1  Introduction
Severe respiratory syndrome coronavirus-2 (SARS-CoV-2) is a novel, highly transmissible betacoronavirus that caused pandemic Coronavirus Disease 2019 (COVID-19) [1]. Outbreaks occurred worldwide, including Italy, where the number of infections was exceptionally high in northern regions [2]. Among betacoronaviruses, SARS-CoV-2 is the third virus causing fatal severe human respiratory disease after SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) [1].
Identification of animal species that can potentially be reservoirs for the virus is strategic to controlling SARS-CoV-2 infection and preventing future outbreaks caused by a mutated form of the virus [3, 4]. From the genetic proximity of SARS-CoV-2 to RaTG13 coronavirus, a bat origin for the current COVID-19 outbreak was hypothesized. Concurrent evidence also proposed pangolins as a potential intermediate reservoir [1, 5, 6, 7, 8]. Concerning the secondary reservoir, susceptibility to the SARS-CoV-2 infection was demonstrated for several domestic animals both in silico [1, 9] and experimentally [10, 11]. SARS-CoV-2 can replicate efficiently in young cats and ferrets. Both these animals can also transmit the infection to other cats via airborne transmission in an experimental setting. Conversely, dogs have shown low susceptibility to experimental infection, while pigs, chickens, and ducks are not susceptible [6]. Besides pets, several studies have demonstrated that numerous wild animals are potential host intermediate for SARS-CoV-2 infection [12].
Mammalian biodiversity is recognized as a risk factor for zoonotic disease emergence [13, 14, 15]. COVID-19, as a zoonotic disease, represents a high treat for regions with high biodiversity; it represents a high treat for regions like Italy, which is among the European countries richest in biodiversity [16, 17] (https://www.isprambiente.gov.it/en/archive/news-and-other-events/ispra-news/year-2015/may/biodiversity-in-italy). Strategies to contain infection are being adopted following the WHO recommendation [18] (https://www.who.int/publications/i/item/clinical-management-of-covid-19). However, given the high biodiversity, great attention is on identifying mammalian species occupying the Italian territory, which may act as potential new primary or secondary reservoir of SARS-CoV-2. Lastly, ecologically speaking, the control of a new virus generation may prevent endangered species extinction and avoid a severe threat to Italy's biodiversity.
Here we present a bioinformatic investigation aimed at identifying the host range of SARS-CoV-2 most diffuse in Italy. In particular, considering that the virus originates from bats [19], we wondered i) if Chiroptera species exist in Italy as potential primary reservoir for the virus, ii) if these bats could infect animals in the wild and domestic environments, and iii) if these animals could be a secondary reservoir in Italy (Figure 1).
Figure 1 Bioinformatic workflow followed for the identification of primary and secondary reservoir in Italy.
Analogously to many viruses [20], infection of SARS-CoV-2 occurs through the binding of viral surface glycoprotein, spike (S) protein, to the angiotensin-converting enzyme 2 (ACE2) [1, 21]. ACE2 is expressed in several cell types, including type II pneumocytes, myocardial cells, cholangiocytes, enterocytes, and oral mucosal epithelium [22]. According to a structural biology view, infection of SARS-CoV-2 depends on an optimal interaction of S protein with the ACE2 receptor. Therefore, species where ACE2 can efficaciously interact with SARS-CoV-2 S protein can be considered valuable reservoirs of the virus.
In this context, starting from the study of the recently solved human ACE2 (hACE2)/S protein complex [9] and considering the sequence similarity of ACE2 in Chiroptera and domestic and wild animal species living in Italy, we followed two bioinformatics approaches to identify new potential primary and secondary reservoirs of SARS-CoV-2 occupying Italian territory (Figure 1).
In silico molecular docking of ACE2 belonging to Chiroptera vs SARS-CoV-2 S protein pointed to Rhinolophus ferrumequinum as a bat living in Italy, that may be a potential primary reservoir of the virus. On the other hand, a sequence similarity search on ACE2 of domestic and wild animals living in Italy indicated 10 species as potential SARS-CoV-2 secondary reservoirs. Molecular docking of ACE2 belonging to these species vs S protein of Bat coronavirus (Bt-CoV/Rp3/2004) suggested that the primary reservoir Rhinolophus ferrumequinum may infect the secondary reservoirs, domestic and wild animals living in Italy, determining a specific risk of SARS-CoV-2 infection.