Detection of alpha and betacoronaviruses in multiple Iberian bat species

Falcón, Ana; Vázquez-Morón, Sonia; Casas, Inmaculada; Aznar, Carolina; Ruiz, Guillermo; Pozo, Francisco; Perez-Breña, Pilar; Juste, Javier; Ibáñez, Carlos; Garin, Inazio; Aihartza, Joxerra; Echevarría, Juan E.

The phylogenetic analysis of Spanish BatCoV was performed using 396nt out of the 512 nt RT-PCR amplified fragments. 116nt fragment information was lost to allowed us include more sequences from other European countries and other continents deposited in GenBanK to perform a meaningful analysis. This small part of the RdRp gene has been previously used, and sufficiently represents the full gene information, for phylogenetic analysis of BatCoV [11, 14, 20] The phylogenetic reconstruction showed 6 different lineages of Spanish BatCoV (Fig. 2) . BatCoV A and B were closely related to other a BatCoV found in China [12] , although they appeared to display certain genetic differentiation (Fig. 2) . Myotis daubentonii-associated CoV H, and Pipistrellus-associated CoV K, clustered respectively with lineages 4 and 3 of a CoV previously described in Germany and are hosted by the same bat species or genera [11] (Fig. 2) . BatCoV L was closely Table 2 . Positive samples described in this work are shown in shaded rectangles and ovals. The new alpha and betacoronavirus groups described in this work are shown in shaded ovals. Amino acid identity was calculated with MEGA 4 using the pairwise deletion option. The alignment comprised the same 396 bp of the RdRp gene used for the phylogenetic reconstruction. Amino acid identities across 132 amino acids are indicated next to the brackets that links every Spanish BatCoV to the phylogenetic neighbours related to cluster HKU7 described in China [10] . BatCoV C, D, E, F, G and I belonged to the same lineage and showed an intriguing new independent cluster (significant posterior probability = 0.95) including BatCoV I 0 (Fig. 2 ). In addition, BatCoV J and M were genetically related to b CoV, although they did not really associate with any of their previously described linages. Sequences corresponding to two different groups of CoV were found in the same bat species (Hypsugo savii) as it was also found for other bat species elsewhere [12] . Spanish BatCoV described here were also classified calculating amino acid distances of these viruses from phylogenetic neighbours and related reference species [21] . Amino acid distance criteria recently described for separating RdRp grouping units (RGU) were adopted [21] . Interestingly, the amino acid distance criteria ([4.8% for alphacoronavirus and [6.3% for betacoronavirus) reinforced the presence of the new group of alphacoronaviruses mentioned above including Spanish BatCoV C, D, E, F, G and I; and additionally showed four new tentative groups (Fig. 2) . BatCoV A and B were included in a new alphacoronavirus group and BatCoV I 0 constituted another independent alphacoronavirus group. BatCoV J, and M represented two new betacoronavirus groups and BatCoV H, K and L remained as part of several established species (Fig. 2) . It has been previously suggested that some CoV associate to certain bat species [10, 11] . However, we found that different bat species from the same colony or location harbour CoV of the same genetic lineage (BatCoV A and B; G and I), indicating a greater diversity and higher complexity than previously described for the ecology of BatCoV. Similar exceptions were found in China and South America [12, 16, 26] and could also be observed with Australian BatCoV (Fig. 2 and Table 2 ).