Strengthening preparedness against global health threats: A paradigm shift based on One Health approaches.

The implementation of preparedness strategies to prevent and mitigate the impact of global health threats poses several challenges. It should promptly identify cross-cutting drivers of pandemic threats, assess context-specific risks, engage multiple stakeholders, and translate complex data from multiple sources into accessible information for action. This requires a coordinated, multidisciplinary and multisectoral effort engaging systems that, most of the time, work in isolation. The One Health (OH) approach promotes the collaboration and communication among different disciplines and sectors, and could be applied across the preparedness phases at national and international level. We discuss here gaps and needs in preparedness strategies, which can benefit from the OH approach, and a set of actionable recommendations, as shared with the G20-2021 with a dedicated Policy Brief. The discussion adds to the current debate about OH operationalization and promotes a paradigm shift towards coordinated prevention and preparedness strategies for early assessment and management of global health threats.

A novel variant of the infectious bronchitis virus resulting from recombination events in Italy and Spain.

Infectious bronchitis is considered to be one of the most devastating diseases in poultry. Control of its spread is typically attempted through biosecurity measures and extensive vaccination. However, the remarkable genetic and antigenic variability of the virus, which originate from both mutations and recombination events, represents an unsolved challenge for this disease. The present study reports on the emergence and spread of recombinant clusters detected in Italy and Spain between 2012 and 2014. A total of 36 Spanish and Italian infectious bronchitis virus (IBV) field strains were investigated and genetically characterized using phylogenetic, molecular, recombination and selection pressure analyses of the complete S1 gene. Based on the partial S1 sequencing, 27 IBV strains originating from Spain and nine from Italy were initially classified as being closely related to the Guandong/Xindadi (XDN) genotype. Phylogenetic analysis of the complete S1 gene revealed that the XDN strains formed a homogeneous clade with the Spanish IBV isolates within the QX genotype, whereas there was higher variability within the Italian strains. Recombination analysis determined that these strains belonged to four groups, which originated from independent recombination events between the QX and 793B IBV genotypes. Our data support the hypothesis of two different scenarios: firstly, in Spain, the large and homogeneous clade probably originated from a single offspring of the recombinant founder, which became dominant and spread throughout the country. Secondly, the nine Italian recombinants, which are characterized by three different recombination patterns, probably represent less fitted strains, because they were less viable with respect to their recombinant parents.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain.

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Novel bocaparvoviruses in rabbits.

Bocaparvovirus is a newly established genus within the family Parvoviridae and has been identified as a possible cause of enteric, respiratory, reproductive/neonatal and neurological disease in humans and several animal species. In this study, metagenomic analysis was used to identify and characterise a novel bocaparvovirus in the faeces of rabbits with enteric disease. To assess the prevalence of the novel virus, rectal swabs and faecal samples obtained from rabbits with and without diarrhoea were screened with a specific PCR assay. The complete genome sequence of the novel parvovirus was reconstructed. The virus was distantly related to other bocaparvoviruses; the three ORFs shared 53%, 53% and 50% nucleotide identity, respectively, to homologous genes of porcine bocaparvoviruses. The virus was detected in 8/29 (28%) and 16/95 (17%) samples of rabbits with and without diarrhoea, respectively. Sequencing of the capsid protein fragment targeted by the diagnostic PCR identified two distinct bocaparvovirus populations/sub-types, with 91.7-94.5% nucleotide identity to each other. Including these novel parvoviruses in diagnostic algorithms of rabbit diseases might help inform their potential pathogenic role and impact on rabbit production and the virological profiles of laboratory rabbits.

West Nile Virus Surveillance in the Lombardy Region, Northern Italy.

In 2013, the circulation of West Nile virus (WNV) was detected in the Lombardy region and the following year a surveillance programme was activated with the aim of early identification of the viral distribution in mosquitoes and wild birds. A total of 50 959 Culex spp. mosquitoes grouped in six hundred and forty-seven pools as well as 1400 birds were screened by RT-PCR for the presence of West Nile virus leading to the identification of the viral genome in 32 mosquito pools and 13 wild birds. The surveillance was able to detect the WNV circulation on an average of 42 days (CI 95% 29.98-53.86; Student's t-distribution) before the occurrence of human West Nile disease (WND) cases in the same area. These results demonstrate the presence of WNV in the Lombardy region and confirm entomological and wild birds surveillance as an effective measure for the early identification of WNV circulation in infected areas, thus providing a useful and cost-effective tool for the public health authorities in the application of measures to prevent human infection.

Detection of the new emerging rabbit haemorrhagic disease type 2 virus (RHDV2) in Sicily from rabbit (Oryctolagus cuniculus) and Italian hare (Lepus corsicanus).

Rabbit haemorrhagic disease virus (RHDV), a member of the genus Lagovirus, causes rabbit haemorrhagic disease (RHD), a fatal hepatitis of rabbits, not previously reported in hares. Recently, a new RHDV-related virus emerged, called RHDV2. This lagovirus can cause RHD in rabbits and disease and mortality in Lepus capensis (Cape hare). Here we describe a case of RHDV2 infection in another hare species, Lepus corsicanus, during a concurrent RHD outbreak in a group of wild rabbits. The same RHDV2 strain infected rabbits and a hare, also causing a RHD-like syndrome in the latter. Our findings confirmed the capability of RHDV2 to infect hosts other than rabbits and improve the knowledge about the epidemiology and the host range of this new lagovirus.

Serosurvey for schmallenberg virus in alpine wild ungulates.

Because Schmallenberg virus (SBV) was first reported in domestic ruminants in Northern Italy in February 2012, we conducted a serosurvey to assess the presence of SBV-specific antibodies in free-ranging alpine ruminants. The tested serum samples were from chamois (23) and red deer (352) hunted from 2007 to 2013. All of the serum samples collected through September, 2012, tested negative, whereas a single chamois serum and 21 red deer sera taken during the 2012-2013 hunting season tested positive for the presence of SBV antibodies. Because this serosurvey is suggestive of an active SBV circulation in Alpine wildlife, targeted surveillance should be performed on wild ruminants to monitor the spread of the virus and to assess the epidemiological role of wildlife at the interface with domestic animals.

Cervus elaphus papillomavirus (CePV1): new insights on viral evolution in deer.

We identified a novel papillomavirus (CePV1) in a fibropapilloma of a 1.5 year old male red deer (Cervus elaphus) shot in the Italian Alps in Brescia province. PV particles were first observed by electron microscopy and PV DNA was then identified by PCR using degenerate primers. Subsequently we cloned the entire genome and determined its complete sequence. CePV1 genome is 8009 bp long and contains all 9 ORFs and the long untranslated regulatory region characteristic for Delta-papillomaviruses. Pairwise nucleotide alignments and phylogenetic analyses based on concatenated E1-E2-L1 ORFs allowed to determine the highest similarity with the Capreolus caprelus papillomavirus CcaPV1. The analysis of the host-parasite phylogenetic tree interactions suggest the co-divergence of CePV1 and C. elaphus while the identified topological incongruences leading us to speculate that CcaPV1 could eventually be the result of an earlier host switch event.

Identification of Mammalian orthoreovirus type 3 in Italian bats.

Summary This study describes the isolation and molecular characterization of Mammalian orthoreovirus (MRV) in microbats. Faecal samples and dead individuals available from rehabilitation centres or collected from known roost sites were virologically tested. In total, 112 carcasses of bats found dead, and 44 faecal samples were analysed. Nineteen viral strains were isolated by in vitro cell culture from faecal and tissue samples of different bat species (Pipistrellus khulii, Tadarida teniotis, Rhinolophus hipposideros and Vespertilio murinus), and they were morphologically identified as reoviruses by negative staining electron microscopy observation. The definitive assignment of all isolates to MRV was confirmed by RT-PCR assays targeting the L1 gene. Through a multiplex RT-PCR assay targeting the S1 gene, we typed 15 of 19 isolates as MRV type 3. Partial L1 (416 bp) and complete S1 (1416 bp) sequences of the isolates were analysed and compared with those of reference strains obtained from GenBank, belonging to the three serotypes. Molecular analysis of the S1 gene revealed that the amino acid residues associated with neurotropism (198-204NLAIRLP, 249I, 350D and 419E) were highly conserved among the Italian bat strains. These results suggest that potentially neurotropic MRV type 3 strains are widespread among Italian bats. Furthermore, the identification of MRV type 3 in bat species such as Pipistrellus Khulii, which is common in urban areas and known for its close contact with humans, underlines the need for vigilance.

Enteric disease in dogs naturally infected by a novel canine astrovirus.

Infection by a novel canine astrovirus was associated with gastroenteritis in two dogs. The virus displayed 70.3 to 73.9% amino acid identity to other canine astroviruses in the full-length capsid. Specific antibodies were detected in the convalescent-phase sera of the dogs, indicating seroconversion. Also, the virus appeared weakly related antigenically to the prototype canine astrovirus isolate ITA/2008/Bari.

A feline rotavirus G3P[9] carries traces of multiple reassortment events and resembles rare human G3P[9] rotaviruses.

The full-length genome sequence of a feline G3P[9] rotavirus (RV) strain, BA222, identified from the intestinal content of an adult cat, was determined. Strain BA222 possessed a G3-P[9]-I2-R2-C2-M2-A3-N1-T3-E2-H3 genomic constellation, differing substantially from other feline RVs. Phylogenetic analyses of each genome segment revealed common origins with selected animal and zoonotic human RVs, notably with rare multi-reassortant human G3P[9] RVs (Ita/PAI58/96 and Ita/PAH136/96). Altogether, the findings suggest that feline RVs are genetically diverse and that human RVs may occasionally originate either directly or indirectly (via reassortment) from feline RVs.

Molecular characterization and phylogenetic analysis of VP1 of porcine enteric picornaviruses isolates in Italy.

Porcine enterovirus (PEV), Porcine Teschovirus and Porcine sapelovirus, belonging to the family Picornaviridae, are ubiquitous and mainly cause asymptomatic infections in pigs. In this study, a total of 40 Italian porcine picornavirus isolates were characterized by sequencing the capsid VP1-encoding gene. This procedure turned out to be a useful diagnostic tool for the molecular identification of porcine enterovirus, teschovirus and sapelovirus strains and for the study of molecular epidemiology and evolution of these viruses confirming the possibility of correlating virus genotype to serotype.

Rotavirus-associated diarrhoea in foals in Greece.

Severe outbreaks of diarrhoeic syndrome occurred in young foals at the same stud farm during two consecutive breeding periods namely spring 2006 and 2007. Rotavirus-like particles were detected by electron microscopy in the faeces of the affected foals and group A rotavirus infection was confirmed by Reverse-Transcription (RT)-PCR with selected sets of rotavirus-specific primers. Sequence analysis of the genes encoding the outer capsid rotavirus proteins VP7 and VP4 enabled classification of the viruses as G3AP[12] and revealed that the viruses were highly similar to recently reported equine rotavirus strains circulating in Europe. All Greek equine rotavirus isolates were genetically identical, suggesting persistence of the same viral strain in the stud farm, over the two consecutive foaling periods.

An outbreak of viral gastroenteritis linked to municipal water supply, Lombardy, Italy, June 2009.

We report an outbreak of viral gastroenteritis linked to municipal drinking water in a town in northern Italy in June 2009. Over one month we identified 299 probable cases of whom 30 were confirmed for at least one of the following viruses: norovirus, rotavirus, enterovirus or astrovirus. Water samples and filters from the water system also tested positive for norovirus and enterovirus. Control measures included treating the water system with chlorine dioxide and filters with peracetic acid, while providing temporary alternative sources of drinking water to the population.

Molecular characterization of a new PToV strain. Evolutionary implications.

Toroviruses are emergent viruses, belonging to the Nidovirales order, that remain mostly ignored, despite they are able to infect different species of domestic animals and humans, causing enteric diseases and diarrhea. Thus far, only five variants of porcine torovirus (PToV) have been identified. In this report we describe the identification and partial characterization of a new strain of porcine torovirus (PToV-BRES) that was detected by RT-PCR in a swine faecal specimen from a farm in Brescia (Italy). The complete genes coding for the nucleocapsid (N), hemagglutinin-esterase (HE) and membrane (M) proteins were amplified, and sequence analysis showed that PToV-BRES is a new PToV strain that, based on the HE gene sequence, is phylogenetically related to P4 strain, that was up to now the only member of a distinct PToV lineage. The nucleocapsid protein from PToV-BRES was expressed in insect cells as a his-tagged protein, purified by affinity chromatography and used to develop an ELISA method to detect antibodies against PToV. This assay was evaluated using a serum collection including 45 samples from three commercial farms from Spain. High antibody prevalence against PToV was observed in the three farms, both in adult animals and in piglets, which could suggest that PToV might be endemic in Spanish porcine population. The ELISA method developed in this work could be useful in future epidemiological surveys about toroviruses.

Genetic analysis of feline panleukopenia viruses from cats with gastroenteritis.

Thirty-nine parvovirus strains contained in faecal samples collected in Italy (n=34) and UK (n=5) from cats with feline panleukopenia were characterized at the molecular level. All viruses were proven to be true feline panleukopenia virus (FPLV) strains by a minor groove binder probe assay, which is able to discriminate between FPLV and the closely related canine parvovirus type 2. By using sequence analysis of the VP2 gene, it was found that the FPLV strains detected in Italy and UK were highly related to each other, with a nucleotide identity of 99.1-100 and 99.4-99.8% among Italian and British strains, respectively, whereas the similarities between all the sequences analysed were 98.6-100%. Eighty-eight variable positions were detected in the VP2 gene of the field and reference FPLV strains, most of which were singletons. Synonymous substitutions (n=57) predominated over non-synonymous substitutions (n=31), and the ratio between synonymous and non-synonymous substitutions (dN/dS) was 0.10, thus confirming that evolution of FPLV is driven by random genetic drift rather than by positive selection pressure. Some amino acid mutations in the VP2 protein affected sites that are thought to be responsible for antigenic and biological properties of the virus, but no clear patterns of segregation and genetic markers, were identified, confirming that FPLV is in evolutionary stasis.

Identification of a porcine calicivirus related genetically to human sapoviruses.

Whether animals may act as reservoirs for human caliciviruses is unclear. By sequence analysis of a short fragment of the RNA-dependent RNA polymerase (RdRp) region, porcine sapovirus (SaV) strains that genetically resemble human SaVs have been detected in piglets, but more-informative sequences (capsid gene) were not available for a precise characterization. In this study, the 3' terminus (the 3' end of open reading frame 1 [ORF1], including the polymerase complex and the complete capsid; ORF2; and the 3' untranslated region) of one such human SaV-like strain, 43/06-18p3/2006/It, was determined, revealing that these viruses are more related genetically to human (47.4 to 54.9% amino acid identity) than to animal (35.2 to 44.7% amino acid identity) SaVs in the capsid gene. In addition, the recombination-prone RdRp-capsid junction region was highly conserved with those of human SaVs of genogroup GI. The presence of porcine viruses similar to human SaVs is a significant finding because of the potential for zoonotic infections or generation of porcine/human recombinants.

Epidemic of diarrhoea caused by porcine epidemic diarrhoea virus in Italy.

There was an epidemic of diarrhoea affecting pigs of all ages in Italy between May 2005 and June 2006. In 63 herds the cause was confirmed as porcine epidemic diarrhoea virus by electron microscopy, immunoelectron microscopy, pcr and serology. Watery diarrhoea without mucus and blood was usually associated with a reduction of feed consumption. In farrowing-to-weaning herds, diarrhoea affected the sows and suckling piglets, and the mortality in newborn piglets was up to 34 per cent. In growers and fatteners the morbidity ranged from 20 to 80 per cent, but there was either no mortality or it was very low. Depending on the size of the herd and the type of operation, the clinical disease lasted for weeks or months.

Molecular characterization of bovine rotavirus strains circulating in northern Italy, 2003-2005.

A total of 232 stools collected from calves with rotavirus infection in herds located in northern Italy from 2003 to 2005 was investigated. Determination of the rotavirus G and P types was carried out using nested RT-PCR. G6 was the most prevalent genotype, accounting for 78.5% of samples, G10 accounted for 9.9% of samples and viruses of G8 type were found in 4.7% of samples. In 3% of samples, viruses were not classified due to concomitant infection with more G type strains, whereas viruses in 3.9% of samples could not be characterized with any of the G-specific primers used in this study. Most common P types were P[11] and P[5], accounting for 65.1% and 25%, respectively. In 2.6% of cases, samples reacted with multiple P-specific primers; no P[1] serotype was identified. The G6P[11] combination was predominant throughout the study period, i.e. 52.5% in 2003, 50% in 2004 and 40% in 2005. The incidence of G6P[5] increased from 13.1% in 2003 to 27% in 2004 and 25.5% in 2005. The G10P[11] combination decreased markedly from 18% in 2003 to 2.6% in 2004, rising again to 7.3% in 2005. G8P[11] viruses were similarly present in 2003 (5%) and 2004 (4.3%), declining slightly in 2005 (1.8%).