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.

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.

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.

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).