Despite several data of influenza infection in dogs, the first natural outbreak of canine influenza virus, closely related to H3N8 equine subtype, dates back to 2004 in Florida. Subsequent studies highlighted the role of dogs in adaptation of H5N1 to mammals and the susceptibility of dogs to different subtypes of influenza. A prevalence study was carried out on 562 sera collected from pet and kennel dogs in the South of Italy. A c-ELISA test was employed and c-ELISA-positive, c-ELISA-doubtful and random c-ELISA-negative samples were also tested in subtype-specific HI test using H3N8 and H3N2 strains. c-ELISA detected a positivity of 3.56%. HI performed with the H3N8 revealed 2 positive samples and when performed with the H3N2, HI revealed 47 positive samples. c-ELISA showed to be a sensitive and specific technique. HI is a specific method only when the test antigen is homologous to the circulating virus and, because nonspecific-hemagglutination inhibitors may be present in dog sera, false positives can result. The study underlines that dogs, for their close contact with humans, must be a target for testing. Furthermore because it remains to be determined how long antibodies to influenza virus persist in canine sera, the observed prevalence might be underestimated.

The correct diagnosis of canine minute virus is critical in dog breeding. In this study, the Bland Altman test was used to compare the performance of two susceptibility-testing methods, namely polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA). The agreement between IFA and PCR in monocytes revealed a mean difference of -1752.16 with 95% confidence and an interval ranging from -3229.80 to -274.53 (SD=2325.62). The agreement between IFA and PCR in Walter Reed canine cells (WRCC) revealed a mean difference of -2396.55 with 95% confidence and an interval ranging from -3774.63 to -1018.48 (SD=2168.93). The Bland Altman test confirmed the overall accuracy of PCR vs IFA and the plot showed that all points were not randomly arranged in the range of average ±1.96×SD of the differences.

The fusion machinery for herpesvirus entry in the host cells involves the interactions of viral glycoproteins with cellular receptors, although additional viral and cellular domains are required. Extensive areas of the plasma membrane surface consist of lipid rafts organized into cholesterol-rich microdomains involved in signal transduction, protein sorting, membrane transport and in many processes of viruses infection. Because of the extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to the lipid rafts, we investigated the effect of cholesterol depletion by methyl-- cyclodextrin (MCD) on caprine herpesvirus 1 (CpHV.1) in three important phases of virus infection such as binding, entry and post-entry. MCD treatment did not prejudice virus binding to cells, while a dose-dependent reduction of the virus yield was observed at the virus entry stage, and 30 mM MCD reduced infectivity evidently. Treatment of MDBK after virus entry revealed a moderate inhibitory effect suggesting that cholesterol is mainly required during virus entry rather than during the post-entry stage. Alteration of the envelope lipid composition affected virus entry and a noticeable reduction in virus infectivity was detected in the presence of 15 mM MCD. Considering that the recognition of a host cell receptor is a crucial step in the start-up phase of infection, these data are essential for the study of CpHV.1 pathogenesis. To date virus receptors for CpHV.1 have not yet been identified and further investigations are required to state that MCD treatment affects the expression of the viral receptors.

Pestiviruses have a worldwide distribution where ruminants farming is extensive and infection in their hosts can vary from subclinical manifestations to severe clinical signs. Although biomolecular methods are successfully employed for pestiviruses identification, they require the presence of the virus at the time of sampling. Because persistent infection is unusual in goats and acute infection is transient, for a retrospective assessment serology is the most useful approach to evaluate pestiviruses spread among ruminants. The prevalence of pestiviruses in the Italian goat population was the main target of the study and the possible influence of the co-habitation with cattle on the seropositivity of goats was taken into account. A total of 7096 sera from healthy goats were tested using an indirect ELISA. The positive samples were confirmed with a virus neutralization (VN) test and were screened for BDV too. The ELISA assay identified 57 farms (33.13%) with positive goats and an overall seropositivity of 1.63%, higher in Calabria (1.74%, 95% CI 1.68% to 1.79%) than in Apulia (1.59%, 95% CI 1.52% to 1.66%). A higher variability among farms was observed, with a significant influence of multi-species (goat and cow) grazing. Despite the huge economic losses, the impact on small ruminant productions in Italy has not yet been assessed and pestivirus infection is largely underestimated and scarcely considered. Nevertheless, the focus on pestiviruses of small ruminants should be strengthened. Considering that pestivirus infections are often subclinical, serological surveillance, the prerequisite for the implementation of control programmes, should be strongly recommended and should be considered in any pestivirus eradication programme, especially in areas such as the Southern Italy, where small ruminants farming is predominant.

In recent years, bats have been found to harbour many viruses, raising several questions about their role as reservoirs and potential disseminators of zoonotic viruses. We investigated the presence of six virus families in bats in three regions of Central‐ Southern Italy. Astroviruses were identified in seven of 13 bat species. Sequence analysis revealed marked genetic heterogeneity among the astroviruses identified, with nucleotide identity ranging between 60.26% and 87.62%. Astrovirus diversity was not associated with the bat species, the geographic areas or the bat colony, suggesting the circulation of several astrovirus strains in Italian ecosystems. Genetic diversification and interspecies transmission appear common in bat astroviruses and could provide, potentially, the bases for transmission to humans and other mammals. Yet overemphasizing this risk might have detrimental consequences for bat conservation and preservation of the important ecosystem services bats provide

Virus entry into and release from epithelial cells are polarized as a result of the distribution of the viral receptors. In order to establish the polarity of entry and release of CCoV from epithelial cells, the interactions of the virus with A72 and CrFK cells grown on permeable supports was evaluated, and the amount of infective virus in the apical and in the basolateral media was determined and compared. Infection of A72 cells after different times post seeding demonstrated that CCoV grow after infection from both apical and basolateral sides. In CrFK cells, CCoV was observed in both compartments only in the later phase of the infection. To establish the reciprocal binding of CCoV on plasma membrane, A72 cells on a permeable support were preincubated with a mAb specific for CCoV. Infection from the apical side was blocked by mAb applied to that side; in contrast, such treatment on the basolateral side had no effect on the infectious process. Similarly, the low levels of CCoV observed after basolateral exposure to virus was abolished following mAb treatment of that side. The identification of CCoV into the basolateral medium could play an important role in the viral pathogenesis.

Coronaviruses are enveloped RNA viruses that have evolved complex relationships with their host cells, and modulate their lipid composition, lipid synthesis and signalling. Lipid rafts, enriched in sphingolipids, cholesterol and associated proteins, are special plasma membrane microdomains involved in several processes in viral infections. The extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to lipid rafts. Because cholesterol-rich microdomains appear to be a general feature of the entry mechanism of non-eneveloped viruses and of several coronaviruses, the purpose of this study was to analyse the contribution of lipids to the infectivity of canine coronavirus (CCoV). The CCoV life cycle is closely connected to plasma membrane cholesterol, from cell entry to viral particle production. The methyl-β-cyclodextrin (MβCD) was employed to remove cholesterol and to disrupt the lipid rafts. Cholesterol depletion from the cell membrane resulted in a dose-dependent reduction, but not abolishment, of virus infectivity, and at a concentration of 15 mM, the reduction in the infection rate was about 68 %. MβCD treatment was used to verify if cholesterol in the envelope was required for CCoV infection. This resulted in a dose-dependent inhibitory effect, and at a concentration of 9 mM MβCD, infectivity was reduced by about 73 %. Since viral entry would constitute a target for antiviral strategies, inhibitory molecules interacting with viral and/or cell membranes, or interfering with lipid metabolism, may have strong antiviral potential. It will be interesting in the future to analyse the membrane microdomains in the CCoV envelope.

Canine herpesvirus 1 (CaHV-1) is known to cause reproductive disorders in adult dogs and neonatal mortality in puppies. Seroprevalence for CaHV-1 has been investigated worldwide, but specific epidemiological studies have not been documented in Italy. Serum samples from 865 dogs were screened for CaHV-1 using a sero-neutralization assay (SN). The CaHV-1 positive sera and 100 CaHV-1 negative sera were also tested using an in-house Immunofluorescence (IF) test. Thirteen bitches with reproductive disorders and 3 bitches with no history of reproductive diseases were examined clinically in order to identify CaHV-1 associated lesions and CaHV-1 DNA in vaginal swabs by PCR. An overall seroprevalence of 14.6% was observed in SN, and of 18.6% in IF. The correlation between SN test and IF was moderate and the SN assay demonstrated a greater sensitivity, with a few exceptions. None of the vaginal swabs tested positive for CaHV-1 DNA. The differences in the sero-positive rates between SN and IF were not statistically significant (P = 0.16) by the chi-squared test. Using the SN test as the reference standard, the sensitivity and specificity of IF were 29% and 95%, respectively. These results suggest that CaHV-1 is common in canine population and may pose a threat to neonatal survival and canine fertility in breeding kennels in Italy. Vaccination of breeding bitches should be recommended if there is a history of reproductive disorders.

Since the first identification of the virus in 1971, the disease caused by canine coronavirus (CCoV) has not been adequately investigated, and the role that the virus plays in canine enteric illness has not been well established. Only after the emergence in 2002 of SARS in human has new attention been focused on coronaviruses. As a consequence of the relatively high mutation frequency of RNA-positive stranded viruses, CCoV has evolved and, with the biomolecular techniques developed over the last two decades, new virus strains, serotypes, and subtypes have been identified in infected dogs. Considering the widespread nature of CCoV infections among dog populations, several studies have been carried out, focusing upon the epidemiological relevance of these viruses and underlining the need for further investigation into the biology of CCoVs and into the pathogenetic role of the infections. This paper reports the evolutionary processes of CCoVs with a note onto recent diagnostic methods.