Special Issue "SARS-CoV-2: Epidemiology and Pathogenesis": Editorial.

Since its emergence in 2019 in Wuhan City, Hubei Province, China, SARS-CoV-2 has spread across hundreds of countries and all continents [...].

SARS-CoV-2 antibodies in dogs and cats in a highly infected area of Brazil during the pandemic

SARS-CoV-2 was a worldwide threat during the COVID-19 pandemic, and the state of Mato Grosso had the second highest mortality rate in Brazil, with 427. 4 deaths/100,000 inhabitants. However, no large-scale study among dogs and cats in such highly infected areas of Brazil has so far been conducted. Accordingly, the present study reports on a serosurvey among dogs and cats in Cuiabá, capital of Mato Grosso from November 2020 to July 2021, where the human mortality rate was 605/100,000 at that time. Overall, 33/762 dogs (4.3%) and 4/182 cats (2.2%) were found to be seropositive for SARS-CoV-2 through ELISA, and 3/762 dogs (0.4%) and 3/182 cats (1.6%) were seropositive through the serum neutralization test. Cats presented higher seroprevalence with higher titers of neutralizing antibodies. Although N-protein based ELISA may be a good screening test, cross-reactivity with other canine coronaviruses may impair its diagnostic use among dogs.

SARS-CoV-2 antibodies in dogs and cats in a highly infected area of Brazil during the pandemic.

SARS-CoV-2 was a worldwide threat during the COVID-19 pandemic, and the state of Mato Grosso had the second highest mortality rate in Brazil, with 427. 4 deaths/100,000 inhabitants. However, no large-scale study among dogs and cats in such highly infected areas of Brazil has so far been conducted. Accordingly, the present study reports on a serosurvey among dogs and cats in Cuiabá, capital of Mato Grosso from November 2020 to July 2021, where the human mortality rate was 605/100,000 at that time. Overall, 33/762 dogs (4.3%) and 4/182 cats (2.2%) were found to be seropositive for SARS-CoV-2 through ELISA, and 3/762 dogs (0.4%) and 3/182 cats (1.6%) were seropositive through the serum neutralization test. Cats presented higher seroprevalence with higher titers of neutralizing antibodies. Although N-protein based ELISA may be a good screening test, cross-reactivity with other canine coronaviruses may impair its diagnostic use among dogs.

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.

A Deletion Encompassing the Furin Cleavage Site in the Spike Encoding Gene Does Not Alter SARS-CoV-2 Replication in Lung Tissues of Mink and Neutralization by Convalescent Human Serum Samples.

SARS-CoV-2 has been shown to lose the furin polybasic cleavage site (FCS) following adaptation on cell culture. Deletion occurring in this region, which may include also the FCS flanking regions, seem not to affect virus replication in vitro; however, a chimeric SARS-CoV-2 virus without the sole FCS motif has been associated with lower virulence in mice and lower neutralization values. Moreover, SARS-CoV-2 virus lacking the FCS was shed to lower titers from experimentally infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. In this study, we investigated the replication kinetics and cellular tropism of a SARS-CoV-2 isolate carrying a 10-amino acid deletion in the spike protein spanning the FCS in lung ex vivo organ cultures of mink. Furthermore, we tested the neutralization capabilities of human convalescent SARS-CoV-2 positive serum samples against this virus. We showed that this deletion did not significantly hamper neither ex vivo replication nor neutralization activity by convalescent serum samples. This study highlights the importance of the preliminary phenotypic characterization of emerging viruses in ex vivo models and demonstrates that mink lung tissues are permissive to the replication of a mutant form of SARS-CoV-2 showing a deletion spanning the FCS. Notably, we also highlight the need for sequencing viral stocks before any infection study as large deletions may occur leading to the misinterpretation of results.

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.

Neutralization of SARS-CoV-2 Variants by Serum from BNT162b2 Vaccine Recipients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved rapidly, leading to viral lineages characterized by multiple mutations in the spike protein, which could potentially confer to the virus the ability to avoid the vaccine-induced immune response, making the vaccines less effective or ineffective. Here, we initially evaluated the neutralization capabilities in vitro by serum neutralization (SN) of six serum samples collected from recipients of the BNT162b2 vaccine against 11 SARS-CoV-2 isolates belonging to the major SARS-CoV-2 lineages that had been circulating in Italy. Then, we considered 30 additional serum samples by SN assay against the dominant B.1.617.2 (Delta) variant. A B.1 lineage isolate was used as a reference. In the first analysis, significant differences when compared with the reference strain (p > 0.05) were not evidenced; instead, when the panel of 30 sera was tested against the B.1.617.2 (Delta) variant, a significant (p = 0.0015) 2.38-fold reduction in neutralizing titres compared with the reference after the first vaccine dose was demonstrated. After the second vaccine dose, the reduction was not significant (p = 0.1835). This study highlights that the BNT162b2 vaccine stimulates a humoral response able to neutralize all tested SARS-CoV-2 variants, thus suggesting a prominent role in mitigating the impact of the SARS-CoV-2 pandemic in real-world conditions. Long-term follow-up is currently ongoing.

Erasing the Invisible Line to Empower the Pandemic Response.

A challenging debate has arisen on the role of veterinary expertise in facing the SARS-CoV-2 pandemic. It seems totally unreasonable that in most countries, veterinary diagnostic and tracing forces were not deployed at the start to perform strategic tasks, which could have mitigated the outcome of this dramatic health emergency. Erasing the invisible line between human and veterinary virology will empower the response to future pandemics.

SARS-CoV-2 RNA Persistence in Naso-Pharyngeal Swabs.

Since February 2020, Italy has been seriously affected by the SARS-CoV-2 pandemic. To support the National Health Care system, naso-pharyngeal/oropharyngeal swabs collected from suspected cases of Teramo province, Abruzzo region, are tested at Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale, for the presence of SARS-CoV-2 RNA. Out of 12,446 tested individuals, 605 returned positive results at least once, with prevalence significantly higher in men. A reduction of the level of viral RNA in the first swab per each positive patient collected over time was also observed. Moreover, 81 patients had at least one positive sample and two final negative tests: positivity in swabs lasted from 14 to 63 days, with a median value of 30 days. This shows the potential for the virus to coexist with patients for a long time, although we highlighted intermittent positivity in several cases. The evolution of the SARS-CoV-2 epidemiological situation and knowledge on viral shedding should be closely monitored, to interpret the findings correctly and adjust accordingly the surveillance activities.

Long-term persistence of neutralizing SARS-CoV-2 antibodies in pets.

We monitored the severe acute respiratory syndrome coronavirus 2 antibody response in seven dogs and two cats by using two multispecies ELISA tests, plaque reduction neutralisation test and virus neutralization. SARS-CoV-2 neutralizing antibodies in pets persisted up to 10 months since the first positive testing, thus replicating observations in COVID-19 human patients.

Whole-Genome Sequences of SARS-CoV-2 Lineage B.1.525 Strains (Variant η) Detected from Patients in the Abruzzo Region (Central Italy) during Spring 2021.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are emerging worldwide. Here, we report the complete genome sequences of 13 severe acute SARS-CoV-2 strains belonging to lineage B.1.525 (variant η).

Epidemiological Significance of SARS-CoV-2 RNA Dynamic in Naso-Pharyngeal Swabs.

From 16 March to 15 December 2020, 132,357 naso-pharyngeal/oropharyngeal swabs were collected in the province of Teramo, Abruzzo Region, Italy, and tested for the presence of SARS-CoV-2 genomic RNA by a commercially available molecular assay. A total of 12,880 swabs resulted positive. For 8212 positive patients (4.150 women and 4.062 men) the median age was statistically different between women (median: 49.55 ± 23.9 of SD) and men (median: 48.35 ± 23.5 of SD) while no differences were found in the comparison between the cycle threshold for the N protein-encoding gene (CT N) median values and gender. Differences were observed in the CT N gene median values of swabs collected from March to September as well as in the pairwise comparison between September and October and between November and December. The CT N gene median values observed in specific periods characterizing the SARS-CoV-2 epidemic in 2020 were also compared with the incidence of COVID-19 cases; a strong inverse correlation was highlighted (Pearson correlation coefficient = -0.978). Our findings confirm the usefulness of the CT N values as an indirect detection parameter to monitor viral loads in the population.

Multiple detection and spread of novel strains of the SARS-CoV-2 B.1.177 (B.1.177.75) lineage that test negative by a commercially available nucleocapsid gene real-time RT-PCR.

Several lineages of SARS-CoV-2 are currently circulating worldwide. During SARS-CoV-2 diagnostic activities performed in Abruzzo region (central Italy) several strains belonging to the B.1.177.75 lineage tested negative for the N gene but positive for the ORF1ab and S genes (+/+/- pattern) by the TaqPath COVID-19 CE-IVD RT-PCR Kit manufactured by Thermofisher. By sequencing, a unique mutation, synonymous 28948C > T, was found in the N-negative B.1.177.75 strains. Although we do not have any knowledge upon the nucleotide sequences of the primers and probe adopted by this kit, it is likely that N gene dropout only occurs when 28948C > T is coupled with 28932C > T, this latter present, in turn, in all B.1.177.75 sequences available on public databases. Furthermore, epidemiological analysis was also performed. The majority of the N-negative B.1.177.75 cases belonged to two clusters apparently unrelated to each other and both clusters involved young people. However, the phylogeny for sequences containing the +/+/- pattern strongly supports a genetic connection and one common source for both clusters. Though, genetic comparison suggests a connection rather than indicating the independent emergence of the same mutation in two apparently unrelated clusters. This study highlights once more the importance of sharing genomic data to link apparently unrelated epidemiological clusters and to, remarkably, update molecular tests.

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.

Novel SARS-CoV-2 Variants in Italy: The Role of Veterinary Public Health Institutes.

Novel SARS-CoV-2 variants with potential impacts on diagnostics, antivirals, and vaccines are spreading in Italy. In this editorial, we highlight the role that veterinary public health institutes may have in this global crisis, as their expertise in genomic/antigenic surveillance and animal studies are crucial to tackle SARS-CoV-2 pandemic.