Vittorio Sarchese

Detection of hepatitis E virus (HEV) in goats

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 also occur in animals and are recognized as zoonotic pathogens. In this study, by screening goat faecal samples collected from six small farms located in the province of Teramo (Abruzzo region, Italy), HEV RNA was found with an overall prevalence of 9.2% (11/119). Upon sequence analysis of a 0.8kb portion of the ORF2 gene, four strains were grouped with animal and human genotype 3 HEVs, subtype c, with the highest match (94.2-99.4% nt identity) to a wild boar strain, WB/P6-15/ITA, identified in the same geographical area in which the six goat farms were located. Further investigations are needed in order to assess if goat may represent an additional active host for HEV.

A novel feline norovirus in diarrheic cats.

Abstract By screening a collection of fecal samples from young cats housed in three different shelters in South Italy, noroviruses (NoVs) were found in 3/48 (6.2%) specimens of animals with enteritis signs while they were not detected in samples collected from healthy cats (0/57). Upon sequence analysis of the short RNA-dependent RNA polymerase (RdRp) region, the three strains displayed the highest nucleotide (nt) and amino acid (aa) identities to the prototype GIV.2 strain lion/Pistoia/387/06/ITA (91.0–93.0% nt and 97.0–98.0% aa). The sequence of ~3.4-kb portion at the 3′ end of the genome of a NoV strain, TE/77-13/ITA, was determined. In the full-length ORF2, encoding the VP1 capsid protein, the virus was genetically closest to the canine GVI.2 NoV strains C33/Viseu/2007/PRT and FD53/2007/ITA (81.0–84.0% nt and 93.0–94.0% aa identities), suggesting a recombination nature, with the cross-over site being mapped to the ORF1-ORF2 junction. Based on the full-length VP1 amino acid sequence, we classified the novel feline NoV, together with the canine strains Viseu and FD53, as a genotype 2, within the genogroup GVI. These findings indicate that, as observed for GIV NoV, GVI strains may infect both the canine and feline host. Unrestricted circulation of NoV strains in small carnivores may provide the basis for quick genetic diversification of these viruses by recombination. Interspecies circulation of NoVs in pets must also be considered when facing outbreaks of enteric diseases in these animals.

Seroprevalence for 2117-like vesiviruses in Italian household dogs

In 2003, a novel calicivirus, the vesivirus (VeV) strain 2117, was identified incidentally as a contaminant in Chinese Hamster Ovary (CHO) cell cultures by a German pharmaceutical company. Similar contaminations have been documented in three additional episodes, in bio-reactors used for production of recombinant drugs. More, recently 2117-like VeVs have also been identified at high prevalence in the stools from asymptomatic kennel dogs and only sporadically in diarrhoeic and healthy household dogs. In this study, antibodies for 2117-like viruses were detected in 21.5% of sera from household dogs, indicating that they are common viruses in the canine host.

Seroprevalence for norovirus genogroup II, IV and VI in dogs

Molecular and serological data suggest that noroviruses (NoVs) might be transmitted between humans and domestic carnivores. In this study we screened an age-stratified collection of canine sera (n=516) by using an ELISA assay based on virus-like particles (VLPs) of human NoVs GII.4 and GIV.1 and carnivore NoVs GIV.2 and GVI.2. Antibodies against GII.4 and GIV.1 human NoVs and GIV.2 and GVI.2 NoVs from carnivores were identified in dog sera (13.0%, 67/516) suggesting their exposure to homologous and heterologous NoVs. Analysis of the trends of age-class prevalence showed a gradual increase in the positive rate from 9.0% and 7.0%, in young dogs <1year of age to 15.0% in dogs older than 12 years, for GII.4 and GVI.2 NoVs, respectively. A significant difference in the IgG distribution by age classes was observed for GIV.1 NoVs, with the highest rate of antibodies (7.0%) in the age group <1year and the lowest (1.0%) in the age-classes 7-9 (P=0.049). High correlation between the reactivity to GII.4 and GVI.2 NoVs was observed, likely due to conserved epitopes in the capsid structure.

Serological and molecular investigation for hepatitis E virus (HEV) in captive non-human primates, Italy

Hepatitis E virus (HEV) is the leading cause of human enterically-transmitted viral hepatitis occurring around the world both as outbreaks and as sporadic cases. Non-human primates (NHPs) have been experimentally infected with HEV, but few studies have been reported about natural infection in wild-living and zoo monkeys. In order to provide a more complete picture on the epidemiology of HEV in NHPs living in controlled environment, we investigated the presence of HEV by screening serologically and molecularly a historical collection of 86 sera from seven different species of primates housed at the Zoological Garden (Bioparco) of Rome, Italy. By using an enzyme-linked immunosorbent assay based on the recombinant capsid protein of a Gt3 HEV strain, IgG antibodies were detected in three macaques (4.8%; 3/62) and in a white-crowned mangabey (16.6%; 1/6), with an overall prevalence of 4.6% (4/86). This positivity was confirmed when assessed the sera by western blotting. Rescreening the sera for IgM and viral RNA, all the samples resulted negative. Also, HEV RNA was not found when 17 stool samples were analyzed by RT-PCR. Although these results suggest that none of the monkeys housed at the Bioparco of Rome in the 17-year time frame spanning 2001 to 2017 developed acute or at least sub-acute HEV disease, the detection of IgG antibodies demonstrated that animals living in this setting were exposed to HEV or to antigenically related viruses.

Seroprevalence of sapovirus in dogs using baculovirus-expressed virus-like particles

Caliciviruses of the Sapovirus genus have been recently detected in dogs. Canine sapoviruses (SaVs) have been identified in the stools of young or juvenile animals with gastro-enteric disease at low prevalence (2.0-2.2%), but whether they may have a role as enteric pathogens and to which extent dogs are exposed to SaVs remains unclear. Here, we report the expression in a baculovirus system of virus like-particles (VLPs) of a canine SaV strain, the prototype virus Bari/4076/2007/ITA. The recombinant antigen was used to develop an enzyme-linked immunosorbent assay (ELISA). By screening an age-stratified collection of serum samples from 516 dogs in Italy, IgG antibodies specific for the canine SaV VLPs were detected in 40.3% (208/516) of the sera. Also, as observed for SaV infection in humans, we observed a positive association between seropositivity and age, with the highest prevalence rates in dogs older than 4 years of age.

Potential role of wolf (Canis lupus) as passive carrier of European brown hare syndrome virus (EBHSV)

European brown hare syndrome virus (EBHSV) was detected in a faecal swab collected from a wolf carcass in Northern Italy. The full-length genome of the EBHSV WOLF/17/2016/ITA strain was determined. In the VP60 capsid gene, the wolf strain displayed the highest genetic identity (99.2-99.1% nucleotide and 99.6-99.7% amino acid) with two EBHSV strains recently found in the intestinal content of a red fox and in the spleen and liver of a hare in Northern Italy. This finding poses interrogatives on the potential role of carnivores as EBHSV passive carriers, favoring the introduction and spread of the virus among different hare populations.

Serological and molecular investigation of 2117-like vesiviruses in cats

Vesivirus 2117 was first discovered as a contaminant in Chinese hamster ovary (CHO) cell cultures used for human drug production. Similar vesiviruses (VeVs) have been detected recently in dogs. In order to address the hypothesis that cats may also be exposed to 2117-like VeVs, in this study, we screened 236 feline sera using an enzyme-linked immunosorbent assay (ELISA) based on a recombinant VP1 protein from the canine VeV Bari/212/07/ITA. IgG antibodies against the 2117-like VeV were detected in 37.3% of the sera tested. Also, by screening cat faecal specimens, the RNA of a 2117-like VeV was detected in a clinically healthy cat.

First molecular identification of kobuviruses in wolves (Canis lupus) in Italy

Canine kobuviruses (CaKoVs) were first identified in diarrhoeic and asymptomatic dogs in 2011 in the USA. Subsequent studies have demonstrated a worldwide distribution of these viruses, but it is not clear if CaKoVs play a role as enteric pathogens of dogs. More recently, CaKoV RNA has been detected in wild carnivores, including red fox, golden jackal, side-striped jackal and spotted hyena. In this study, we addressed the hypothesis that wolves are susceptible to CaKoV infections. A total of 185 wolf stool samples were collected from necropsied animals and from transects in the Liguria, Piemonte and Valle D’Aosta regions of Italy, and CaKoV RNA was identified in two of these specimens. Both samples were obtained from necropsied wolves, with a prevalence rate of 4.9% (2/41). Sequence analysis of the full-length VP1 region showed that these strains displayed the highest nucleotide (nt) sequence identity (86.3-98.5%) to canine strains identified in the UK and Africa, and to kobuviruses that were previously detected in other African wild carnivores. This suggests that genetically related CaKoV strains circulate in domestic and wild carnivores, with interspecies transmission being not uncommon among carnivores of different ecosystems.