease viral DNA by nearly 8 log10. These findings confirm that PHA767491 is highly effective not only against simplexviruses (HSV-1 and HSV-2), but also against the varicellovirus CpHV-1. Experiments will be necessary to assess whether PHA767491 is suitable for treatment of vaginal lesions in CpHV-1-goat model. This could provide hints for the therapy of genital alphaherpesvirus infections in humans.

Pestiviruses are responsible for widespread diseases affecting cattle, pigs and other ruminants, presenting a wide range of clinical manifestations, with significant impact on animal production. Given the recent various reports of a relatively high number of new strains and atypical genomic variants, in the present study, ninety-seven genomic sequences from southern Italy have been evaluated applying the palindromic nucleotide substitutions method, based on 5’-UTR secondary structure alignment and computing genetic distance among strains in the internal ribosome entry site. Sequence analysis revealed a highly heterogeneous virus population, indicating the introduction of virus variants of Bovine viral diarrhea virus and Border disease virus species from foreign countries. The application of different analytical procedures was useful to avoid interpretation difficulties. Circulation of heterogeneous virus populations showed the need for more accurate epidemiological investigations and stringent veterinary controls to protect animal health and welfare.

Feline calicivirus (FCV) is a contagious viral pathogen that usually causes a mild, self-limiting respiratory disease. More recently, highly virulent FCV strains have emerged and have been associated with severe systemic infection, referred to as virulent systemic disease (VSD). The objective of this study is to report VSD cases in Italian cats along with the molecular characterization of two detected FCV strains. Three client-owned cats showed clinical signs resembling to those described for VSD cases. The cats were subjected to molecular investigations for detection of FCV and other feline pathogens. Histopathology and immunohistochemistry were performed on internal organs of one cat; molecular characterization of two detected FCV strains was obtained through sequence and phylogenetic analyses. Putative VS-FCV strains were detected in all three cats, which were co-infected with feline panleukopenia virus. The cat submitted to histopathology and immunohistochemistry displayed severe histological changes and FCV antigens in internal organs. Two Italian FCV strains, for which amplification of ORF2 was successful, were strictly related and formed a unique phylogenetic cluster. These viruses did not show consistent changes in the amino acid sequences with respect to reference VS-FCVs. The results of our study confirm that VS-FCV strains are circulating in Italy and that VSD diagnosis is complicated since both genetic and clinical markers have not been identified so far.

BRD is associated with infectious agents, but management and transport-stress are trigger factors. Metaphylactic administration of antimicrobial reduces colonization of respiratory tract by pathogens, but the development of antibiotic-resistance raises public health concerns leading to propose new control strategies. The study analyzed nasopharyngeal swabs of 231 imported cattle, 10% of 49 trucks, transported from France to southern Italy and, through Real-time PCR identified the prevalence of the involved pathogens speculating on strategies to reduce the impact of BRD. The samples were tested by Real-time PCR, for the detection of bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus (BPiV), bovine adenovirus (BAdV), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Yates-corrected chi squared, or Fisher's exact test were used to compare both animal-health status and positivity/negativity to pathogens, and the relationship between presence/absence of clinical signs and Real-time PCR-positivity. H. somni and BCoV were the most frequently identified pathogens. In BRD-diagnosed cattle, BAdV was detected in 13.8% (19/138), BRSV in 14.5% (20/138) and BPiV in 4.3% (6/138). Healthy cattle were mostly positive for H. somni (89.2%, 83/93). A statistically significant association was observed between clinical signs and positivity to M. haemolytica (p value = 0.016). Although mass-medication and vaccination are used for BRD control, it still remains a primary health problem. Our results highlight that the nasopharyngeal microbiota could be affected by transport and that strategies to enhance calf immunity for reducing BRD-risk development would be more effective if applied at farm of origin prior to loading.

Astroviruses (AstVs) have been identified in the stools of calves with enteritis and in the brain tissues of bovines with encephalitis but their pathogenic role has not been clarified. In this study, we report the detection and characterization of bovine and water buffalo AstV strains identified in young bovine and buffalo calves with enteritis in Italy between 2012 and 2015. By negative staining transmission electron microscopy (TEM) observation, AstV-like particles were identified in the stools of the animals and AstV RNA was confirmed molecularly. The sequence (~3.2-kb) at the 3′ end of the genome was determined for two bovine and two buffalo AstVs. Sequence and phylogenetic analysis on the partial ORF1b and full-length ORF2 revealed a marked genetic diversity although the viruses were distantly related to other AstV identified from ruminants. Gathering sequence information on ruminant AstVs is important to understand the extent of inter-species circulation and for the development of reliable, specific diagnostic tools., Highlights • Astroviruses in ruminants are neglected enteric viruses. • Recently, neurotropic strains have been identified in ruminants. • Astrovirus was detected in outbreaks of enteritis in cattle and buffalo in Italy. • Genome sequencing of the viruses unveiled marked genetic diversity. • Implementing the diagnostics is pivotal to conceive surveillance studies.

The emergence of human alphaherpesvirus strains (i.e. HHV-1 and -2) resistant to commonly used antiviral drugs has prompted the research for alternative, biologically active anti-herpetic agents. Natural-product and synthetic α-hydroxytropolones (αHTs) have been identified as lead therapeutic agents for a number of infections, including HHV-1 and -2, and several veterinary herpesviruses, i.e. bovine alphaherpesvirus 1 (BoHV-1), equine alphaherpesvirus 1 (EHV-1) and feline alphaherpesvirus 1 (FHV-1). In the present study we evaluated the activity in vitro of two natural and two synthetic α-hydroxytropolones (αHTs) against Caprine alphaherpesvirus 1 (CpHV-1) which is regarded as a useful homologous animal model for the study of HSV-2 infection, chiefly for the assessment of antiviral drugs in in vivo studies. AlphaHTs were able to decrease significantly CpHV-1 viral titres up to 4.25 log10 TCID50/50 μl and suppressed extensively CpHV-1 nucleic acids up to 8.71 log10 viral DNA copy number/10 μl. This study demonstrated the efficacy of αHTs against CpHV-1 in vitro, adding to their activity observed against the human and animal alphaherpesviruses in vitro. The activity of αHTs against CpHV-1 appeared similar but not identical to the patterns of activity observed against other alphaherpesviruses, suggesting virus-related variability in terms of response to specific αHT molecules. These findings open several perspectives in terms of future studies using the CpHV-1 homologous animal model, for the development of therapeutic tools against herpesviruses.

Caprine herpesvirus 1 (CpHV-1) infection in goats induces genital vesicular-ulcerative lesions that strictly resemble the lesions induced by herpesvirus 2 in the human host. The immunosuppressive drug Mizoribine (MIZ) was found to increase the antiviral activity of Acyclovir (ACV) against herpesvirus infections, raising interesting perspectives on new combined therapeutic strategies. In this study the anti-CpHV-1 activity in vitro of ACV alone or in combination with MIZ was characterized. When applied alone at non-toxic concentrations, ACV had a slight effect on CpHV-1 replication while in combination with MIZ a dose-dependent inhibition of the virus yield was observed with an IC50 of ACV of 28.5 µM. These findings suggest that combined therapy of ACV and MIZ is potentially exploitable in the treatment of genital infection by herpesviruses.

Unlike the original canine parvovirus type 2 (CPV-2), CPV-2 variants have gained the ability to replicate in vivo in cats but there is limited information on the disease patterns induced by these variants in the feline host. During 2008, two distinct cases of parvoviral infection were diagnosed in our laboratories. A CPV-2a variant was identified in a 3-month-old Persian kitten displaying clinical sign of feline panleukopenia (FPL) (acute gastroenteritis and marked leukopenia) and oral ulcerations, that died eight days after the onset of the disease. Two pups living in the same pet shop as the cat were found to shed a CPV-2a strain genetically identical to the feline virus and were likely the source of infection. Also, non-fatal infection by a CPV-2c strain occurred in a 2.5-month-old European shorthair kitten displaying non-haemorrhagic diarrhoea and normal white blood cell counts. By sequence analysis of the major capsid protein (VP2) gene, the feline CPV-2c strain showed 100% identity to a recent canine type-2c isolate. Both kittens had been administered multivalent vaccines against common feline pathogens including FPL virus. Whether and to which extent the FPL vaccines can protect cats adequately from the antigenic variants of CPV-2 should be assessed.

Hobi-like pestivirus, a new tentative species within genus Pestivirus, was firstly detected in foetal bovine serum batches and later associated to respiratory distress and reproductive failures in cattle. In the present study, the cross-antibody response between bovine viral diarrhoea virus 1 (BVDV-1) and the emerging pestivirus was evaluated in the sheep model. Ten sheep were immunised against BVDV-1 or Hobi-like pestivirus using inactivated preparations and the induced antibody responses were evaluated against the homologous and heterologous viruses. The results showed that heterologous antibody titres were significantly lower than the homologous ones, thus suggesting the need to develop specific vaccines against the emerging pestiviral species.

The analysis of cytokines secreted by antigen-specific lymphocytes is hampered in goats by the paucity of species-specific reagents yet it is crucial to study immune responses to infections. To overcome this limit, two commercial kits designed to measure soluble bovine IL-4 (by ELISA) and frequencies of bovine IFN-gamma secreting cells (by ELISPOT) were tested for cross-reactivity in goats. In addition, an ELISA specific to bovine/ovine IL-4 and employing two monoclonal antibodies, clones CC313 and CC314, was tested as well. Concanavalin A-stimulated caprine peripheral blood mononuclear cells (PBMCs) cultures were studied and they exhibited high levels of soluble IL-4 and high frequencies of IFN-gamma secreting cells. In addition, the two IL-4 ELISAs detected similar amounts of cytokine. To start defining the cytokine response triggered by caprine herpesvirus 1 (CpHV-1) infection, PBMC cultures were setup from goats naturally or experimentally infected with CpHV-1. High frequencies of IFN-gamma producing cells and low, when detectable, levels of soluble IL-4 were observed in CpHV-1-specific PBMC cultures from both groups of infected goats. Thus, the availability of cross-reactive research tools can expand cytokine studies in goats and can implement the research on immunity to other caprine infections.

Four outbreaks of infectious canine hepatitis (ICH) occurring in Italy between 2001 and 2006 are reported. Three outbreaks were observed in animal shelters of southern Italy, whereas a fourth outbreak involved two purebred pups imported from Hungary few days before the onset of clinical symptoms. In all outbreaks canine adenovirus type 1 (CAV-1) was identified by virus isolation and PCR. In three outbreaks, other canine viral pathogens were detected, including canine distemper virus, canine parvovirus or canine coronavirus. The present study shows that CAV-1 is currently circulating in the Italian dog population and that vaccination is still required.