Group A Rotavirus Associated with Encephalitis in Red Fox.

In 2011, a group A rotavirus was isolated from the brain of a fox with encephalitis and neurologic signs, detected by rabies surveillance in Italy. Intracerebral inoculation of fox brain homogenates into mice was fatal. Genome sequencing revealed a heterologous rotavirus of avian origin, which could provide a model for investigating rotavirus neurovirulence.

Porcine Epidemic Diarrhea Virus and Discovery of a Recombinant Swine Enteric Coronavirus, Italy.

Porcine epidemic diarrhea virus (PEDV) has been detected sporadically in Italy since the 1990s. We report the phylogenetic relationship of swine enteric coronaviruses collected in Italy during 2007-2014 and identify a drastic shift in PEDV strain variability and a new swine enteric coronavirus generated by recombination of transmissible gastroenteritis virus and PEDV.

A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences.

A panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics.

Experimental infection of house sparrows (Passer domesticus) with West Nile virus isolates of Euro-Mediterranean and North American origins.

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5-6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.

Different evolutionary trends of swine H1N2 influenza viruses in Italy compared to European viruses.

European H1N2 swine influenza viruses (EU H1N2SIVs) arose from multiple reassortment events among human H1N1, human H3N2, and avian influenza viruses. We investigated the evolutionary dynamics of 53 Italian H1N2 strains by comparing them with EU H1N2 SIVs. Hemagglutinin (HA) phylogeny revealed Italian strains fell into four groups: Group A and B (41 strains) had a human H1 similar to EU H1N2SIVs, which probably originated in 1986. However Group B (38 strains) formed a subgroup that had a two-amino acid deletion at positions 146/147 in HA. Group C (11 strains) contained an avian H1 that probably originated in 1996, and Group D (1 strain) had an H1 characteristic of the 2009 pandemic strain. Neuraminidase (NA) phylogeny suggested a series of genomic reassortments had occurred. Group A had an N2 that originated from human H3N2 in the late 1970s. Group B had different human N2 that most likely arose from a reassortment with the more recent human H3N2 virus, which probably occurred in 2000. Group C had an avian-like H1 combined with an N2 gene from one of EU H1N2SIVs, EU H3N2SIVs or Human H3N2. Group D was part of the EU H3N2SIVs clade. Although selection pressure for HA and NA was low, several positively selected sites were identified in both proteins, some of which were antigenic, suggesting selection influenced the evolution of SIV. The data highlight different evolutionary trends between European viruses and currently circulating Italian B strains and show the establishment of reassortant strains involving human viruses in Italian pigs.

West Nile virus: characterization and diagnostic applications of monoclonal antibodies.

BACKGROUND: Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. METHODS: Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity. RESULTS: All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs. CONCLUSIONS: MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.

A nested PCR approach for unambiguous typing of pestiviruses infecting cattle.

An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle.

Astroviruses as causative agents of poultry enteritis: genetic characterization and longitudinal studies on field conditions.

Astroviruses (AstVs) are nonenveloped RNA small round viruses (SRVs) with a genome of 6.8-7.9 kb. Known avian AstVs are spread worldwide; they have been associated with poult enteritis and mortality syndrome in the United States and reported in Italy in intensive turkey and guinea fowl flocks. Nevertheless, their real prevalence and their pathogenic role in avian enteritis affecting Italian flocks is far from clear. Negative staining electron microscopy (nsEM) is used for the routine diagnosis of avian enteric SRVs, although it cannot distinguish morphologically similar particles. Enzyme-linked immunosorbent assay (ELISA), reverse-transcription PCR (RT-PCR), and genomic sequencing are now used for this specific purpose. We analyzed 329 samples of chicken, turkey, and guinea fowl intestinal contents from Italian poultry flocks. Most samples were from enteritis outbreaks, but we also included samples from three longitudinal studies (one on 11 broiler flocks and the other two on a guinea fowl flock). We first examined the samples with nsEM. SRVs, including AstVs, are often associated with rotaviruses and were the most commonly detected morphotypes in avian enteric diseases. We then analyzed 124 of the samples with an RT-PCR targeting the open reading frame (ORF)-1b of AstV. This gene codes for an RNA-dependent polymerase. We then sequenced and genetically analyzed the RT-PCR positive samples. Phylogenetic analysis distinguished three defined clusters: the first included guinea fowl AstVs and turkey AstVs-2; the second, chicken AstVs; and the third was formed by avian nephritis viruses (ANVs). No strains clustered with turkey AstVs-1. The results indicate that ORF-1b presents certain genetic variability, even among AstVs from the same species. In longitudinal studies, samples retrieved from the same shed were homogeneous, with some exceptions suggesting possible coexistence of different genetic types in the same unit. The finding of ANV-like viruses in commercial guinea fowls underlines the genetic variability of AstVs and strengthens the hypothesis of a varied intraherd situation.

Parapoxvirus infections of red deer, Italy.

To characterize parapoxviruses causing severe disease in wild ruminants in Stelvio Park, Italy, we sequenced and compared the DNA of several isolates. Results demonstrated that the red deer isolates are closely related to the parapox of red deer in New Zealand virus.

Atypical pestivirus and severe respiratory disease in calves, Europe.

In 2010, a HoBi-like pestivirus was isolated from clinically affected calves in Italy. This European virus reproduced a milder form of disease under experimental conditions and was genetically related to previously reported HoBi-like strains. Isolation of this novel virus from a clinical outbreak may have implications for cattle health and prophylactic programs.

Phylogenetic relationships of Western Mediterranean West Nile virus strains (1996-2010) using full-length genome sequences: single or multiple introductions?

In recent years, West Nile virus (WNV) has re-emerged in the Western Mediterranean region. As a result, the number of complete WNV genome sequences available from this region has increased, allowing more detailed phylogenetic analyses, which may help to understand the evolutionary history of WNV circulating in the Western Mediterranean. To this aim, the present work describes six new complete WNV sequences from recent outbreaks and surveillance in Italy in 2008-2009 and in Spain in 2008 and 2010. Comparison with other sequences from different WNV clusters within lineage 1 (clade 1a) confirmed that all Western Mediterranean WNV isolates obtained since 1996 (except one from Tunisia, collected in 1997) cluster in a single monophyletic group (here called 'WMed' subtype). The analysis differentiated two subgroups within this subtype, which appear to have evolved from earlier WMed strains, suggesting a single introduction in the area, and further dissemination and evolution. Close similarities between WNV variants circulating in consecutive years, one in Spain, between 2007 and 2008, and another in Italy between 2008 and 2009, suggest that the virus possibly overwinters in Western Mediterranean sites. The NS3(249)-proline genotype, recently proposed as a virulence determinant for WNV, has arisen independently at least twice in the area. Overall, these results indicate that the frequent recurrence of outbreaks caused by phylogenetically homogeneous WNV in the Western Mediterranean since 1996 is consistent with a single introduction followed by viral persistence in endemic foci in the area, rather than resulting from independent introductions from exogenous endemic foci.

Recombinant canine coronaviruses related to transmissible gastroenteritis virus of Swine are circulating in dogs.

Four canine coronavirus type II (CCoV-II) strains were identified in the guts and internal organs of pups which had died of acute gastroenteritis. The CCoV-II strains were strictly related to porcine transmissible gastroenteritis virus (TGEV) in the N-terminal domain of the spike protein, whereas in the other parts of the genome, a higher genetic relatedness to recent CCoV-II isolates was observed. Experimental infection of dogs with a TGEV-like isolate induced mild gastroenteritis without any systemic involvement. By virus neutralization tests, antigenic differences between reference and TGEV-like CCoVs were found. Our data support the potential recombinant origin of the TGEV-like CCoVs.

Encephalomyocarditis virus infection in an Italian zoo.

A fatal Encephalomyocarditis virus (EMCV) infection epidemic involving fifteen primates occurred between October 2006 and February 2007 at the Natura Viva Zoo. This large open-field zoo park located near Lake Garda in Northern Italy hosts one thousand animals belonging to one hundred and fifty different species, including various lemur species. This lemur collection is the most relevant and rich in Italy. A second outbreak between September and November 2008 involved three lemurs. In all cases, the clinical signs were sudden deaths generally without any evident symptoms or only with mild unspecific clinical signs. Gross pathologic changes were characterized by myocarditis (diffuse or focal pallor of the myocardium), pulmonary congestion, emphysema, oedema and thoracic fluid. The EMCV was isolated and recognized as the causative agent of both outbreaks. The first outbreak in particular was associated with a rodent plague, confirming that rats are an important risk factor for the occurrence of the EMCV infection.

Epidemic of infectious laryngotracheitis in Italy: characterization of virus isolates by PCR-restriction fragment length polymorphism and sequence analysis.

Between May 2007 and October 2008, 34 outbreaks of mild to moderate forms of infectious laryngotracheitis (ILT) occurred in commercial broiler flocks in Italy. Affected birds showed watery eyes, conjunctivitis, nasal discharge, reduction of feed and water consumption, and gasping with expectoration of blood-stained mucus. The mortality rate was < 10%. Gross lesions consisted of conjunctivitis, excess of mucus, blood, or presence of diphtheritic membranes in trachea. A real-time PCR assay was performed to confirm the presence of ILT virus (ILTV) DNA in tracheal tissue homogenates. Twenty-three ILTV isolates were propagated on the chorion-allantoic membrane of embryonated chicken eggs showing typical plaques. PCR combined with restriction fragment length polymorphism and gene sequencing of isolates showed a high genetic correlation between field strains and chicken embryo origin vaccines.

Characterization of a new genotype and serotype of infectious bronchitis virus in Western Africa.

Between 2002 and 2007, more than 1000 chickens from commercial farms, live bird markets and backyard farms in Nigeria and Niger were tested for the presence of the infectious bronchitis virus (IBV) genome. Phylogenetic analysis of full-length sequences of the spike 1 (S1) gene revealed a new genotype of IBV that we refer to as 'IBADAN'. The minimum genetic distance to the closest 'non-IBADAN' strains (UK/7/93 at the nucleotide level; H120 and M41 at the amino acid level) reached 24 and 32 % at the nucleotide and amino acid levels, respectively. The full genome of the IBADAN reference strain (NGA/A116E7/2006) had a genetic distance of 9.7-16.4 % at the nucleotide level with all available fully sequenced strains. As IBV S1 plays a major role in antigenicity, the antigenic relatedness of NGA/A116E7/2006 was compared with strains of other serotypes. NGA/A116E7/2006 did not cross-react with antisera against IT02, M41, D274, Connecticut or 793/B strains in virus neutralization assays. NGA/A116E7/2006 cross-reacted with the QX-like strain ITA/90254/2005 but only to a low level (antigenic relatedness of 33 %), suggesting that IBADAN also represents a new serotype. A comparison of S1 sequences identified several amino acids that may play a role in IBV antigenicity. Despite the absence of obvious clinical signs in poultry infected by IBADAN strains, it is important to test the cross-protection of current vaccine strains.

A candidate modified-live bovine coronavirus vaccine: safety and immunogenicity evaluation.

A modified-live vaccine against the respiratory form of bovine coronavirus (BCoV) infection was developed by progressive attenuation of a respiratory strain (438/06-TN). The vaccine was found to be safe as four colostrum-deprived newborn calves remained healthy after oronasal administration of ten doses of the vaccine. The immunogenicity of the vaccine was assessed by intramuscular injection of one vaccine dose to 30 BCoV-antibody negative 2-3-month-old calves. At 30 days post-vaccination, all vaccinated calves displayed high antibody titres against BCoV. Sequence analysis of the S gene of wild-type and cell-adapted 438/06-TN strain detected 10 nucleotide changes, 9 of which were non-synonymous.

Severe outbreak of bovine coronavirus infection in dairy cattle during the warmer season.

A severe outbreak of enteric and respiratory disease associated with bovine coronavirus (BCoV) infection is described. The outbreak occurred in a dairy herd of southern Italy in the first decade of September 2006, when summer temperatures were still recorded, affecting calves, heifers and adult cows, with a marked decrease in milk production. By virus isolation and RT-PCR targeting the S gene, BCoV was identified as the etiological agent of the outbreak, whereas bacteriological, parasitological and toxicological investigations failed to detect other causes of disease. BCoV strains with 99-100% nucleotide identity in the S gene were isolated from nasal, ocular and rectal swabs, thus proving the absence of separate clusters of virus on the basis of tissue tropism. Sequence analysis of the haemagglutination-esterase and spike proteins of the strain detected in one rectal sample (339/06) showed a high genetic relatedness with recent BCoV isolates (98-99% amino acid identity), with several unique amino acid substitutions in the S protein. The BCoV outbreak described in this paper presents interesting aspects: (i) the occurrence of a severe form of disease in the warmer season; (ii) the simultaneous presence of respiratory and enteric disease; (iii) the involvement of young as well as adult cattle.

Serological and molecular evidence that canine respiratory coronavirus is circulating in Italy.

Canine respiratory coronavirus (CRCoV) is a group II coronavirus that was firstly identified in lung samples of dogs with canine infectious respiratory disease (CIRD) in UK in 2003. We report for the first time the identification of CRCoV in Italy, together with serological evidence that the virus has been circulating in the Italian dog population as from 2005. Serological investigations on 216 dog sera, carried out by an ELISA test using the strictly related bovine coronavirus (BCoV) as antigen, revealed an overall CRCoV seroprevalence of 32.06% in the last 2 years. RT-PCR targeting the S-gene of CRCoV was carried out on 109 lung samples collected from carcasses of dogs submitted for diagnostic investigations. Positive results were obtained from the lungs of a dog of the Apulia region that was co-infected with canine parvovirus type 2. Sequence analysis of the S-gene fragment amplified by RT-PCR (595bp) showed similarity to group II coronaviruses, with the highest nucleotide identity (98%) to the only CRCoV strain currently available in the GenBank database (strain T101). The results of the present study show that CRCoV is present also in continental Europe, although further studies are required to determine the real pathogenic potential of the virus.

Genetic and antigenic characterization of infectious bursal disease viruses isolated in Italy during the period 2002-2005.

During the period 2002-2005, 109 infectious bursal disease virus (IBDV) field strains were isolated from bird flocks located in various parts of Italy. Out of these strains, 91 were isolated from broilers, 12 from pullets, and six from backyard flocks. Forty-two IBDV strains were further investigated and characterized on the basis of the geographical origin, source, and clinical signs. Antigenic and genetic characterizations were carried out using a monoclonal antibody (MAb)-based antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA) or a virus neutralization assay and a reverse transcription, amplification, and direct sequencing of a genome fragment encoding the VP2 variable domain. The viruses were compared with reference IBDV strains, F52/70 (classical, 1970), 89163 (typical very virulent [vv]IBDV, 1989), 91168 (antigenically modified vvIBDV, 1991) and 94432 (antigenically modified vvIBDV, 1994) among others. All 42 strains were genetically characterized, and the comparison of their nucleotide sequences revealed the presence of six clusters having 100% identity, named group 1, 2, 3, 4, 5, and 6. Twelve strains, representative of each molecular group and/or with interesting amino acid sequence, were also antigenically characterized. The antigenic characterization showed six strains--151573, 157185 (group 1), 192294 (group 2), 77882 (group 3), 217 (group 4), and 192304--with the profile typical of vvIBDV (lack of binding of MAbs 3 and 4). Two strains, 77165 and 204875 (group 6), were also related to vvIBDV but did not react with MAb 5. Three isolates exhibited a profile of cell culture-adapted viruses and classical strains but with some differences: strain 157776 reacted with all MAbs; strain 168026 with all MAbs except MAb 4, which weakly neutralized it; and strain 72293 with all MAbs except MAb 9, which is rather unusual. The last strain, 213622, showed a very uncommon antigenic profile with missing or reduced binding of MAbs 3, 4, 5, 6, 8, and 9. Genetic characterization revealed 37 strains identified as vvIBDV viruses divided in 26 isolates (including groups 1, 2, 3, and 4) with the four amino acids residues typical of vvIBDV (222A, 256I, 294I, 299S) and 11 isolates (including groups 5, 6, and 213622) with some other amino acid exchanges. Four isolates (72293, 168026, 196783, and 222220) presented an amino acid sequence closely related to attenuated classical viruses whereas the last isolate (157776) exhibited a rather different sequence with some mutations typical of vvIBDV and others for cell culture-adapted viruses. Results of the antigenic and genetic characterization revealed that the majority of viruses (n = 37) were related to vvIBDV strains but, among these, 11 strains presented antigenically and genetically modified characteristics and originated, in major part, from the area where viruses have been circulating for a long time. The remaining viruses (n = 5) were related but not identical to attenuated classical viruses and came from areas where vaccination with intermediate strains is applied.

Serosurvey of roe deer, chamois and domestic sheep in the central Italian Alps.

Roe deer (Capreolus capreolus), chamois (Rupicapra rupricapra rupicapra), and domestic sheep in the Orobie Alps, Italy, were serologically tested for antibodies to selected pathogens that may be transmitted across species. Antibodies against Brucella spp. and bovine herpesvirus 1 (roe deer and chamois only) were not detected in any species. In roe deer, antibodies were detected against Toxoplasma gondii (13%) and Neospora caninum (3%). Chamois tested positive for antibodies to T. gondii (5%), N. caninum (21%), bovine respiratory syncytial virus (BRSV) (41%), bovine parainfluenza type-3 virus (17%), pestiviruses (18%), and Mycoplasma conjunctivae (17%). In the sheep, particularly high antibody prevalence rates were found for T. gondii (78%), Chlamydophila spp. (20%), pestiviruses (90%), BRSV (82%), and M. conjunctivae (81%).