Molecular Detection and Genetic Characterization of Bovine Kobuvirus (BKV) in Diarrhoeic Calves in a Central Italy Herd

Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.

The Delta variant wave in Tunisia: Genetic diversity, spatio-temporal distribution and evidence of the spread of a divergent AY.122 sub-lineage.

Introduction: The Delta variant posed an increased risk to global public health and rapidly replaced the pre-existent variants worldwide. In this study, the genetic diversity and the spatio-temporal dynamics of 662 SARS-CoV2 genomes obtained during the Delta wave across Tunisia were investigated. Methods: Viral whole genome and partial S-segment sequencing was performed using Illumina and Sanger platforms, respectively and lineage assignemnt was assessed using Pangolin version 1.2.4 and scorpio version 3.4.X. Phylogenetic and phylogeographic analyses were achieved using IQ-Tree and Beast programs. Results: The age distribution of the infected cases showed a large peak between 25 to 50 years. Twelve Delta sub-lineages were detected nation-wide with AY.122 being the predominant variant representing 94.6% of sequences. AY.122 sequences were highly related and shared the amino-acid change ORF1a:A498V, the synonymous mutations 2746T>C, 3037C>T, 8986C>T, 11332A>G in ORF1a and 23683C>T in the S gene with respect to the Wuhan reference genome (NC_045512.2). Spatio-temporal analysis indicates that the larger cities of Nabeul, Tunis and Kairouan constituted epicenters for the AY.122 sub-lineage and subsequent dispersion to the rest of the country. Discussion: This study adds more knowledge about the Delta variant and sub-variants distribution worldwide by documenting genomic and epidemiological data from Tunisia, a North African region. Such results may be helpful to the understanding of future COVID-19 waves and variants.

SARS-CoV-2 in Namibian Dogs.

The pandemic of coronavirus disease 19 (COVID-19) has focused the attention of researchers, and especially public opinion, on the role of the human-animal-environment interface in disease emergence. At the beginning of the COVID-19 pandemic, media reports regarding the role of pets in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused significant concern and social anxiety. Although nowadays proven negligible in developed countries, essentially no studies have been performed in low-income African areas where companion animals are often raised differently from high income countries, and the contact patterns occurring in these scenarios could affect the epidemiological scenario. An extensive molecular biology survey was performed from March 2022 to September 2022 on Namibian dogs residing in urban and rural areas, showing a low but not negligible SARS-CoV-2 prevalence (1%; 95CI: 0.33-2.32%) of 5 out of 500. In only one instance (i.e., a 4-year-old female Labrador) was there a clear association that could be established between the infections of the owner and animal. In all other cases, no evidence of human infection could be obtained and no episodes of COVID-19 were reported by the owners. Although no consistent evidence of pet-to-pet transmission was proven in the present study, a cautionary principle suggests intensive and dedicated investigation into companion animal populations, especially when animal contact is frequent and a particularly susceptible population is present.

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.

SARS-CoV-2 Delta VOC in a Paucisymptomatic Dog, Italy.

Since the very beginning of the COVID-19 pandemic, SARS-CoV-2 detection has been described in several animal species. A total of 625 outbreaks in animals have been reported globally, affecting 17 species in 32 countries and the human source of infection has been recognized including pet owners, zookeepers, and farmers. In this report, we describe the case of a paucisymptomatic dog in Italy infected with SARS-CoV-2 from a household with three confirmed human cases of COVID-19 living in Pesaro (Marche region, Italy). The dog showed high viral RNA titers in the nasal and oropharyngeal swabs. In the nasal swab, SARS-CoV-2 RNA lasted for a least a week. By sequencing, the strain was assigned to the AY.23 lineage (PANGO), one of the sub-lineages of the major SARS-CoV-2 Delta variant of concern (VOC). Although we did not process the swabs of the three human cases, we strongly suspect a human origin for the dog infection. In this regard, AY.23 sequences, although never released thus far in the Marche region, were detected in the neighboring regions. Our findings highlight once more the need for a One Health approach for SARS-CoV-2 surveillance, management, and control, thus preventing viral spillover from animals to humans.

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.

Emergence and Spread of SARS-CoV-2 Lineages B.1.1.7 and P.1 in Italy.

Italy's second wave of SARS-CoV-2 has hit hard, with more than three million cases and over 100,000 deaths, representing an almost ten-fold increase in the numbers reported by August 2020. Herein, we present an analysis of 6515 SARS-CoV-2 sequences sampled in Italy between 29 January 2020 and 1 March 2021 and show how different lineages emerged multiple times independently despite lockdown restrictions. Virus lineage B.1.177 became the dominant variant in November 2020, when cases peaked at 40,000 a day, but since January 2021 this is being replaced by the B.1.1.7 'variant of concern'. In addition, we report a sudden increase in another documented variant of concern-lineage P.1-from December 2020 onwards, most likely caused by a single introduction into Italy. We again highlight how international importations drive the emergence of new lineages and that genome sequencing should remain a top priority for ongoing surveillance in Italy.

Infection sustained by lineage B.1.1.7 of SARS-CoV-2 is characterised by longer persistence and higher viral RNA loads in nasopharyngeal swabs.

Following the announcement on December 2020 about the emergence of a new variant (VOC 202012/ 01, B.1.1.7 lineage) in the United Kingdom, a targeted surveillance was put in place in the Abruzzo region (Italy), which allowed detection of 313 persons affected by lineage B.1.1.7, up to the 20th of February 2021. We investigated the results of RT-PCR on nasopharyngeal swabs tested from December 2020 to February 2021 to verify any difference on the viral load and persistence between people infected by lineage B.1.1.7 and others. Statistically significant lower values of CT associated with the detection of the N protein encoding gene (CT N) were observed in persons with lineage B.1.1.7 infection (median CT N = 15.8)in comparison to those infected by other lineages (median CT N = 16.9). A significantly longer duration of the persistence of SARS-CoV-2 RNA in nasopharyngeal swabs was observed in persons with lineage B.1.1.7 infection (16 days) in comparison to those infected by other lineages (14 days).

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.

A "One-Health" approach for diagnosis and molecular characterization of SARS-CoV-2 in Italy.

The current pandemic is caused by a novel coronavirus (CoV) called SARS-CoV-2 (species Severe acute respiratory syndrome-related coronavirus, subgenus Sarbecovirus, genus Betacoronavirus, family Coronaviridae). In Italy, up to the 2nd of April 2020, overall 139,422 confirmed cases and 17,669 deaths have been notified, while 26,491 people have recovered. Besides the overloading of hospitals, another issue to face was the capacity to perform thousands of tests per day. In this perspective, to support the National Health Care System and to minimize the impact of this rapidly spreading virus, the Italian Ministry of Health involved the Istituti Zooprofilattici Sperimentali (IZSs), Veterinary Public Health Institutes, in the diagnosis of SARS-CoV-2 by testing human samples. The Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise is currently testing more than 600 samples per day and performing whole genome sequencing from positive samples. Sequence analysis of these samples suggested that different viral variants may be circulating in Italy, and so in Abruzzo region. CoVs, and related diseases, are well known to veterinarians since decades. The experience that veterinarians operating within the Public Health system gained in the control and characterization of previous health issues of livestock and poultry including avian flu, bluetongue, foot and mouth disease, responsible for huge economic losses, is certainly of great help to minimize the impact of this global crisis.