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.

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.

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.

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.

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.

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.

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.

Exploring the Enteric Virome of Cats with Acute Gastroenteritis.

Viruses are a major cause of acute gastroenteritis (AGE) in cats, chiefly in younger animals. Enteric specimens collected from 29 cats with acute enteritis and 33 non-diarrhoeic cats were screened in PCRs and reverse transcription (RT) PCR for a large panel of enteric viruses, including also orphan viruses of recent identification. At least one viral species, including feline panleukopenia virus (FPV), feline enteric coronavirus (FCoV), feline chaphamaparvovirus, calicivirus (vesivirus and novovirus), feline kobuvirus, feline sakobuvirus A and Lyon IARC polyomaviruses, was detected in 66.1% of the samples.. Co-infections were mainly accounted for by FPV and FCoV and were detected in 24.2% of the samples. The virome composition was further assessed in eight diarrhoeic samples, through the construction of sequencing libraries using a sequence-independent single-primer amplification (SISPA) protocol. The libraries were sequenced on Oxford Nanopore Technologies sequencing platform. A total of 41 contigs (>100 nt) were detected from seven viral families infecting mammals, included Parvoviridae, Caliciviridae, Picornaviridae, Polyomaviridae, Anelloviridae, Papillomaviridae and Paramyxoviridae, revealing a broad variety in the composition of the feline enteric virome.

Exploring the Astrovirome of Shellfish Matrices Using Nanopore Sequencing.

Astroviruses are important human enteric pathogens transmissible with contaminated food and water. Astroviruses have also been identified in mammals, birds, lower vertebrates and invertebrates. The genetic diversity of human and animal astroviruses poses a challenge for diagnostics and taxonomy. As a proof of concept, we used a panastrovirus consensus primer set, able to amplify in a nested RT-PCR protocol a 400-nt-long fragment of the RNA-dependent RNA polymerase of most members of the Astroviridae family, in conjunction with a nanopore sequencing platform, to generate information on the astrovirome in filter-feeding mollusks. Amplicons generated from bivalve samples were used to generate libraries for deep sequencing. In three samples, only one unique RdRp sequence type was obtained. However, in seven samples and in three barcodes with eleven pooled samples, we identified a variety of known and unknown RdRp sequence types, in most cases distantly related to astrovirus sequences available in the databases. In total, 37 different sequence contigs were generated. Avian-origin astrovirus sequences were predominant, likely due to contamination of shellfish harvesting waters by marine birds. Astroviruses of the aquatic eco-system were also identified, whereas human astroviruses were not detected.

Serological and Molecular Survey on Domestic Dog Hepadnavirus in Household Dogs, Italy.

The discovery of hepadnaviruses in cats (domestic cat hepadnavirus, DCH) and of a DCH-like virus in dogs has raised several questions regarding the role of these viruses in pets, with particular emphasis on their potential impact on animal health and epidemiology, as well as possible zoonotic implications. In this study, by screening an age-stratified collection of 600 canine serum samples for DCH with an ELISA assay based on the recombinant core antigen (DCHCAg), specific antibodies were found with an overall prevalence of 10.0% (60/600), with a higher prevalence in younger and older dogs. By retesting the canine DCHCAbs-positive sera with an ELISA test based on the recombinant surface protein of DCH (DCHSAg), a total of 18 sera (30%, 18/60) also contained IgG anti-DCHSAg. All the sera were also assessed molecularly using either a consensus hepadnavirus PCR or a specific real-time PCR for DCH. Hepadnavirus DNA was detected in four seronegative dogs, with a prevalence rate of 0.7% (4/600). On sequence analysis of the polymerase region amplified with pan-hepadnavirus primers, the amplicons displayed the highest nucleotide identity (97.3-99.6%) to DCH sequences detected in cats and to the domestic dog hepadnavirus recently identified in a canine serum sample from Italy.

Detection at high prevalence of newlavirus (protoparvovirus) in the carcasses of red foxes.

Wildlife conservation also relies on the study of animal virome. We identified the DNA of a novel fox protoparvovirus, newlavirus, with high (71%) prevalence in the carcasses of red foxes. On genome sequencing, high genetic diversity and possible recombination was observed, suggesting complex evolutionary dynamics in wildlife.

Detection and Characterization of a Novel Picornavirus in European Badger (Meles meles).

The recent development of unbiased metagenomic next-generation sequencing has provided a richer view of the wild animal virome making it necessary to expand the knowledge about virus diversity in wildlife, as well as to monitor their potential transmission to domestic animals or humans. In the present study, by screening collections of enteric specimens from wild animals, a novel picornavirus was identified in the intestinal content of a badger (Meles meles). By enrichment with a sequence-independent single-primer amplification (SISPA) approach and deep sequencing with Oxford Nanopore Technologies (ONT) platform, the genome sequence of a novel picornavirus strain, Badger/3A-2019/ITA, was reconstructed. On comparison based on the polyprotein sequences, the virus was distantly related (58.7% and 59.7% sequence identity at the nucleotide and amino acid level, respectively) to the feline picornavirus strain FFUP1, identified in 2012 in Portugal and classified into genus Sakobovirus within the species Sakobuvirus A. Upon phylogenetic, pairwise homology, and distance analyses performed on the P1, 2Chel, 3Cpro, and 3Dpol proteins and the complete genomic sequence, the badger picornavirus may be considered a member of a new sakobuvirus species, which we propose as Sakobuvirus B.

Current Knowledge of Hepatitis E Virus (HEV) Epidemiology in Ruminants.

Hepatitis E virus (HEV) infection represents an emerging public health concern worldwide. In industrialized countries, increasing numbers of autochthonous cases of human HEV infection are caused by zoonotic transmission of genotypes 3 and 4, mainly through the consumption of contaminated raw or undercooked meat of infected pigs and wild boars, which are considered the main reservoirs of HEV. However, in the last few years, accumulating evidence seems to indicate that several other animals, including different ruminant species, may harbor HEV. Understanding the impact of HEV infection in ruminants and identifying the risk factors affecting transmission among animals and to humans is critical in order to determine their role in the epidemiological cycle of HEV. In this review, we provide a summary of current knowledge on HEV ecology in ruminants. A growing body of evidence has revealed that these animal species may be potential important hosts of HEV, raising concerns about the possible implications for public health.

Detection and characterization of bopiviruses in domestic and wild ruminants.

Highly divergent picornaviruses (PVs) classified in the genus Bopivirus have been recently discovered on faecal samples from sheep and goats in Hungary and from fallow and red deer in Australia. In this study, we investigated the epidemiology of these novel viruses in domestic and wild ruminants from Northwestern Italian Alps by testing archival faecal samples collected from 128 sheep, 167 goats, 61 red deer (Cervus elaphus), 77 roe deer (Capreolus capreolus), 43 chamois (Rupicapra rupicapra) and 32 Alpine ibex (Capra ibex). Bopivirus RNA was detected in a total of 19 animals, including 14 sheep (10.9%), 2 red deer (3.3%), 1 roe deer (1.3%), 1 chamois (2.3 %) and 1 Alpine ibex (3.3 %), but not in goats. Upon sequence analysis of the 3DRdRp region, the sequences generated from chamois, roe deer, Alpine ibex and ovine faecal samples showed the highest nucleotide identity (96.8-100%) to bopiviruses detected in goats and sheep from Hungarian farms, whereas strains found in red deer displayed the closest relatedness (90.8%-91.2%) to bopiviruses identified in fallow and red deer in Australia. The nearly complete genome sequence of strains 12/2020/ITA (ON497046) and 14-73/2020/ITA (ON497047) detected in an Alpine ibex and in a sheep, respectively, was determined by combining a modified 3'-RACE protocol with Oxford Nanopore Technologies sequencing platform. On phylogenetic analysis based on the complete polyprotein, both strains segregated into the candidate species Bopivirus B along with ovine and caprine strains detected in Hungary (90.0-94.6% nucleotide and 94.6-98.0% amino acid identities). The findings of this study expand the host range of these novel viruses and hint to a possible virus circulation between domestic ruminants and wild animals.

Emerging Respiratory Viruses of Cats.

In recent years, advances in diagnostics and deep sequencing technologies have led to the identification and characterization of novel viruses in cats as protoparviruses and chaphamaparvoviruses, unveiling the diversity of the feline virome in the respiratory tract. Observational, epidemiological and experimental data are necessary to demonstrate firmly if some viruses are able to cause disease, as this information may be confounded by virus- or host-related factors. Also, in recent years, researchers were able to monitor multiple examples of transmission to felids of viruses with high pathogenic potential, such as the influenza virus strains H5N1, H1N1, H7N2, H5N6 and H3N2, and in the late 2019, the human hypervirulent coronavirus SARS-CoV-2. These findings suggest that the study of viral infections always requires a multi-disciplinary approach inspired by the One Health vision. By reviewing the literature, we provide herewith an update on the emerging viruses identified in cats and their potential association with respiratory disease.

A novel hepadnavirus in domestic dogs.

Hepadnaviruses have been identified in several animal species. The hepadnavirus prototype, human hepatitis B virus (HBV), is a major public health problem associated with chronic liver diseases and hepatocellular carcinoma. Recently, a novel hepadnavirus, similar to HBV, was identified in domestic cats. Since several pathogens can be shared between cats and dogs, we hypothesized that dogs could also harbor hepadnaviruses and we tested a collection of canine sera with multiple molecular strategies. Overall, hepadnavirus DNA was identified in 6.3% (40/635) of canine serum samples, although the viral load in positive sera was low (geometric mean of 2.70 × 102 genome copies per mL, range min 1.36 × 102-max 4.03 × 104 genome copies per mL). On genome sequencing, the canine hepadnaviruses revealed high nucleotide identity (about 98%) and similar organization to the domestic cat hepadnavirus. Altered hepatic markers were found in hepadnavirus-positive dogs, although the role of hepadnavirus in canine health remains to be elucidated.

Detection of antibodies against domestic cat hepadnavirus using baculovirus-expressed core protein.

A novel orthohepadnavirus (domestic cat hepadnavirus [DCH]) similar to human hepatitis B virus has been recently detected in serum and liver samples from domestic cats with chronic hepatitis and hepatocellular carcinoma. Molecular investigations by independent research groups around the world have revealed positivity rates ranging from 6.5% to 12.5% in blood samples and up to 14.0% in liver tissue. In this study, we screened an age-stratified collection of feline sera (n = 256) by using an antibody detection enzyme-linked immunosorbent assay based on the recombinant core antigen of DCH (DCHc). Specific antibodies (DCHc Abs) were detected with a prevalence of 25.0%. The DNA of DCH was detected in 35.9% (23/64) of seropositive cats and only in 1.0% (2/192) of seronegative animals. Based on the serological (IgG and IgM anti-DCHc) and virological status, the possible stages of DCH infection were predicted.

Feline chaphamaparvovirus in cats with enteritis and upper respiratory tract disease.

Feline chaphamaparvovirus (FeChPV) is a novel parvovirus, first discovered in a multi-facility feline shelter in Canada in 2019, during an outbreak of acute gastro-enteritis (AGE) in cats, and detected at high prevalence (47.0%) in faecal samples. Whether this finding was anecdotal or similar viruses are common components of feline virome is still unclear. Also, the potential impact of this virus on feline health is uncertain. Herewith, a case-control study was performed to investigate whether this novel parvovirus may play a role as enteric pathogen, screening samples collected from cats with and without AGE signs. Furthermore, we extended the research by testing archival paired oropharyngeal and ocular samples collected from cats with or without upper respiratory tract disease (URTD). FeChPV DNA was detected at high prevalence rate (36.8%, 14/38) in clinical cases, representing the most frequently identified enteric virus, followed by feline panleukopenia parvovirus (23.7%, 9/38), feline coronavirus (5.3%, 2/38), feline kobuvirus (5.3%, 2/38) and noroviruses (5.3%, 2/38). The different prevalence rates of FeChPV between the case and control group were statistically significant, suggesting a possible association of the virus with acute gastro-enteric disease. The virus was also detected at low rate in the respiratory samples of cats with (3.3%, 6/183) or without URTD (4.3%, 6/140), although there was no significant association between FeChPV and URTD. The complete VP encoding gene was determined for five viruses and the nearly full-length genome was reconstructed for three viruses, namely 313R/2019/ITA, 284R/2019/ITA and 49E/2019/ITA. In the NS1-based tree, the Italian strains clustered tightly with the two FeChPV prototypes detected in Canada, within a monophyletic cluster related to but clearly distinct from canine chaphamaparvovirus, currently classified in the species Carnivore chaphamaparvovirus 1 (CaChPV-1).

Detection and Characterization of Feline Calicivirus Associated with Paw and Mouth Disease.

Feline calicivirus (FCV) infection in cats can led to several diverse clinical presentations, ranging from mild upper respiratory signs to virulent systemic disease. Herein, we report a paw and mouth disease case in a 7-year-old household cat due to an FCV infection. An asymptomatic cat living in the same household was also infected with FCV. Clinical and pathological investigations were combined with the molecular and phenotypical characterization of the FCV strains. The RNA of the FCV was detected using qualitative and quantitative reverse transcription (RT)-PCR assays, and FCV antigen was confirmed by immunohistochemistry. After the whole genome analysis, the strains detected in the two cats appeared to be genetically diverse from FCVs previously detected in association with paw and mouth disease and with virulent systemic disease. Interestingly, the isolates obtained in this study were resistant to low pH conditions and slightly susceptible to bile salts, but they were susceptible to a trypsin treatment, revealing a phenotype pattern that is different from that which has been observed for respiratory FCVs.