Paslahepevirus balayani (Hepatitis E Virus) in Italian Nonungulate Wildlife: Molecular Detection and Characterization of an Isolate from a Crested Porcupine (Hystrix cristata).

Paslahepevirus balayani (hepatitis E virus [HEV]) is the causative agent of hepatitis E, a worldwide zoonosis involving a wide range of hosts among domestic and wild animals. This species is characterized by a great genomic heterogeneity and includes eight genotypes, HEV-1 to HEV-8. The HEV-3 genotype is one of the most common types circulating in Italy in humans and Suidae. Although domestic and wild Sus scrofa and deer (Cervidae) are recognized as the main reservoirs of HEV, several other wild species are potential carriers. A total of 228 liver samples were collected from nonungulate wild animals, found dead, in the framework of the regional passive surveillance program in Umbria and Marche regions (central Italy) during 2018-20. These were tested using real-time reverse-transcriptase PCR (RT-PCR) for detection of RNA of HEV-1 to HEV-4 and confirmed by nested RT-PCR assay. One of the 11 samples collected from crested porcupines (Hystrix cristata) tested positive for the presence of HEV RNA; all other samples were negative. Sequence analysis based on the full-length genome revealed that this isolate, 49434/UM/2018 (accession no. OL658617), belongs to the HEV-3e subtype. These findings suggest a potential role of crested porcupines as a carrier of HEV infection.

Development of molecular and antigenic-based rapid tests for the identification of African swine fever virus in different tissues.

African swine fever (ASF) is a severe haemorrhagic infectious disease affecting suids, thus representing a great economic concern. Considering the importance of the early diagnosis, rapid point of care testing (POCT) for ASF is highly demanded. In this work, we developed two strategies for the rapid onsite diagnosis of ASF, based on Lateral Flow Immunoassay (LFIA) and Recombinase Polymerase Amplification (RPA) techniques. The LFIA was a sandwich-type immunoassay exploiting a monoclonal antibody directed towards the p30 protein of the virus (Mab). The Mab was anchored onto the LFIA membrane to capture the ASFV and was also labelled with gold nanoparticles for staining the antibody-p30 complex. However, the use of the same antibody for capturing and as detector ligand showed a significant competitive effect for antigen binding, so required an experimental design to minimize reciprocal interference and maximize the response. The RPA assay, employing primers to the capsid protein p72 gene and an exonuclease III probe, was performed at 39 °C. The limit of detection of the method was assessed using a plasmid encoding the target gene and resulted in 5 copy/µL. The new LFIA and RPA were applied for ASFV detection in the animal tissues usually analysed by conventional assays (i.e., real-time PCR), such as kidney, spleen, and lymph nodes. A simple and universal virus extraction protocol was applied for sample preparation, followed by DNA extraction and purification for the RPA. The LFIA only required the addition of 3% H2O2 to limit matrix interference and prevent false positive results. The two rapid methods (25 min and 15 min were needed to complete the analysis for RPA and LFIA, respectively) showed high diagnostic specificity (100%) and sensitivity (93% and 87% for LFIA and RPA, respectively) for samples with high viral load (Ct < 27). False negative results were observed for samples with low viral load (Ct > 28) and/or also containing specific antibodies to ASFV, which decreased antigen availability and were indicative of a chronic, poorly transmissible infection. The simple and rapid sample preparation and the diagnostic performance of the LFIA suggested its large practical applicability for POC diagnosis of ASF.

Use of multivariate analysis to evaluate antigenic relationships between US BVDV vaccine strains and non-US genetically divergent isolates.

Bovine viral diarrhea virus (BVDV) comprises two species, BVDV-1 and BVDV-2. But given the genetic diversity among pestiviruses, at least 22 subgenotypes are described for BVDV-1 and 3-4 for BVDV-2. Genetic characterization is generally accomplished through complete or partial sequencing and phylogeny, but it is not a reliable method to define antigenic relationships. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to define antigenic relatedness can be difficult to discern for BVDV isolates within the same BVDV species. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between US vaccine strains and field isolates from Switzerland, Italy, Brazil, and the UK. Polyclonal sera were generated against six BVDV strains currently contained in vaccine formulations, and each serum was used in VNs to measure the titers against seven vaccine strains (including the six homologous strains) and 23 BVDV field isolates. Principal component analysis (PCA) was performed using VN titers, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among various isolates suggesting antigenic relatedness. As expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. Notably, a number of clusters representing antigenically related BVDV-1 subgroups contain isolates of different subgenotypes. The multivariate analysis may be a method to better characterize antigenic relationships among BVDV isolates that belong to the same BVDV species and do not have distinct antigenic differences. This might be an invaluable tool to ameliorate the composition of current vaccines, which might well be important for the success of any BVDV control program that includes vaccination in its scheme.

Global Distribution and Genetic Heterogeneity of Border Disease Virus.

Border disease virus (BDV) belongs to the genus Pestivirus of the family Flaviviridae. Interspecies transmission of BDV between sheep, cattle, and pigs occurs regularly, sometimes making diagnosis a challenge. BDV can yield substantial economic losses, including prenatal and postnatal infections in lambs, which are the primary source of infection and maintenance of the virus in the population. Since BDV is antigenically and genetically related to bovine viral diarrhea virus (BVDV), it might pose a significant risk to cattle, influencing BVDV eradication campaigns. Similarly, the presence of BDV in swine herds due to pestivirus spillover between small ruminants and pigs might cause uncertainty in classical swine fever virus (CSFV) diagnostics. Therefore, knowledge of BDV epidemiology in different geographical regions will help prevent its spread and optimize control measures. Previous epidemiological studies have shown that various BDV genotypes are predominant in different countries. This review provides an overview of the spread of BDV world-wide in different host species.

Evaluation of Safety and Efficacy of an Inactivated Marker Vaccine against Bovine alphaherpesvirus 1 (BoHV-1) in Water Buffalo (Bubalus bubalis).

Recent studies have explored the seropositivity of Bovine alphaherpesvirus 1 (BoHV-1) in water buffaloes, suggesting the urgency for developing strategies to eradicate the virus involving both cattle and water buffaloes. However, in Europe, the glycoprotein E (gE) deleted marker vaccines against BoHV-1 are commercially available only for the cattle industry. This study, for the first time, evaluated the safety and efficacy of a commercial inactivated gE-deleted marker vaccine in water buffalo. Five animals devoid of BoHV-1-neutralizing antibodies were vaccinated via intramuscular route. Five additional animals served as an unvaccinated control group. Sixty days after the first immunization, all animals were experimentally infected with a virulent BoHV-1via intranasal route. A detectable BoHV-1-humoral immune response was observed in the vaccinated group on post-vaccination day 30, whereas the antibodies appeared on post-challenge day 10 in the control group. Moreover, the vaccinated animals neither show viral shedding nor clinical signs compared to the control upon challenge. However, post-challenge, the BoHV-1-specific humoral and cell-mediated immune responses were significantly more increased in vaccinated animals than the control animals. Overall, the present study provides evidence of both the safety and efficacy of an inactivated gE-deleted marker vaccine against BoHV-1 in water buffaloes.

Hepatitis E virus (HEV) genotype 3 diversity: Identification of a novel HEV subtype in wild boar in Central Italy.

In the last decade in Europe, the number of autochthonous cases of hepatitis E has significantly increased. Most of the cases arise from foodborne infections caused by the zoonotic hepatitis E virus (HEV) genotypes HEV-3 and HEV-4. Several human cases have been linked to consumption of raw or undercooked animal products of both pork (liver sausages) and wild boar meat. In this study, the occurrence of HEV infection was investigated in 611 livers and 88 paired lungs from wild boars collected during the hunting seasons of 2016-2020 in the Umbria-Marche Apennines (Central Italy). Using real-time reverse transcription polymerase chain reaction, 15 liver samples (2.45%) and one lung sample were found to be positive for HEV RNA. The phylogenetic tree built on the partial ORF2 gene revealed that the detected HEV strains belonged to HEV-3f (n = 5), HEV-3e (n = 1) and HEV-3c (n = 1) subtypes. Interestingly, 8 strains were genetically placed in a different cluster, further away from all other subtypes. To corroborate this finding, four complete genomes were obtained by next generation sequencing. The full genome of the HEV strains clustered together with another wild boar strain previously detected in Southern Italy in 2015 but the strains were divergent from all the HEV-3 strains classified in any subtype defined so far. Thus, these strains represent a novel subtype that might have originated in Italy, which we have tentatively named HEV-3n.

Serological Cross-Reactivity Between Bovine alphaherpesvirus 2 and Bovine alphaherpesvirus 1 in a gB-ELISA: A Case Report in Italy.

In this study, we demonstrated for the first time in Italy, the serological cross-reactivity between Bovine alphaherpesvirus 2 (BoHV-2) and Bovine alphaherpesvirus 1 (BoHV-1). Five months after arriving at a performance test station in Central Italy, a 6-month-old calf, which was part of a group of 57 animals, tested positive for BoHV-1 in a commercial gB-ELISA test. It was immediately transferred to the quarantine unit and subjected to clinical observation and serological and virological investigations. During this period, the calf showed no clinical signs. The results from laboratory investigations demonstrated the presence of antibodies via competitive glycoprotein B (gB) ELISAs, indirect BoHV-1 ELISAs, and indirect BoHV-2 ELISAs. Furthermore, the plaque reduction assay provided evidence for the presence of antibodies only for BoHV-2, whereas the virus neutralization test showed negative results for both BoHV-1 and BoHV-5. These findings strongly suggest the occurrence of a serological cross-reactivity between BoHV-2 and BoHV-1. Interference of BoHV-2 antibodies in serological BoHV-1 diagnostics should be considered during routine IBR tests, especially when animals are kept in a performance test station.

Near-complete nucleotide sequence of a border disease virus genotype BDV-7 isolate from Italy.

To gain further insight into the genomic features of border disease virus (BDV), we determined the nearly complete genome sequence of isolate TO/121/04 from an aborted ovine fetus. Its genome contains a single open reading frame (ORF), which comprises 11,681 nucleotides encoding a polyprotein of 3893 amino acids. Phylogenetic analysis of the near full-length genome sequence showed that the BDV isolate differed significantly from all ovine pestiviruses identified so far, thus re-affirming the presence in Italy of this novel genetic group, termed BDV-7.

African Swine Fever Circulation among Free-Ranging Pigs in Sardinia: Data from the Eradication Program.

African swine fever virus (ASFV), the cause of a devastating disease affecting domestic and wild pigs, has been present in Sardinia since 1978. In the framework of the regional ASF eradication plan, 4484 illegal pigs were culled between December 2017 and February 2020. The highest disease prevalence was observed in the municipality with the highest free-ranging pig density, and culling actions drastically reduced ASFV circulation among these animals. ASFV-antibody were detected in 36.7% of tested animals, which were apparently healthy, thus, the circulation of low-virulence ASFV isolates was hypothesized. ASFV genome was detected in 53 out of 2726 tested animals, and virus isolation was achieved in two distinct culling actions. Two ASFV haemadsorbing strains were isolated from antibody-positive apparently healthy pigs: 55234/18 and 103917/18. Typing analysis revealed that both isolates belong to p72 genotype I, B602L subgroup X; phylogenetic analysis based on whole genome sequencing data showed that they were closely related to Sardinian ASFV strains collected since 2010, especially 22653/Ca/2014. Our data suggested the absence of immune-escaped ASFV variants circulating among free-ranging pigs, indicating that other elements contributed to virus circulation among these animals. Understanding factors behind disease persistence in endemic settings might contribute to developing effective countermeasures against this disease.

Cross-Reactivity Antibody Response after Vaccination with Modified Live and Killed Bovine Viral Diarrhoea Virus (BVD) Vaccines.

Pestivirus A or bovine viral diarrhoea virus (BVDV) type 1 is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. The objective of the present study was to verify whether animals immunised with four commercial vaccines also developed a protective humoral immunity against other viral subgenotypes than those contained in each vaccine. Four groups of 25 bovines each were formed and vaccinated according to the manufacturer's instructions of the commercial vaccines. On sera collected 28 days after the last vaccination, virus neutralisation tests (VNT) were performed using homologous and heterologous viruses and enzyme-linked immunosorbent assay (ELISA) methods. Finally, the VNT results were comparatively evaluated through a statistical analysis. Serological results highlighted that, although with a different degree of efficiency, the four vaccines resulted in not developing a solid antibody-mediated cross-immunity against all the strains used.

Evaluation of Three Different Vaccination Protocols against EHV1/EHV4 Infection in Mares: Double Blind, Randomized Clinical Trial.

EHV1 and EHV4 are the most important herpesviruses in horses. Repeated cases of abortion in mares regularly vaccinated, prompted us to investigate the immune response after vaccination with the same inactivated vaccine, but with three different protocols. Eighteen mares were chosen and randomly divided in three study groups (G1-G2-G3) and a control group (Ctrl). For serologic and PCR investigations nasal swabs, sera and blood were collected. The protocol used in G3 (4 doses) increased the titer recorded by ELISA and seroneutralization (SN). Poor agreement and no correlation were observed in titer values between ELISA and SN and between SN and PCR. A very weak positive correlation between ELISA and PCR was obtained. Seven out of 18 nasal swabs were positive by PCR; none showed viremia and no abortion occurred, regardless of vaccination status and despite active circulation of EHV-1 in the farm at the time of the study. The study was conducted in field conditions, in a susceptible population with a known history of infection and abortion, and among the three protocols, the one proposed in the G1 was the least efficient while the one proposed for the G3, seems to have induced a higher antibody titer in both SN and ELISA.

Highly pathogenic Bovine Respiratory Syncytial virus variant in a dairy herd in Italy.

Bovine respiratory syncytial virus (BRSV) is an economically significant pathogen in cattle production worldwide. Usually, it is detected in outbreaks of respiratory disease, most often during the winter period. During the middle of October 2018, a serious outbreak of respiratory disease occurred in a cattle farm comprising about 300 heads located in Central Italy. The herd was affected by a severe flu-like syndrome unresponsive to any antibiotic treatment. Within 3 weeks, 39 adult animals died, and 12 abortions occurred. Direct and indirect laboratory tests were performed to detect the main pathogens causing the respiratory disease of the affected cattle. The results of laboratory investigations provided evidence of an acute and severe BRSV syndrome characterized by unusual mortality. In order to investigate the molecular underpinnings of this syndrome, phylogenetic analysis of the BRSV strain detected from the outbreak was carried out. The sequence analysis showed that the strain was genetically divergent from BRSV strains previously identified in Italy, as it showed high sequence similarity of more than 97% with strains isolated during a major BRSV epizootic that occurred in Sweden, Norway and Denmark during 2010-2011. The infection of the herd in Italy with this BRSV strain was likely due to the introduction of animals imported into Italy from abroad.

The evolution of African swine fever virus in Sardinia (1978-2014) as revealed by whole-genome sequencing and comparative analysis.

African swine fever (ASF) is a highly contagious and lethal viral disease of pigs and wild boars, which is enzootic in many African countries and on the Italian island of Sardinia, where it has been present since 1978. Previous genetic analyses of Sardinian ASF virus (ASFV) isolates have revealed that they all belong to p72 genotype I, with only minor sequence variations. However, these studies examined only a few selected genes. To distinguish between these closely related isolates and better investigate ASFV evolution in Sardinia, we sequenced the complete genomes of 12 Sardinian ASFV isolates collected between 1978 and 2012, and compared them with 47/Ss/2008 and 26544/OG10. Most of the observed changes occurred in a time-dependent manner; however, their biological significance remains unclear. As a whole, our results demonstrate the remarkable genetic stability of these strains, supporting a single-source introduction of the virus.

Phylogenetic analysis of small ruminant lentiviruses in Germany and Iran suggests their expansion with domestic sheep.

Small ruminant lentiviruses (SRLVs) are found in sheep in Germany and Iran. SRLVs have been classified into four genotypes: A-C and E. Genotype A has been subdivided into 20 subtypes. Previous studies suggested that, first, the ancestors of genotype A are those SRLVs found in Turkey, second, the evolution of SRLVs is related to the domestication process, and, third, SRLV infection was first observed in sheep in Iceland and the source of that infection was a flock imported from Germany. This study generated, for the first time, partial SRLV sequence data from German and Iranian sheep, enhancing our knowledge of the genetic and evolutionary relationships of SRLVs, and their associations with the domestication process. Based on 54 SRLV sequences from German and Iranian sheep, our results reveal: (1) SRLV subtypes A4, A5, A11, A16 and A21 (new) are found in German sheep and A22 (new) in Iranian sheep. (2) Genotype A has potentially an additional ancestor (A22), found in Iran, Lebanon and Jordan. (3) Subtype A22 is likely an old version of SRLVs. (4) The transmission routes of some SRLVs are compatible with domestication pathways. (5) This study found no evidence of Icelandic subtype A1 in German sheep.

Evaluation of Passive Immunity Induced by Immunisation Using Two Inactivated gE-deleted Marker Vaccines against Infectious Bovine Rhinotracheitis (IBR) in Calves.

Different types of vaccines against Infectious Bovine Rhinotracheitis (IBR) are commercially available. Among these, inactivated glycoprotein E (gE)-deleted marker vaccines are commonly used, but their ability to induce passive immunity is poorly known. Here, we evaluated the passive immunity transferred from dams immunised with commercial inactivated gE-deleted marker vaccines to calves. We vaccinated 12 pregnant cattle devoid of neutralising antibodies against Bovine alphaherpesvirus 1 (BoHV-1) and divided them into two groups with 6 animals each. Both groups were injected with a different inactivated gE-deleted marker vaccine administrated via intranasal or intramuscular routes. An additional 6 pregnant cattle served as the unvaccinated control group. After calving, the number of animals in each group was increased by the newborn calves. In the dams, the humoral immune response was evaluated before calving and, subsequently, at different times until post-calving day 180 (PCD180). In addition, the antibodies in colostrum, milk, and in serum samples from newborn calves were evaluated at different times until PCD180. The results indicated that inactivated glycoprotein E (gE)-deleted marker vaccines are safe and produce a good humoral immune response in pregnant cattle until calving and PCD180. Moreover, results showed that, in calf serum, passive immunity persists until PCD180.

Serological relationship between a novel ovine pestivirus and classical swine fever virus.

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.

Survival of African swine fever virus (ASFV) in various traditional Italian dry-cured meat products.

A number of animal diseases can be transmitted to pigs via pork meat and pork products imported from infected areas. Therefore, feeding of swill to pigs is regulated or prohibited in many swine-rearing countries. African swine fever is one of the major porcine diseases recognized as significant in this transmission pathway. Assessment of disease risks associated with pork products requires knowledge about the viral load in the original material and for how long infectious virus can be recovered from the resulting product. In this work, we present the effect of the dry curing process on the inactivation of African swine fever virus (ASFV) in three different Italian dry-cured meat products prepared from experimentally infected pigs slaughtered at the peak of viremia. The meat products were processed using commercial methods and industrial procedures currently being followed in Italy. Samples collected at predetermined intervals during processing were analyzed for virus survival by virus isolation and animal inoculation. ASFV was detected by in vivo experiments for up to 18, 60, and 83 days of curing in Italian salami, pork belly, and loin, respectively. These data provide valuable information for the pork processing industry when planning the export of these products.

Complete genome sequences of a cytophatic/noncytophatic pair of bovine viral diarrhea virus subtype 1a viruses.

The complete genome sequences of both biotypes of a pair of bovine viral diarrhea viruses isolated from a bovid affected by mucosal disease were determined by next generation sequencing. The cytopathic virus possessed a 423-base insertion derived from bovine poly ubiquitin in the NS2/3 coding region and one nucleotide change. Both biotypes showed an additional glycosylation site in their N-terminus.

Chimeric DNA/LNA-based biosensor for the rapid detection of African swine fever virus.

African swine fever (ASF) virus is a DNA virus responsible for a severe haemorrhagic fever in pigs, which (still in the absence of vaccination strategies) results in high mortality rates. Herein, we present a biosensor-based method for the detection of ASF viral DNA in the blood of pigs. The biosensor exploits a single-strand DNA probe with locked nucleic acid nucleotides (LNA) substitutions as the complementary recognition element for the conserved region of vp72 gene of ASF virus. The biosensor was calibrated using qPCR-quantified ASF viral DNA extracted from the blood of pigs experimentally infected with the virulent Italian isolate 49/08, genotype I. Globally, the proposed biosensor showed good sensitivity and specificity, with the limits of detection (LOD) and quantification (LOQ) being 178 and 245 copies/µL of genomic ASF viral DNA, respectively. The reversible nature of the interaction between the DNA/LNA probe and the target DNA sequence granted multiple rapid analyses, with up to 40 analyses per single surface possible, and a single test requiring approximately 5 min. When applied to non-amplified DNA extracts from the blood of field-infected pigs, the assay discriminated between ASFV-infected and ASFV non-infected animals, and allowed the rapid quantification of ASF viral DNA, with values falling in the range 373-1058 copies/µL of genomic ASFV DNA. In this range, excellent correlation was observed between the results of this biosensor and OIE-approved qPCR. This method represents a promising screening assay for preliminary ASF diagnosis, having the major advantages in the relative rapidity, ease-of-use, the reusability of the sensing surface, and low cost per single test.

Highlighting priority areas for bovine viral diarrhea control in Italy: A phylogeographic approach.

The prevalence and genetic diversity of bovine viral diarrhea virus (BVDV) in a geographic area are largely influenced by live animal trade and management practices. Despite control and eradication programs currently underway in several European countries, the risk of BVDV spread within and among countries is still present. BVDV-1 is the predominant type circulating in European cattle population. In this study, a phylogeographic analysis was applied to the BVDV-1 highest prevalent subtypes in Italy to reconstruct the origin and spatial-temporal distribution and to trace main viral flows between different locations to highlight priority areas for BVDV control. A comprehensive dataset of BVDV-1b (n = 173) and 1e (n = 172) 5' UTR sequences was analysed, including both novel and published sequences from Italy and from European countries bordering and/or with commercial cattle flows with Italy. A common phylogeographic pattern was observed for BVDV-1b and 1e subtypes: interspersion from multiple Italian areas and European countries was widespread until the end of the last century, whereas significant local clusters were observed starting from 2000. These findings support a continuous viral flow among different areas over long time scales with no evidence of significant geographical structure, while local transmission networks are limited to more recent years. Northern Italy has been confirmed as the area of origin of the main clades of both BVDV subtypes at national level, acting both as a crucial area for introduction and a maintenance source for other areas. Piedmont, Central and Southern Italian regions contributed to limited geographical distribution and local BVDV-1b and 1e persistence. On the whole, priority control measures for BVDV-1b and 1e in Italy should be focused on: i) implementation of BVDV systematic control in all Northern Italian regions to break the viral flow from larger to smaller animal populations; and ii) breaking the dynamics of infections in regions with self-maintenance of BVDV by voluntary control programs.