Low pathogenic human coronaviruses during the first waves of COVID-19 in Italy.

Low-pathogenic human coronaviruses (HCoVs) infect the upper respiratory tract and cause mild, cold-like respiratory illness. Although several studies have shown evidence of the global distribution of HCoVs, information about their distribution in Italy are often focused only on hospitalized children and elderly with respiratory symptoms. In this study, a total of 916 swab samples collected during the first two SARS-CoV-2 pandemic waves in Abruzzo region (central Italy) was selected for molecular screening of low pathogenic HCoVs by real-time RT-PCR. We identified low-pathogenic HCoV in nine samples. Positive samples underwent whole genome sequencing for genome characterization; indeed, we also report the whole genome sequence of a HCoV-229E strain.

A cell-adapted SARS-CoV-2 mutant, showing a deletion in the spike protein spanning the furin cleavage site, has reduced virulence at the lung level in K18-hACE2 mice.

Here we investigated the virulence properties of a unique cell-adapted SARS-CoV-2 mutant showing a ten-amino acid deletion encompassing the furin cleavage site of the spike protein (Δ680SPRAARSVAS689; Δ680-689-B.1) in comparison to its parental strain (wt-B.1) and two Delta variants (AY.122 and AY.21) of concern. After intranasal inoculation, transgenic K18-hACE2 mice were monitored for 14 days for weight change, lethality, and clinical score; oral swabs were daily collected and tested for the presence of N protein subgenomic RNA. At 3 and 7 dpi mice were also sacrificed and organs collected for molecular, histopathological, and immune response profile investigations. The Δ680-689-B.1-infected mice exhibited reduced shedding, lower virulence at the lung level, and milder pulmonary lesions. In the lung, infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi (IL-4, IL-27, and IL-28) and 7 dpi (IL-4, IL-27, IL-28, IFN-γ and IL-1α).

Characterization of Epizootic Hemorrhagic Disease Virus Serotype 8 in Naturally Infected Barbary Deer (Cervus elaphus barbarus) and Culicoides (Diptera: Ceratopogonidae) in Tunisia.

Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Molecular epidemiology of Canine circovirus in domestic dogs and wildlife in Namibia, Africa.

Canine circovirus (CanineCV) is a DNA virus affecting domestic dogs and other wild carnivore species. Despite the potential implications for dogs' health and wildlife conservation, data on CanineCV presence, epidemiology and genetic features from Africa is still poor. In the present study, biological specimens collected between 2020 and 2022 from a total of 32 jackals and 575 domestic dogs were tested for the presence of CanineCV DNA to evaluate its frequency. Furthermore, sequencing was conducted on positive samples to characterize the strains and compare them with publicly available sequences through phylogenetic analysis. A high CanineCV prevalence was observed both in jackals (43.75%; 95 CI: 28.17% - 60.67%) and domestic dogs (27.13%; 95 CI: 23.66% - 30.91%). All aside from one Namibian strain formed an independent clade, suggestive of extremely rare introduction events, followed by local persistence, circulation, and evolution. Remarkably, different recombination events were observed involving strains from both jackals and domestic dogs, which testify to the likely strain exchange between these populations. Distinctive amino acid residues were also observed in jackals. The limitations of the considered host populations however prevent a definitive conclusion on host adaptation, biological, and clinical features. Further studies should be performed to expand our current knowledge of the CanineCV disease scenario in Namibia, other African regions, and associated host species in Africa.

First influenza D virus full-genome sequence retrieved from livestock in Namibia, Africa.

Influenza D virus (IDV) was first isolated in 2011 in the USA and has since been shown to circulate in cattle, pigs, sheep, wild boar, and camels. In Africa, there is limited data on the epidemiology of IDV and, so, we investigated the presence of IDV among domestic ruminants and wild animals in Namibia by screening nasal swabs using an IDV-specific molecular assay. IDV RNA was detected in bovines (n=2), giraffes (n=2) and wildebeest (n=1). The hemagglutinin-esterase-fusion (HEF) gene from one of the bovine and the wildebeest samples was successfully sequenced as well as the full genome for the second bovine sample. Phylogenetic analysis of the HEF gene positioned the African virus variants within the D/OK lineage but with a long branch. The African variant had an amino acid diversity of 2.41% and most likely represents a distinct genotype within the lineage. Notably, the polymerase acidic protein gene (PA) was more closely related to a different lineage (D/660), indicative of potential reassortment. This is the first genetic characterization of IDV in Africa and it adds important data to our understanding of the global IDV distribution.

Detection and Genetic Characterization of Canine Adenoviruses, Circoviruses, and Novel Cycloviruses From Wild Carnivores in Italy.

Wild carnivores are known to play a role in the epidemiology of several canine viruses, including canine adenoviruses types 1 (CAdV-1) and 2 (CAdV-2), canine circovirus (CanineCV) and canine distemper virus (CDV). In the present study, we report an epidemiological survey for these viruses in free ranging carnivores from Italy. A total of 262 wild carnivores, including red foxes (Vulpes vulpes), wolves (Canis lupus) and Eurasian badgers (Meles meles) were sampled. Viral nucleic acid was extracted and screened by real-time PCR assays (qPCR) for the presence of CAdVs and CanineCV DNA, as well as for CDV RNA. CAdV-1 DNA was detected only in red foxes (4/232, 1.7%) whilst the wolves (0/8, 0%) and Eurasian badgers (0/22, 0%) tested negative. CanineCV DNA was detected in 4 (18%) Eurasian badgers, 4 (50%) wolves and 0 (0%) red foxes. None of the animals tested positive for CDV or CAdV-2. By sequence and phylogenetic analyses, CAdV-1 and CanineCV sequences from wild carnivores were closely related to reference sequences from domestic dogs and wild carnivores. Surprisingly, two sequences from wolf intestines were identified as cycloviruses with one sequence (145.20-5432) displaying 68.6% nucleotide identity to a cyclovirus detected in a domestic cat, while the other (145.201329) was more closely related (79.4% nucleotide identity) to a cyclovirus sequence from bats. A continuous surveillance in wild carnivores should be carried out in order to monitor the circulation in wildlife of viruses pathogenic for domestic carnivores and endangered wild species.

Feline Morbillivirus in Southern Italy: Epidemiology, Clinico-Pathological Features and Phylogenetic Analysis in Cats.

Feline morbillivirus (FeMV) was isolated for the first time in 2012 with an association with chronic kidney disease (CKD) suggested. This study aimed at investigating in cats from southern Italy FeMV prevalence and risk factors for exposure to FeMV, including the relationship with CKD; sequencing amplicons and analyzing phylogeny of PCR positive samples. Blood serum, K3EDTA blood and urine samples from 223 cats were investigated. Ten carcasses were also evaluated. FeMV RNA was detected in 2.4% (5/211) blood and 16.1% (36/223) urine samples. One carcass tested positive by qPCRFeMV from kidney, urinary bladder, and submandibular lymph nodes. Antibodies against FeMV were detected in 14.5% (28/193) cats. We followed up 27 cats (13 FeMV positive cats) and documented in some cases urine shedding after up to 360 days. Older and foundling cats and cats living in rescue catteries, were more frequently infected with FeMV. A significant correlation between FeMV and higher serum creatinine values or low urine specific gravity was found. FeMV positivity was significantly associated with retroviral infection, and the presence of some clinical signs apart from CKD clinicopathological markers. Our study highlights the possibility of a link between FeMV exposure and CKD and a general impairment of feline health.

Genome Sequences of Three SARS-CoV-2 P.1 Strains Identified from Patients Returning from Brazil to Italy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. We report the complete sequences of three SARS-CoV-2 P.1 strains obtained from nasopharyngeal swab specimens from three patients returning from Brazil to Italy.

West Nile Virus Lineage 1 in Italy: Newly Introduced or a Re-Occurrence of a Previously Circulating Strain?

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008-2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory active.

Circulation of diverse protoparvoviruses in wild carnivores, Italy.

Protoparvovirus is a monophyletic viral genus that includes the species Carnivore protoparvovirus-1 infecting domestic and wild carnivores. In this paper, the results of an epidemiological survey for Carnivore protoparvovirus-1 in wild carnivores in Italy are reported. Overall, 34 (11.4%) out of 297 tested animals were positive for Carnivore protoparvovirus-1, but the frequency of detection was much higher in intestine (54%) than in spleen samples (2.8%), thus suggesting that the intestine is the best sample to collect from wild animals for parvovirus detection. Feline panleukopenia virus (FPV) was detected in red foxes (Vulpes vulpes) (2.8%, 7/252) and Eurasian badgers (Meles meles) (10%, 1/10), whilst canine parvovirus (CPV) was found in wolves (54.3%, 19/35), Eurasian badgers (60%, 6/10) and one beech marten (Martes foina) (100%, 1/1), with more than one parvovirus type detected in some animals. Protoparvoviral DNA sequences from this study were found to be related to CPV/FPV strains detected in Asia and Europe, displaying some amino acid changes in the main capsid protein VP2 in comparison with other parvovirus strains from wildlife. In particular, the two most common mutations were Ile418Thr and Ala371Gly, which were observed in 6/12 (50%) and 5/12 (41.7%) of the CPV sequences from this study. Continuous surveillance for parvoviruses in wild carnivores and genetic analysis of the detected strains may help obtain new insight into the role of these animals in the evolution and epidemiology of carnivore parvoviruses.

Epidemiology, pathological aspects and genome heterogeneity of feline morbillivirus in Italy.

Feline morbillivirus (FeMV) is an emerging morbillivirus first described in cats less than a decade ago. FeMV has been associated with chronic kidney disease of cats characterized by tubulointerstitial nephritis (TIN), although this aspect is still controversial and not demonstrated with certainty. To investigate FeMV prevalence and genomic characteristics, an epidemiological survey was conducted in a total number of 127 household cats originating from two Italian regions, Abruzzi and Emilia-Romagna. A total number of 69 cats originating from three feline colonies were also enrolled for the study. Correlation with TIN was investigated by employing a total number of 35 carcasses. Prevalence of FeMV RNA was higher in urine samples collected from cats of colonies (P = 31.8%, CI 95% 22.1-43.6) compared to household cats (P = 8.66%, CI 95% 4.9-14.9) and in young and middle-aged cats while prevalence of FeMV Abs was higher in old cats. Sequences obtained straight from infected biological samples, either partial or complete, cluster into two clades within FeMV genotype 1, distantly related to FeMV genotype 2. Immunohistochemistry analysis of kidney sections of FeMV RNA positive cats revealed immunoreactivity within epithelial cells of renal tubuli and inflammatory cells. However, statistically significant association between FeMV and renal damages, including TIN, was not demonstrated (p= 0.0695, Fisher exact test). By virus histochemistry performed with FeMV-negative feline tissues and a FeMV isolate, tropism for different cellular types such as inflammatory cells residing in blood vessels of kidney and brain, airway epithelial cells, alveolar macrophages and to a lesser extent, the central nervous system, was demonstrated. Additional studies are warranted in order to establish viral tropism and immune response during the early phases of infection and to disentangle the role of FeMV in co-infection processes.