Immunization with Usutu virus and with a chimeric West Nile virus (WNV) harboring Usutu-E protein protects immunocompetent adult mice against lethal challenges with different WNV lineage 1 and 2 strains.

West Nile virus (WNV) and Usutu virus (USUV), two antigenically related flaviviruses co-circulating in Europe, can cause severe neurological disease in animals and humans. The immune response against USUV and WNV and their immunopathogenesis are still poorly investigated. Here we present results upon sequential infections of adult immunocompetent CD-1 and BALB/c mice primed with two different doses (high dose, HD or low dose, LD) of an USUV isolate and challenged with HD or LD of three different WNV isolates. CD-1 and BALB/c LD USUV-primed mice, regardless of the dose, are largely protected from lethal WNV challenges despite showing no detectable neutralizing antibodies. Furthermore, mice immunized with a chimeric virus harboring the E protein of USUV within the WNV backbone (WNVE-USUV) are protected against a lethal challenge with WNV. We believe these findings could contribute to understanding the dynamics of the interaction during sequential infection of these two flaviviruses.

The envelope protein of Usutu virus attenuates West Nile virus virulence in immunocompetent mice.

West Nile virus (WNV) and Usutu virus (USUV) are the two most widespread mosquito-borne flaviviruses in Europe causing severe neuroinvasive disease in humans. Here, following standardization of the murine model with wild type (wt) viruses, we engineered WNV and USUV genome by reverse genetics. A recombinant virus carrying the 5' UTR of WNV within the USUV genome backbone (r-USUV5'-UTR WNV) was rescued; when administered to mice this virus did not cause signs or disease as wt USUV suggesting that 5' UTR of a marked neurotropic parental WNV was not per se a virulence factor. Interestingly, a chimeric virus carrying the envelope (E) protein of USUV in the WNV genome backbone (r-WNVE-USUV) showed an attenuated profile in mice compared to wt WNV but significantly more virulent than wt USUV. Moreover, except when tested against serum samples originating from a live WNV infection, r-WNVE-USUV showed an identical antigenic profile to wt USUV confirming that E is also the major immunodominant protein of USUV.

Seroprevalence of African horse sickness in selected donkey populations in Namibia.

BACKGROUND AND AIM: African horse sickness (AHS) is a non-contagious viral disease of horses and other equids caused by an arbovirus belonging to the Reoviridae family and genus Orbivirus. AHS is an endemic disease that is responsible for the death of a high number of horses every year in Namibia. At present, there is no information on the prevalence and distribution of AHS virus (AHSV) serotypes in the different regions of Namibia. Therefore, this survey aimed to fill this knowledge gap by investigating the AHSV seroprevalence in Namibian donkeys. MATERIALS AND METHODS: A total of 260 blood samples (20 samples for each region) were randomly collected from donkeys aged between 3 and 5 years. Sera were screened for AHSV-specific immunoglobulin G antibodies using acommercial competitive enzyme-linked immunosorbent assay kit and samples positive to AHSV antibodies were further tested by serum neutralization (SN) assay to evaluate the AHSV serotype-specific immune response. RESULTS: Seroprevalence of antibodies against AHSV in Namibian donkeys was 63.5%. The AHSV prevalence was significantly higher in the northern region (64%) than in the southern region (36%). A significantly (p<0.05) higher number of donkeys had antibodies against AHSV-6 (37.8%) and AHSV-9 (37.8%). The AHSV-2, AHSV-6, and AHSV-9 prevalence were higher (p<0.05) in the northern regions compared to the southern regions. None of the donkeys in this study, however, tested positive for AHSV-8. CONCLUSION: Results of the current study indicate that all AHSV serotypes have either circulated previously or are circulating in Namibia except for AHSV-8. In particular, AHSV-1, -2, -3, -4, -5, -6, and -9 serotypes have circulated or are circulating in the northern region of Namibia, while AHSV-1, -4, -5, -6, -7, and -9 have infected donkeys in the south. AHSV-9 and AHSV-6 were the most prevalent serotypes detected in donkeys in this study. SN results showed that several donkeys from Kavango East, Kavango West, and Ohangwena regions had been exposed to multiple serotypes, indicating the possibility of cocirculation of several strains in Namibia.

Detection of Astrovirus in a Cow with Neurological Signs by Nanopore Technology, Italy.

In this study, starting from nucleic acids purified from the brain tissue, Nanopore technology was used to identify the etiological agent of severe neurological signs observed in a cow which was immediately slaughtered. Histological examination revealed acute non-suppurative encephalomyelitis affecting the brainstem, cerebrum, cerebellum, and medulla oblongata, while by using PCR-based assays, the nucleic acids of major agents for neurological signs were not detected. By using Nanopore technology, 151 sequence reads were assigned to Bovine Astrovirus (BoAstV). Real-time RT-PCR and in situ hybridization (ISH) confirmed the presence of viral RNA in the brain. Moreover, using the combination of fluorescent ISH and immunofluorescence (IF) techniques, it was possible to detect BoAstV RNA and antigens in the same cells, suggesting the active replication of the virus in infected neurons. The nearly whole genome of the occurring strain (BoAstV PE3373/2019/Italy), obtained by Illumina NextSeq 500, showed the highest nucleotide sequence identity (94.11%) with BoAstV CH13/NeuroS1 26,730 strain, an encephalitis-associated bovine astrovirus. Here, we provide further evidence of the role of AstV as a neurotropic agent. Considering that in a high proportion of non-suppurative encephalitis cases, which are mostly indicative of a viral infection, the etiologic agent remains unknown, our result underscores the value and versatility of Nanopore technology for a rapid diagnosis when the PCR-based algorithm gives negative results.

Diagnosis and characterization of canine distemper virus through sequencing by MinION nanopore technology.

Prompt identification of the causative pathogen of an infectious disease is essential for the choice of treatment or preventive measures. In this perspective, nucleic acids purified from the brain tissue of a dog succumbed after severe neurological signs were processed with the MinION (Oxford Nanopore Technologies, Oxford UK) sequencing technology. Canine distemper virus (CDV) sequence reads were detected. Subsequently, a specific molecular test and immunohistochemistry were used to confirm the presence of CDV RNA and antigen, respectively, in tissues. This study supports the use of the NGS in veterinary clinical practice with potential advantages in terms of rapidity and broad-range of molecular diagnosis.

Identification and genetic characterization of bovine enterovirus by combination of two next generation sequencing platforms.

Prompt and accurate diagnosis is warranted for infectious diseases of domestic animals which may have a significant impact on animal production or clinical practice. In this study, the identification and genetic characterization of a bovine enterovirus (BEV) strain isolated from a calf with diarrhea, are described. Two different next generation sequencing platforms were employed. Shotgun metagenomic accomplished by MinION sequencing (Oxford Nanopore Technologies) allowed the identification of BEV RNA from a cell-culture isolate. BEV was then confirmed by a specific real time RT-PCR assay. To achieve the whole genome of this isolate, sequence reads obtained by MinION were coupled with those originating from NextSeq500 (Illumina). Genomic relatedness and phylogeny with extant BEV strains is also reported. Overall, this manuscript highlights the use of the portable MinION sequence technology as a tool for support diagnostics in veterinary practice.

Outbreak of porcine epidemic diarrhoea virus (PEDV) in Abruzzi region, central-Italy.

Here we report and characterize a porcine epidemic diarrhea (PED) outbreak which occurred in a swine fattening farm in the province of Teramo, Abruzzi region (central Italy), in January 2016. PED virus (PEDV) identification was determined by real-time RT-PCR performed on RNAs purified from fecal samples collected from two symptomatic pigs. Whole genome sequence (PEDV 1842/2016) was also obtained by next generation sequencing straight from RNA purified from one fecal sample. Genome comparison with extant global PEDV strains revealed a high nucleotide identity with recently reported European and American S-INDEL PEDVs. Efficient sequencing, share of genomic data combined with the implementation of epidemiological tools would be the ideal approach for study and analysis of transboundary infectious diseases as PED.

Analysis of bluetongue serotype 3 spread in Tunisia and discovery of a novel strain related to the bluetongue virus isolated from a commercial sheep pox vaccine.

Bluetongue (BT), is one of the OIE-listed major diseases of ruminants. Following the official report of BT virus serotype 3 (BTV-3) in a sheep in Cap Bon (Tunisia), blood and serum samples of ruminants were collected from some areas of Tunisia to further investigate the presence of this virus in the country. A quantitative real time RT-PCR has been first developed for the detection and quantitation of BTV-3 RNA from field specimens. Out of 62 collected blood samples, 23 were shown to be positive for BTV-3 RNA. Isolation on cell cultures was also possible from six samples. Genome sequencing revealed the circulation of two unrelated western strains of BTV-3, one circulating in Cap Bon and neighboring areas, and the other circulating nearby the border with Libya. The presence of a putative novel BTV serotype (BTV-Y TUN2017) in sheep introduced from Libya to Tunisia, genomically related to the BTV strain contaminating a commercially-available sheep pox vaccine and to BTV-26, has been also demonstrated. This finding highlights the pressing need for a prompt production and release of a novel inactivated BTV-3 vaccine to be used in case of emergence or proactively in the areas of Southern Europe at major risk of BTV introduction. The assessment of a novel vaccine will certainly exalt the role and importance of surveillance activities and collaboration with Northern African countries.

Correction to: Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy.

CORRECTION: After Publication of the article [1], it has been brought to our attention that an author's name has been spelt incorrectly. The correct spelling should be "Massimo Ciccozzi", but it was previously included as "Massimo Cicozzi". The original version has now been revised to reflect this.

Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii. METHODS: Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB). RESULTS: Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs. CONCLUSIONS: This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity.

Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of epizootic haemorrhagic disease virus (EHDV) antibodies.

Epizootic haemorrhagic disease (EHD) is a vector-borne infectious viral disease of domestic and wild ruminants. EHD could spread from infected northern African countries in free territories like the EU; therefore, the availability of diagnostic assays would represent key components for adequate surveillance and control programs. In this study, the gene encoding the VP7 protein of EHD virus (EHDV) was expressed into a baculovirus-infected insect cell system. With this unpurified protein we developed a home-made competitive ELISA (cELISA) and a total number of 275 serum samples, originating from domestic and wild ruminants, were tested. 74/275 were previously shown to be positive for EHDV antibodies by a commercially available ELISA kit. A "very good" agreement was demonstrated when compared to a commercial ELISA kit (Cohen's kappa value=0.832). Samples which caused disagreement between the two assays originated from wildlife which highlights the need for further validation by using serum samples from wild animals.

Genome Sequence of Canine Adenovirus Type 1 Isolated from a Wolf (Canis lupus) in Southern Italy.

Canine adenovirus type 1 (CAdV-1), a DNA virus of the family Adenoviridae, causes infectious canine hepatitis, a highly contagious disease primarily affecting canids. In this report, we describe the isolation and whole-genome sequence of a CAdV-1 isolate from the liver of a free-ranging wolf (Canis lupus).

Whole-Genome Sequence of a Suid Herpesvirus-1 Strain Isolated from the Brain of a Hunting Dog in Italy.

Suid herpesvirus-1 (SHV-1), a DNA virus of the family Herpesviridae, causes a severe and fatal disease in a wide range of mammals. Here, we report the whole-genome sequence of an SHV-1 isolated in Italy in 2014 from the brain of a hunting dog that suffered from an acute and severe disease.

Lethal distemper in badgers (Meles meles) following epidemic in dogs and wolves.

Canine distemper virus (CDV) represents an important conservation threat to many wild carnivores. A large distemper epidemic sustained by an Arctic-lineage strain occurred in Italy in 2013, mainly in the Abruzzi region, causing overt disease in domestic and shepherd dogs, Apennine wolves (Canis lupus) and other wild carnivores. Two badgers were collected by the end of September 2015 in a rural area of the Abruzzi region and were demonstrated to be CDV-positive by real time RT-PCR and IHC in several tissues. The genome of CDV isolates from badgers showed Y549H substitution in the mature H protein. By employing all publicly available Arctic-lineage H protein encoding gene sequences, six amino acid changes in recent Italian strains with respect to Italian strains of dogs from 2000 to 2008, were observed. A CDV strain belonging to the European-wildlife lineage was also identified in a fox found dead in the same region in 2016, proving co-circulation of an additional CDV lineage.

Genome characterization of feline morbillivirus from Italy.

Feline morbillivirus (FeMV) has been recently identified by RT-PCR in the urine sample of a nephropathic cat in Italy. In this report, we describe the whole genome sequence of strain Piuma/2015 obtained by combination of sequence independent single primer amplification method (SISPA) and next generation sequencing (NGS) starting from RNA purified from the infected urine sample. The existence in Germany and Turkey of FeMVs from cats divergent from Piuma/2015, suggests the presence of FeMV heterogeneity in Europe as it has been described previously in Japan and China.

Circovirus in domestic and wild carnivores: An important opportunistic agent?

Circoviruses are relatively novel pathogens with increased importance in canids. In this study, we first screened the presence of dog circovirus (DogCV) by molecular methods from a total number of 389 internal organ samples originating from 277 individuals of domestic dogs and wild animals including wolves, foxes and badgers. All the animals originated from Central-Southern Italy, specifically from Abruzzi and Molise regions, areas hosting several natural parks. DogCV was detected in 9/34 wolves (P=26.4%; IC 95%: 14.6-43.1%), 8/209 dogs (P=3.8%; IC 95%: 1.9-7.3%), 0/24 foxes (P=0%; IC 95%: 0-13.8%), 1/10 badgers (P=10%; IC 95%: 1.79-40.4%). However, all DogCV positive animals were shown to be infected at least by an additional key pathogen, including canine distemper virus (CDV) and canine parvovirus type 2. All wolves, but one, presenting DogCV in the internal tissues suffered from CDV infection. The DNA purified from 17 DogCV infected organs was used for whole genome sequencing and phylogenetic analysis.

First report of feline morbillivirus in Europe.

Feline morbillivirus was detected in urine samples of a 15 year old cat suffering from severe nephropathy. Viral RNA was not detected in blood and faecal samples and also the most common pathogens associated to cat kidney failure were not found. This report describes the first evidence of feline morbillivirus in Europe.

Toscana virus genome stability: data from a meningoencephalitis case in Mantua, Italy.

In July of 2013, samples from a patient with a neurological syndrome were collected from Mantua hospital and sent to the National Reference Laboratory for Arboviruses (National Institute of Health, Rome). On the basis of the symptoms, serological and molecular assays were performed to diagnose either West Nile virus (WNV) or Toscana virus (TOSV) infection. Molecular and serological tests confirmed TOSV infection. Virus isolation was obtained from cerebrospinal fluid. A full genome sequence was determined from this TOSV strain with next-generation sequencing using Ion Torrent technology. Nucleotide and amino acidic sequences grouped phylogenetically with lineage TOSV A and showed a low genome variability.

Genetic characterization and evolution of H1N1pdm09 after circulation in a swine farm.

Following the emergence of the A(H1N1)pdm09 in humans, this novel influenza virus was reverse transmitted from infected people to swine population worldwide. In this study we investigated the molecular evolution of A(H1N1)pdm09 virus identified in pigs reared in a single herd. Nasal swabs taken from pigs showing respiratory distress were tested for influenza type A and A(H1N1)pdm09 by real-time RT-PCR assays. Virus isolation from positive samples was attempted by inoculation of nasal swabs samples into specific pathogen free embryonated chicken eggs (ECE) and complete genome sequencing was performed on virus strains after replication on ECE or from original swab sample. The molecular analysis of hemagglutinin (HA) showed, in four of the swine influenza viruses under study, a unique significant amino acid change, represented by a two-amino acid insertion at the HA receptor binding site. Phylogenetic analysis of HA, neuraminidase, and concatenated internal genes revealed a very similar topology, with viruses under study forming a separate cluster, branching outside the A(H1N1)pdm09 isolates recognized until 2014. The emergence of this new cluster of A(H1N1)pdm09 in swine raises further concerns about whether A(H1N1)pdm09 with new molecular characteristics will become established in pigs and potentially transmitted to humans.

Full-length genomic analysis of porcine rotavirus strains isolated from pigs with diarrhea in Northern Italy.

Group A rotaviruses (RVA) cause acute dehydrating diarrhea in young of man and many animal species, including pigs. Swine RVA has an important economic impact on the farming industry, and pigs represent a potential reservoir for zoonotic transmission of RVA to humans. To investigate the genetic diversity of porcine RVA strains in Italy and identify their possible zoonotic characteristics, 25 RVA-positive feces were collected from diarrheic pigs in Northern Italy, in 2009-2010; all viral strains were characterized by G and P genotyping RT-PCR. Three samples were selected for full genome sequencing. Sequencing of the NSP3 genes of all samples was also performed. Rotavirus diagnosis was carried out by ELISA and electron microscopy. RT-PCR and Sanger sequencing were performed in a one-tube format, using primer sets specific for each of the 11 genome segments. Analysis of the G (VP7) and P (VP4) genotypes showed that all strains identified were typical porcine RVAs (G4, G5, G9; P[6], P[13], P[23]). Full-length genome sequencing was performed on selected G9 isolates. Most segments belonged to the genotype constellation 1 (Wa-like), which is shared by most human RVA strains, but gene types such as I5 (VP6) and A8 (NSP1), which are typical of porcine and rare among human RVAs, were also detected. We identified RVA strains showing the T7 genotype, an NSP3 gene type that was previously reported in unusual strains of possible porcine or bovine origin from children with diarrhea. Recent reports suggested that G9 RVA may have been introduced from swine to human populations involving gene reassortment events. The observation that some of the RVA genotypes from swine in Italy were similar to viruses characterized in children underlines the importance of animal RVA surveillance, to clarify and monitor the role of animals as genetic reservoirs of emerging RVA strains pathogenic for humans.