Genomic characterization of swine influenza viruses in Italy using NGS technology

I sottotipi H1N1, H1N2 e H3N2 di influenza A virus sono largamente diffusi nella popolazione suina di tutto il mondo. Nel presente lavoro è stato sviluppato un protocollo di sequenziamento di c.d. nuova generazione, su piattaforma Ion Torrent PGM, idoneo per l’analisi di tutti i virus influenzali suini (SIV). Per valutare l’evoluzione molecolare dei SIV italiani, sono stati sequenziati ed analizzati mediante analisi genomica e filogenetica un totale di sessantadue ceppi di SIV appartenenti ai sottotipi H1N1, H1N2 e H3N2, isolati in Italia dal 1998 al 2014. Sono stati evidenziati in sei campioni due fenomeni di riassortimento: tutti i SIV H1N2 esaminati presentavano una neuraminidasi di derivazione umana, diversa da quella dei SIV H1N2 circolanti in Europa, inoltre l’emoagglutinina (HA) di due isolati H1N2 era originata dal riassortimento con un SIV H1N1 avian-like. L’analisi molecolare dell’HA ha permesso di rivelare un’inserzione di due amminoacidi in quattro SIV H1N1 pandemici e una delezione di due aminoacidi in quattro SIV H1N2, entrambe a livello del sito di legame con il recettore cellulare. E’ stata inoltre evidenziata un’elevata omologia di un SIV H1N1 con ceppi europei isolati negli anni ’80, suggerendo la possibile origine vaccinale di questo virus. E’ stato possibile, in aggiunta, applicare il nuovo protocollo sviluppato per sequenziare un virus influenzale aviare altamente patogeno trasmesso all’uomo, direttamente da campione biologico. La diversità genetica nei SIV esaminati in questo studio conferma l’importanza di un continuo monitoraggio della costellazione genomica dei virus influenzali nella popolazione suina.Three major swine influenza virus (SIV) subtypes (H1N1, H1N2, and H3N2) are currently identified in pigs worldwide. In this study, a protocol able to sequence all SIV subtypes using the Ion Torrent next generation sequencing technology was developed. Moreover, to investigate the genetic characteristics and the molecular evolution of Italian SIV circulating strains, we conducted genomic and phylogenetic analysis for each segment of sixty-two SIVs isolated in Italy between 1998 and 2014 comprising H1N1, H1N2 and H3N2 strains. Two independent reassortment events in six SIV strains were detected. H1N2 SIVs showed the introduction of a human-like neuraminidase, different from the homologous of circulating H1N2 detected in Europe from swine. Two of these reassortant H1N2 strains contained an avian-like hemagglutinin (HA). Analysis of HA revealed a two-amino acid insertion in four H1N1 pandemic strains and a two-amino acid deletion in four H1N2 strains, both at the HA receptor binding site. High nucleotide identity between an H1N1 of this study and European field strains isolated in the 80s was revealed, suggesting a probable vaccine origin for this strain. Translational benefit of the newly developed protocol was immediately achieved by sequencing a H7N7 high pathogenic avian influenza virus, which caused disease in humans. The genetic diversity of SIVs demonstrated in our investigation confirms the importance of continuous monitoring of SIV genomic constellation

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

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

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

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

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 a commercial 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

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 â\u80\u9cvery goodâ\u80\u9d 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

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

Abstract 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

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 USUVprimed 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