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 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.

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.

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.

Genome Sequences of Three SARS-CoV-2 P.1 Strains Identified from Patients Returning from Brazil to Italy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. We report the complete sequences of three SARS-CoV-2 P.1 strains obtained from nasopharyngeal swab specimens from three patients returning from Brazil to 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.

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.