Epizootic Hemorrhagic Disease Virus Serotype 8, Italy, 2022.

We describe the detection of epizootic hemorrhagic disease virus (EHDV) serotype 8 in cattle farms in Sardinia and Sicily in October-November 2022. The virus has a direct origin in North Africa; its genome is identical (>99.9% nucleotide sequence identity) to EHDV serotype 8 strains detected in Tunisia in 2021.

The panzootic potential of SARS-CoV-2.

Each year, SARS-CoV-2 is infecting an increasingly unprecedented number of species. In the present article, we combine mammalian phylogeny with the genetic characteristics of isolates found in mammals to elaborate on the host-range potential of SARS-CoV-2. Infections in nonhuman mammals mirror those of contemporary viral strains circulating in humans, although, in certain species, extensive viral circulation has led to unique genetic signatures. As in other recent studies, we found that the conservation of the ACE2 receptor cannot be considered the sole major determinant of susceptibility. However, we are able to identify major clades and families as candidates for increased surveillance. On the basis of our findings, we argue that the use of the term panzootic could be a more appropriate term than pandemic to describe the ongoing scenario. This term better captures the magnitude of the SARS-CoV-2 host range and would hopefully inspire inclusive policy actions, including systematic screenings, that could better support the management of this worldwide event.

Feline Panleukopenia Virus in Dogs from Italy and Egypt.

Canine parvovirus and feline panleukopenia virus (FPV) are variants of Carnivore protoparvovirus 1. We identified and characterized FPV in dogs from Italy and Egypt using genomic sequencing and phylogenetic analyses. Cost-effective sequencing strategies should be used to monitor interspecies spread, evolution dynamics, and potential host jumping of FPV.

SARS-CoV-2 surveillance in Italy through phylogenomic inferences based on Hamming distances derived from pan-SNPs, -MNPs and -InDels.

BACKGROUND: Faced with the ongoing global pandemic of coronavirus disease, the 'National Reference Centre for Whole Genome Sequencing of microbial pathogens: database and bioinformatic analysis' (GENPAT) formally established at the 'Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise' (IZSAM) in Teramo (Italy) is in charge of the SARS-CoV-2 surveillance at the genomic scale. In a context of SARS-CoV-2 surveillance requiring correct and fast assessment of epidemiological clusters from substantial amount of samples, the present study proposes an analytical workflow for identifying accurately the PANGO lineages of SARS-CoV-2 samples and building of discriminant minimum spanning trees (MST) bypassing the usual time consuming phylogenomic inferences based on multiple sequence alignment (MSA) and substitution model. RESULTS: GENPAT constituted two collections of SARS-CoV-2 samples. The first collection consisted of SARS-CoV-2 positive swabs collected by IZSAM from the Abruzzo region (Italy), then sequenced by next generation sequencing (NGS) and analyzed in GENPAT (n = 1592), while the second collection included samples from several Italian provinces and retrieved from the reference Global Initiative on Sharing All Influenza Data (GISAID) (n = 17,201). The main results of the present work showed that (i) GENPAT and GISAID detected the same PANGO lineages, (ii) the PANGO lineages B.1.177 (i.e. historical in Italy) and B.1.1.7 (i.e. 'UK variant') are major concerns today in several Italian provinces, and the new MST-based method (iii) clusters most of the PANGO lineages together, (iv) with a higher dicriminatory power than PANGO lineages, (v) and faster that the usual phylogenomic methods based on MSA and substitution model. CONCLUSIONS: The genome sequencing efforts of Italian provinces, combined with a structured national system of NGS data management, provided support for surveillance SARS-CoV-2 in Italy. We propose to build phylogenomic trees of SARS-CoV-2 variants through an accurate, discriminant and fast MST-based method avoiding the typical time consuming steps related to MSA and substitution model-based phylogenomic inference.

Possible Human-to-Dog Transmission of SARS-CoV-2, Italy, 2020.

We detected severe acute respiratory syndrome coronavirus 2 in an otherwise healthy poodle living with 4 family members who had coronavirus disease. We observed antibodies in serum samples taken from the dog, indicating seroconversion. Full-length genome sequencing showed that the canine and human viruses were identical, suggesting human-to-animal transmission.

Insights into SARS-CoV-2, the Coronavirus Underlying COVID-19: Recent Genomic Data and the Development of Reverse Genetics Systems.

The emergence and rapid worldwide spread of a novel pandemic of acute respiratory disease - eventually named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) - across the human population has raised great concerns. It prompted a mobilization around the globe to study the underlying pathogen, a close relative of severe acute respiratory syndrome coronavirus (SARS-CoV) called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Numerous genome sequences of SARS-CoV-2 are now available and in-depth analyses are advancing. These will allow detailed characterization of sequence and protein functions, including comparative studies. Care should be taken when inferring function from sequence information alone, and reverse genetics systems can be used to unequivocally identify key features. For example, the molecular markers of virulence, host range and transmissibility of SARS-CoV-2 can be compared to those of related viruses in order to shed light on the biology of this emerging pathogen. Here, we summarize some recent insights from genomic studies and strategies for reverse genetics systems to generate recombinant viruses, which will be useful to investigate viral genome properties and evolution.

Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

Polymorphisms in the haemagglutinin gene influenced the viral shedding of pandemic 2009 influenza virus in swine.

Interactions between the viral surface glycoprotein haemagglutinin (HA) and the corresponding receptors on host cells is one important aspect of influenza virus infection. Mutations in HA have been described to affect pathogenicity, antigenicity and the transmission of influenza viruses. Here, we detected polymorphisms present in HA genes of two pandemic 2009 H1N1 (H1N1pdm09) isolates, A/California/04/2009 (Ca/09) and A/Mexico/4108/2009 (Mx/09), that resulted in amino acid changes at positions 186 (S to P) and 194 (L to I) of the mature HA1 protein. Although not reported in the published H1N1pdm09 consensus sequence, the P186 genotype was more readily detected in primary infected and contact-naïve pigs when inoculated with a heterogeneous mixed stock of Ca/09. Using reverse genetics, we engineered Ca/09 and Mx/09 genomes by introducing Ca/09 HA with two naturally occurring variants expressing S186/I194 (HA-S/I) and P186/L194 (HA-P/L), respectively. The Ca/09 HA with the combination of P186/L194 with either the Ca/09 or Mx/09 backbone resulted in higher and prolonged viral shedding in naïve pigs. This efficiency appeared to be more likely through an advantage in cell surface attachment rather than replication efficiency. Although these mutations occurred within the receptor-binding pocket and the Sb antigenic site, they did not affect serological cross-reactivity. Relative increases of P186 in publicly available sequences from swine H1N1pdm09 viruses supported the experimental data, indicating this amino acid substitution conferred an advantage in swine.

Influenza A virus PB1-F2 protein expression is regulated in a strain-specific manner by sequences located downstream of the PB1-F2 initiation codon.

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 molecular function during infection has been collected primarily from human and avian viral isolates. As the 2009 H1N1 (H1N1pdm09) strain highlighted, some swine-derived influenza viruses have the capacity to infect human hosts and emerge as a pandemic. Understanding the impact that virulence factors from swine isolates have on both human and swine health could aid in early identification of viruses with pandemic potential. Studies examining PB1-F2 from swine isolates have focused primarily on H1N1pdm09, which does not encode PB1-F2 but was engineered to carry a full-length PB1-F2 ORF to assess the impact on viral replication and pathogenicity. However, experimental evidence of PB1-F2 protein expression from swine lineage viruses has not been demonstrated. Here, we reveal that during infection, PB1-F2 expression levels are substantially different in swine and human influenza viruses. We provide evidence that PB1-F2 expression is regulated at the translational level, with very low levels of PB1-F2 expression from swine lineage viruses relative to a human isolate PB1-F2. Translational regulation of PB1-F2 expression was partially mapped to two independent regions within the PB1 mRNA, located downstream of the PB1-F2 start site. Our data suggest that carrying a full-length PB1-F2 ORF may not be predictive of PB1-F2 expression in infected cells for all influenza A viruses.

Contemporary epidemiology of North American lineage triple reassortant influenza A viruses in pigs.

The 2009 pandemic H1N1 infection in humans has been one of the greatest concerns for public health in recent years. However, influenza in pigs is a zoonotic viral disease well-known to virologists for almost one century with the classical H1N1 subtype the only responsible agent for swine influenza in the United States for many decades. Swine influenza was first recognized clinically in pigs in the Midwestern U.S. in 1918 and since that time it has remained important to the swine industry throughout the world. Since 1988, however, the epidemiology of swine influenza changed dramatically. A number of emerging subtypes and genotypes have become established in the U.S. swine population. The ability of multiple influenza virus lineages to infect pigs is associated with the emergence of reassortant viruses with new genomic arrangements, and the introduction of the 2009 pandemic H1N1 from humans to swine represents a well-known example. The recent epidemiological data regarding the current state of influenza A virus subtypes circulating in the Canadian and American swine population is discussed in this review.

Epidemiological Analyses of the First Incursion of the Epizootic Hemorrhagic Disease Virus Serotype 8 in Tunisia, 2021-2022.

Epizootic hemorrhagic disease (EHD) is a non-contagious arthropod-transmitted viral disease and a World Organization for Animal Health (WOAH)-listed disease of domestic and wild ruminants since 2008. EHDV is transmitted among susceptible animals by a few species of midges of genus Culicoides. During the fall of 2021, a large outbreak caused by the epizootic hemorrhagic disease virus (EHDV), identified as serotype 8, was reported in Tunisian dairy and beef farms with Bluetongue virus (BTV)-like clinical signs. The disease was detected later in the south of Italy, in Spain, in Portugal and, more recently, in France, where it caused severe infections in cattle. This was the first evidence of EHDV-8 circulation outside Australia since 1982. In this study, we analyzed the epidemiological situation of the 2021-2022 EHDV outbreaks reported in Tunisia, providing a detailed description of the spatiotemporal evolution of the disease. We attempted to identify the eco-climatic factors associated with infected areas using generalized linear models (GLMs). Our results demonstrated that environmental factors mostly associated with the presence of C. imicola, such as digital elevation model (DEM), slope, normalized difference vegetation index (NDVI), and night-time land surface temperature (NLST)) were by far the most explanatory variables for EHD repartition cases in Tunisia that may have consequences in neighboring countries, both in Africa and Europe through the spread of infected vectors. The risk maps elaborated could be useful for disease control and prevention strategies.

Validation of a focus reduction neutralization test (FRNT) to rapidly titrate human SARS-CoV-2 neutralizing antibodies by using the CTL Immunospot S6 universal analyzer.

In this paper we describe the validation of a focus reduction neutralization test (FRNT) to quantitate human SARS-CoV-2 neutralizing antibodies by using the CTL Immunospot S6 Universal Analyzer. We employed a previously published protocol and compared its performances to a well-established and traditional serum-neutralization assay (SN). To assess diagnostic sensitivity, a total number of 201 human sera positive by SN for SARS-CoV-2 NAbs were processed: 196/201 tested positive by FRNT50 (97.51 %). A diagnostic specificity of 100 % was obtained by evaluating 206 negative serum samples. Repeatability of the test was evaluated by determining the intra and inter-assay coefficient of variation (CV). A standard deviation of 0.83 and a CV of 13 % were evidenced demonstrating an acceptable reproducibility of the assay. Moreover, a Cohen's Kappa of 0.975 was obtained proving an extremely high level of agreement between the FRNT protocol and the SN. Despite an acceptable correlation between methods (p < 0.05), FRNT demonstrated a statistically significant increase in NAbs titres compared to SN as well as higher data variability and asymmetry. These discrepancies could be attributed to FRNT sensitivity or most probably to the subjective interpretation of SN, although this aspect needs to be further investigated with a more representative number of samples. Basing on our results, it is reasonable to replace the SN with the FRNT assay as, with this, fast processing time (less than 2 days) and operator bias-free results registrations are guaranteed.

Low pathogenic human coronaviruses during the first waves of COVID-19 in Italy.

Low-pathogenic human coronaviruses (HCoVs) infect the upper respiratory tract and cause mild, cold-like respiratory illness. Although several studies have shown evidence of the global distribution of HCoVs, information about their distribution in Italy are often focused only on hospitalized children and elderly with respiratory symptoms. In this study, a total of 916 swab samples collected during the first two SARS-CoV-2 pandemic waves in Abruzzo region (central Italy) was selected for molecular screening of low pathogenic HCoVs by real-time RT-PCR. We identified low-pathogenic HCoV in nine samples. Positive samples underwent whole genome sequencing for genome characterization; indeed, we also report the whole genome sequence of a HCoV-229E strain.

Detection of vesivirus in minks (Neovison vison), Italy 2021.

Vesiviruses are important animal pathogens with a broad host range, and they have also been involved in accidental contamination of cells used for the production of drugs for rare and life-threatening human diseases. A vesivirus (family Caliciviridae) was detected in minks (Neovison vison) with respiratory and neurological signs, during syndromic surveillance for SARS-CoV-2 conducted in Italy. The complete genome (8,397 nucleotides in length) of the vesivirus strain ITA/2021/mink/TE (OR130287) was obtained by combining NGS approach with 5' and 3' RACE protocols. The virus was seemingly more related (95.9-97.2% nt identity in the partial RNA-dependent RNA polymerase) to American vesivirus isolates 9/1980/US, 12/1980/US, and 20/1980/US dating back to the early 1980s than to recent mink strains. These results highlight the importance of gathering information on the virome of animals.

Validation of a real-time PCR assay for the detection of African swine fever virus in fresh pork meat juice.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a disease with detrimental effects on the health, welfare, and production of domestic and wild pigs. The ASF laboratory confirmation is based on the analysis of blood, serum and organ samples. However, testing these samples could not be always convenient, economically feasible or possible. This study describes the validation process of a PCR-based assay targeting a portion of p72 gene, used for the molecular detection of ASFV, from meat juice samples obtained from pigs succumbed to ASFV. More specifically, we investigated the capability of a real-time PCR assay to detect ASFV DNA in meat juices obtained from the diaphragmatic muscle along with the correspondent spleens of 55 ASFV-positive pigs and wild boars sampled from confirmed outbreaks in Romania and from 73 ASFV-negative and regularly slaughtered healthy pigs collected in the Abruzzo region (Italy). The test was able to detect viral DNA in both types of samples, with lower Ct values in spleens (mean=21.11, median=20.61) than meat juices (mean=23.08, median=22.40). However, distributions of Ct values were strongly correlated each other (R2= 0.83, P<0.001). Considering the distribution of the observed Ct values in the 55 positive meat juice samples, a 1:10 dilution would be able to detect 90 % of positive samples, whereas a 1:100 dilution would reduce the detectability to 78 % of more contaminated samples. As meat juice could be obtained easily from muscles and considering the potential use of this test on pooled samples, it could represent a tool to aid the investigation of ASFV spread.

Unveiling the molecular epidemiology of canine distemper virus in Namibia: An expected pathogen showing an unexpected origin.

Objective: Canine distemper virus (CDV) is a highly infectious virus that represents a threat for domestic dogs and several wild species. Despite recognized in several African countries, current knowledge of its molecular epidemiology is scarce and poorly updated. Design: Twenty-two hemagglutinin sequences, obtained from symptomatic Namibian dogs from 2020 to 2023, were analysed through phylogenetic and phylodynamic analysis to characterize the local CDV epidemiology and contextualize it in the international scenario. Results: Two unrelated clades were identified, including strains sampled in different Namibian towns, in the absence of a strong geographical clustering. The ancestors of the two clades were estimated to have originated from South America, likely Brazil, and South Africa, approximately in 2000 and 2006, respectively. While the introduction from South Africa was predictable, the introduction from Brazil was unexpected. The mediation of other African countries, particularly Angola, appears to be the most likely importation pathway. Conclusions: The occurrence of multiple introduction events, likely originating from cross-border illegal animal trade between African countries, and the absence of any geographical clustering within Namibian regions, suggest a need for further investigation into its spreading pattern, as well as improved biosecurity measures to limit foreign viral introduction into the country.

Detection, Characterization and Sequencing of BTV Serotypes Circulating in Cuba in 2022.

In Cuba, despite a high sero-prevalence of bluetongue virus (BTV), circulating serotypes remain unknown. The aim of this study was to identify circulating BTV serotypes in farms throughout the western region of Cuba. Blood samples were collected from 200 young cattle and sheep between May and July 2022 for virological analyses (PCR, viral isolation and virus neutralization) and genome sequencing. The results confirmed viral circulation, with viro-prevalence of 25% for BTV. The virus was isolated from 18 blood samples and twelve BTV serotypes were identified by sequencing RT-PCR products targeting the segment 2 of the BTV genome (BTV-1, 2, 3, 6, 10, 12, 13, 17, 18, 19, 22 and 24). Finally, the full genome sequences of 17 Cuban BTV isolates were recovered using a Sequence Independent Single Primer Amplification (SISPA) approach combined to MinION Oxford Nanopore sequencing technology. All together, these results highlight the co-circulation of a wide diversity of BTV serotypes in a quite restricted area and emphasize the need for entomological and livestock surveillance, particularly in light of recent changes in the global distribution and nature of BTV infections.

Efficacy of an inactivated EHDV-8 vaccine in preventing viraemia and clinical signs in experimentally infected cattle.

Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is a vector-borne viral disease transmitted through Culicoides biting midges. EHDV comprises seven serotypes (1, 2, and 4-8), with EHDV-8 having recently emerged and spread in Europe over the last two years. Such event has raised concerns about the significant threat posed by EHDV-8 to livestock industry. In this study, an inactivated vaccine against EHDV-8 (vEHDV8-IZSAM) was developed. Safety and efficacy of the vaccine were evaluated in calves through clinical, serological, and virological monitoring following experimental challenge. The vaccine was proven safe, with only transient fever and localized reactions observed in a few animals, consistent with adjuvanted vaccine side effects. vEHDV8-IZSAM elicited a robust humoral response, as evidenced by the presence of neutralizing antibodies. After challenge with a virulent isolate, viraemia and clinical signs were evidenced in control animals but in none of the vaccinated animals. This study highlights the potential of vEHDV8-IZSAM as a safe and highly effective vaccine against EHDV-8 in cattle. It offers protection from clinical disease and effectively prevents viraemia. With the recent spread of EHDV-8 in European livestock, the use of an inactivated vaccine could be key in protecting animals from clinical disease and thus to mitigate the economic impact of the disease. Further investigations are warranted to assess the duration of the induced immunity and the applicability of this vaccine in real-world settings. Accordingly, joint efforts between public veterinary institutions and pharmaceutical companies are recommended to scale up vaccine production.

Identification and characterization of two atypical strains of bluetongue virus in sheep, Tunisia.

Bluetongue virus (BTV) is the causative agent of the bluetongue disease (BT), an infectious disease of domestic and wild ruminants that is primarily transmitted by Culicoides biting midges. In recent years, several novel BTV serotypes (often referred to as "atypical" BTVs) have been documented. These strains are usually asymptomatic in animals and seem to be unable to replicate efficiently in the arthropod vector. Here we report the detection of two putative atypical BTV strains in the Governorate of Gafsa, in the southwest region of Tunisia. Specifically, we recognised the recurrence of an atypical BTV strain (BTV-Y TUN2022) and a novel BTV-W TUN2022.

A cell-adapted SARS-CoV-2 mutant, showing a deletion in the spike protein spanning the furin cleavage site, has reduced virulence at the lung level in K18-hACE2 mice.

Here we investigated the virulence properties of a unique cell-adapted SARS-CoV-2 mutant showing a ten-amino acid deletion encompassing the furin cleavage site of the spike protein (Δ680SPRAARSVAS689; Δ680-689-B.1) in comparison to its parental strain (wt-B.1) and two Delta variants (AY.122 and AY.21) of concern. After intranasal inoculation, transgenic K18-hACE2 mice were monitored for 14 days for weight change, lethality, and clinical score; oral swabs were daily collected and tested for the presence of N protein subgenomic RNA. At 3 and 7 dpi mice were also sacrificed and organs collected for molecular, histopathological, and immune response profile investigations. The Δ680-689-B.1-infected mice exhibited reduced shedding, lower virulence at the lung level, and milder pulmonary lesions. In the lung, infection with Δ680-689-B.1 was associated with a significant lower expression of some cytokines at 3 dpi (IL-4, IL-27, and IL-28) and 7 dpi (IL-4, IL-27, IL-28, IFN-γ and IL-1α).