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.

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).

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.

Evaluation of next generation sequencing approaches for SARS-CoV-2.

Within public health control strategies for SARS-CoV-2, whole genome sequencing (WGS) is essential for tracking viral spread and monitoring the emergence of variants which may impair the effectiveness of vaccines, diagnostic methods, and therapeutics. In this manuscript different strategies for SARS-CoV-2 WGS including metagenomic shotgun (SG), library enrichment by myBaits® Expert Virus-SARS-CoV-2 (Arbor Biosciences), nCoV-2019 sequencing protocol, ampliseq approach by Swift Amplicon® SARS-CoV-2 Panel kit (Swift Biosciences), and Illumina COVIDSeq Test (Illumina Inc.), were evaluated in order to identify the best approach in terms of results, labour, and costs. The analysis revealed that Illumina COVIDSeq Test (Illumina Inc.) is the best choice for a cost-effective, time-consuming production of consensus sequences.

Spatial and temporal dynamics of West Nile virus between Africa and Europe.

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses.

Development and validation of an RT-qPCR for detection and quantitation of emerging epizootic hemorrhagic disease virus serotype 8 RNA from field samples.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides-transmitted virus circulating in multiple serotypes. It has become a concern in the European Union as a novel strain of the serotype 8 (EHDV-8) of clear Northern African origin, has been recently discovered in symptomatic cattle in Italy (islands of Sardinia and Sicily), Spain, and Portugal. Current molecular typing methods targeting the S2 nucleotide sequences -coding for the outermost protein of the virion VP2- are not able to detect the novel emerging EHDV-8 strain as they enrolled the S2 sequence of the unique EHDV-8 reference strain isolated in Australia in 1982. Thus, in this study, we developed and validated a novel typing assay for the detection and quantitation of the novel EHDV-8 RNA from field samples, including blood of ruminants and insects. This molecular tool will certainly support EHDV-8 surveillance and control.

Characterization of Epizootic Hemorrhagic Disease Virus Serotype 8 in Naturally Infected Barbary Deer (Cervus elaphus barbarus) and Culicoides (Diptera: Ceratopogonidae) in Tunisia.

Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central-Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

Novel Amplicon-Based Sequencing Approach to West Nile Virus.

West Nile virus is a re-emerging arbovirus whose impact on public health is increasingly important as more and more epidemics and epizootics occur, particularly in America and Europe, with evidence of active circulation in Africa. Because birds constitute the main reservoirs, migratory movements allow the diffusion of various lineages in the world. It is therefore crucial to properly control the dispersion of these lineages, especially because some have a greater health impact on public health than others. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to West Nile virus. This study was carried out on different strains from lineage 1 and 2 from Senegal and Italy. The presented protocol/approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for West Nile genomic surveillance.

Complete Genome of African Swine Fever Virus Genotype II in Central Italy.

We report here the whole-genome sequence of the African swine fever virus (ASFV) genotype II, strain 20355/RM/2022_Italy, identified in a wild boar in the city of Rome (Lazio region, Italy) in April 2022.

Molecular Characterization of the First African Swine Fever Virus Genotype II Strains Identified from Mainland Italy, 2022.

African swine fever (ASF) is responsible for important socio-economic effects in the global pig industry, especially for countries with large-scale piggery sectors. In January 2022, the African swine fever virus (ASFV) genotype II was identified in a wild boar population in mainland Italy (Piedmont region). This study describes the molecular characterization, by Sanger and next-generation sequencing (NGS), of the first index case 632/AL/2022 and of another isolate (2802/AL/2022) reported in the same month, in close proximity to the first, following multiple ASF outbreaks. Phylogenetic analysis based on the B646L gene and NGS clustered the isolates 632/AL/2022 and 2802/AL/2022 within the wide and most homogeneous p72 genotype II that includes viruses from European and Asian countries. The consensus sequence obtained from the ASFV 2802/AL/2022 isolate was 190,598 nucleotides in length and had a mean GC content of 38.38%. At the whole-genome level, ASF isolate 2802/AL/2022 showed a close genetic correlation with the other representative ASFV genotype II strains isolated between April 2007 and January 2022 from wild and domestic pigs in Eastern/Central European (EU) and Asian countries. CVR subtyping clustered the two Italian ASFV strains within the major CVR variant circulating since the first virus introduction in Georgia in 2007. Intergenic region I73R-I329L subtyping placed the Italian ASFV isolates within the variant identical to the strains frequently identified among wild boars and domestic pigs. Presently, given the high sequence similarity, it is impossible to trace the precise geographic origin of the virus at a country level. Moreover, the full-length sequences available in the NCBI are not completely representative of all affected territories.

Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations.

West Nile Virus Lineage 2 Overwintering in Italy.

In January 2022, West Nile virus (WNV) lineage 2 (L2) was detected in an adult female goshawk rescued near Perugia in the region of Umbria (Italy). The animal showed neurological symptoms and died 15 days after its recovery in a wildlife rescue center. This was the second case of WNV infection recorded in birds in the Umbria region during the cold season, when mosquitoes, the main WNV vectors, are usually not active. According to the National Surveillance Plan, the Umbria region is included amongst the WNV low-risk areas. The necropsy evidenced generalized pallor of the mucous membranes, mild splenomegaly, and cerebral edema. WNV L2 was detected in the brain, heart, kidney, and spleen homogenate using specific RT-PCR. Subsequently, the extracted viral RNA was sequenced. A Bayesian phylogenetic analysis performed through a maximum-likelihood tree showed that the genome sequence clustered with the Italian strains within the European WNV strains among the central-southern European WNV L2 clade. These results, on the one hand, confirmed that the WNV L2 strains circulating in Italy are genetically stable and, on the other hand, evidenced a continuous WNV circulation in Italy throughout the year. In this report case, a bird-to-bird WNV transmission was suggested to support the virus overwintering. The potential transmission through the oral route in a predatory bird may explain the relatively rapid spread of WNV, as well as other flaviviruses characterized by similar transmission patterns. However, rodent-to-bird transmission or mosquito-to-bird transmission cannot be excluded, and further research is needed to better understand WNV transmission routes during the winter season in Italy.

Full Genome Characterization of Respiratory Syncytial Virus Causing a Fatal Infection in an Immunocompromised Patient in Tunisia.

Human orthopneumovirus (HRSV) is a virus belonging to the Pneumovirus genus that causes lower respiratory tract infections (LRTI) in infants worldwide. In Tunisia, thousands of infants hospitalized for LRTI are found to be positive for HRSV but no whole genome sequences of HRSV strains circulating in this country are available thus far. In this study, five nasal swab samples collected at different time points from a three-month-old female baby with severe immunodeficiency that was hospitalized for acute bronchiolitis were investigated by next generation sequencing. The Tunisian sequences from this study originated from samples collected in 2021, belong to the ON1 genotype of HRSV-A, and are clustered with European sequences from 2019 and not from 2020 or 2021. This is most likely related to local region-specific transmission of different HRSV-A variants due to the COVID-19 related travel restrictions. Overall, this is the first report describing the whole genome sequence of HRSV from Tunisia. However, more sequence data is needed to better understand the genetic diversity and transmission dynamic of HRSV.

Epidemiological and genomic findings of the first documented Italian outbreak of SARS-CoV-2 Alpha variant of concern.

From 24 December 2020 to 8 February 2021, 163 cases of SARS-CoV-2 Alpha variant of concern (VOC) were identified in Chieti province, Abruzzo region. Epidemiological data allowed the identification of 14 epi-clusters. With one exception, all the epi-clusters were linked to the town of Guardiagrele: 149 contacts formed the network, two-thirds of which were referred to the family/friends context. Real data were then used to estimate transmission parameters. According to our method, the calculated Re(t) was higher than 2 before the 12 December 2020. Similar values were obtained from other studies considering Alpha VOC. Italian sequence data were combined with a random subset of sequences obtained from the GISAID database. Genomic analysis showed close identity between the sequences from Guardiagrele, forming one distinct clade. This would suggest one or limited unspecified viral introductions from outside to Abruzzo region in early December 2020, which led to the diffusion of Alpha VOC in Guardiagrele and in neighbouring municipalities, with very limited inter-regional mixing.

Epizootic Haemorrhagic Disease Virus Serotype 8 in Tunisia, 2021.

Epizootic haemorrhagic disease (EHD) is a Culicoides-borne viral disease caused by the epizootic haemorrhagic disease virus (EHDV) associated with clinical manifestations in domestic and wild ruminants, primarily white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus). In late September 2021, EHDV was reported in cattle farms in central/western Tunisia. It rapidly spread throughout the country with more than 200 confirmed outbreaks. We applied a combination of classical and molecular techniques to characterize the causative virus as a member of the serotype EHDV-8. This is the first evidence of EHDV- 8 circulation since 1982 when the prototype EHDV-8 strain was isolated in Australia. This work highlights the urgent need for vaccines for a range of EHDV serotypes.

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.

Characterization and In Vitro Efficacy against Listeria monocytogenes of a Newly Isolated Bacteriophage, ɸIZSAM-1.

Listeria monocytogenes is a bacterial pathogen responsible of listeriosis, a disease that in humans is often related to the contamination of ready-to-eat foods. Phages are candidate biodecontaminants of pathogenic bacteria thanks to their ability to lyse prokaryotes while being safe for eukaryotic cells. In this study, ɸIZSAM-1 was isolated from the drain-waters of an Italian blue cheese plant and showed lytic activity against antimicrobial resistant Listeria monocytogenes strains. This phage was subjected to purification and in vitro efficacy tests. The results showed that at multiplicities of infection (MOIs) ≤ 1, phages were able to keep Listeria monocytogenes at low optical density values up to 8 h, with bacterial counts ranging from 1.02 to 3.96 log10 units lower than the control. Besides, ɸIZSAM-1 was further characterized, showing 25 principal proteins (sodium dodecyl sulfate polyacrylamide gel electrophoresis profile) and a genome of approximately 50 kilo base pairs. Moreover, this study describes a new approach to phage isolation for applications in Listeriamonocytogenes biocontrol in food production. In particular, the authors believe that the selection of phages from the same environments where pathogens live could represent a new approach to successfully integrating the control measures in an innovative, cost effective, safe and environmentally friendly way.

SARS-CoV-2 replicates in respiratory ex vivo organ cultures of domestic ruminant species.

There is strong evidence that severe acute respiratory syndrome 2 virus (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, originated from an animal reservoir. However, the exact mechanisms of emergence, the host species involved, and the risk to domestic and agricultural animals are largely unknown. Some domestic animal species, including cats, ferrets, and minks, have been demonstrated to be susceptible to SARS-CoV-2 infection, while others, such as pigs and chickens, are not. Importantly, the susceptibility of ruminants to SARS-CoV-2 is unknown, even though they often live in close proximity to humans. We investigated the replication and tissue tropism of two different SARS-CoV-2 isolates in the respiratory tract of three farm animal species - cattle, sheep, and pigs - using respiratory ex vivo organ cultures (EVOCs). We demonstrate that the respiratory tissues of cattle and sheep, but not of pigs, sustain viral replication in vitro of both isolates and that SARS-CoV-2 is associated to ACE2-expressing cells of the respiratory tract of both ruminant species. Intriguingly, a SARS-CoV-2 isolate containing an amino acid substitution at site 614 of the spike protein (mutation D614G) replicated at higher magnitude in ex vivo tissues of both ruminant species, supporting previous results obtained using human cells. These results suggest that additional in vivo experiments involving several ruminant species are warranted to determine their potential role in the epidemiology of this virus.

Genomic Epidemiology of the First Wave of SARS-CoV-2 in Italy.

Italy was one of the first countries to experience a major epidemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with >1000 cases confirmed by 1 March 2020. However, virus genome sequence data is sparse and there has been only limited investigation of virus transmission across the country. Here, we provide the most extensive study to date of the genomic epidemiology of SARS-CoV-2 in Italy covering the first wave of infection. We generated 191 new full-length genomes, largely sampled from central Italy (Abruzzo), before, during, and after the enforcement of a nationwide "lockdown" (8 March-3 June). These were combined with 460 published SARS-CoV-2 sequences sampled across Italy. Phylogenetic analysis including global sequence data revealed multiple independent introductions into Italy, with at least 124 instances of sequence clusters representing longer chains of transmission. Eighteen of these transmission clusters emerged before the nation-wide lockdown was implemented on 8 March, and an additional 18 had evidence for transmission between different Italian regions. Extended transmission periods between infections of up to 104 days were observed in five clusters. In addition, we found seven clusters that persisted throughout the lockdown period. Overall, we show how importations were an important driver of the first wave of SARS-CoV-2 in Italy.

Genetic Diversity of Rift Valley Fever Strains Circulating in Namibia in 2010 and 2011.

Outbreaks of Rift Valley fever (RVF) occurred in Namibia in 2010 and 2011. Complete genome characterization was obtained from virus isolates collected during disease outbreaks in southern Namibia in 2010 and from wildlife in Etosha National Park in 2011, close to the area where RVF outbreaks occurred in domestic livestock. The virus strains were sequenced using Sanger sequencing (Namibia_2010) or next generation sequencing (Namibia_2011). A sequence-independent, single-primer amplification (SISPA) protocol was used in combination with the Illumina Next 500 sequencer. Phylogenetic analysis of the sequences of the small (S), medium (M), and large (L) genome segments of RVF virus (RVFV) provided evidence that two distinct RVFV strains circulated in the country. The strain collected in Namibia in 2010 is genetically similar to RVFV strains circulating in South Africa in 2009 and 2010, confirming that the outbreaks reported in the southern part of Namibia in 2010 were caused by possible dissemination of the infection from South Africa. Isolates collected in 2011 were close to RVFV isolates from 2010 collected in humans in Sudan and which belong to the large lineage containing RVFV strains that caused an outbreak in 2006-2008 in eastern Africa. This investigation showed that the RVFV strains circulating in Namibia in 2010 and 2011 were from two different introductions and that RVFV has the ability to move across regions. This supports the need for risk-based surveillance and monitoring.