Download Since the early 70`s a variety of pathological conditions in domestic

Since the early 70’s a variety of pathological conditions in domestic and wild animals has been attributed to
Coronaviruses (CoVs) infections. Currently six different CoV strains are known to infect humans. Two of these
belonged to Beta-CoV genus, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East
respiratory syndrome coronavirus (MERS-CoV), and caused severe respiratory diseases with case fatality rates of
9% and 36% respectively. The evolutionary origin of SARS-CoV, which was firstly detected in 2002, involved bat
hots, possibly with civets as intermediate host and the source of human infection. The origin of the MERS-CoV is
currently not well known although more recent studies point for camels as possible reservoirs or intermediate
hosts. Bats have also been suspected as the source of MERS-CoV because of genetic likenesses between BetaCoV found in bats and the MERS virus in humans. Recently, a CoV originated from an South African bat (SA bat)
was identified as highly related to MERS-CoV, and a fragment of a CoV showing 100% of identity to MERS-CoV
was found in a fecal sample from an Egyptian tomb bat (Taphozous perforatus). In this study, we described the
detection and full genome sequencing of CoVs from two Italian bats (IT bats) of different species, Pipistrellus
kuhlii and Hypsugo savii. Bat carcasses were obtained from the Modena Wildlife Recovery Center (North Italy).
During necropsy, no macroscopic lesions suggesting infectious diseases but traumatic injuries were observed.
Pools of viscera (lung, heart, spleen and liver) and intestine were tested separately using a pan-coronavirus onestep RT-PCR, which used neoCoV specific primes that amplified 180bp of the RdRp gene. For phylogenetic
analysis, full genome sequencing was conducted directly on pancoronavirus positive intestines using the Ion
Torrent NGS technology. Complete genome sequences of MERS-CoV and Alfa- and Beta-CoVs from bats, human
and camels (n. 131) were downloaded from ViPR system and a multiple sequence alignment was calculated
using the MUSCLE algorithm. Maximum likelihood phylogenetic tree was performed using the ViPR
implementation of the RAxML algorithm with the GTR model. Genetic relationships between Italian and SA bats
and MERS-CoV were confirmed by comparison of the sequence distances of MERS-CoV and bat-BCoV 2c (SA bat
and IT bats) using SSE v1.2. IT bats BCoVs showed 80% overall nucleotide identity across the whole genome to
MERS-CoV. The nucleotide distances respect to MERS-CoV were higher in the genomic region encoding the S
protein for all bat sequences including SA bat and in the ORF3 and ORF5 coding regions for the Italian sequences
(fig.1A). Phylogenetic tree of the complete genome showed a monophyletic group formed by MERS sequences
originated from humans and camels that are related to bat sequences. The most related sequences are those
originated from SA, Italy and China (fig. 1B). Bats, with extensive geographical distribution and capability of flight
have been documented as natural hosts of large number of diverse viruses such as lyssaviruses, paramyxoviruses
and filoviruses. The genetic diversity of CoVs in bats exceeds that known for other hosts, which is compatible
with bats being the major reservoir of mammalian CoVs. The emergence of MERS-CoV probably involved genetic
exchanges between different viral ancestors that may have occurred either in bat ancestors or in camels acting
as mixing vessels for viruses from different hosts