Bat-coronavirus. Results of both ParaFit and PACo analyses of RdRp gene sequences showed evidence of coevolution between coronaviruses (CoVs) and their bat hosts (ParaFitGlobal = 390.8896, P = 0.001; m2 global value = 57.136, P ≤ 0.001). Fifty-one of the 60 individual host-parasite links were significant with a ParaFit1 or ParaFit2 P value ≤ 0.05.
MERS related coronaviruses (MERSr-CoVs) were detected in bats from Europe, Africa, and Asia. All of these bats belonged to the family Vespertilionidae (Fig. 1). Bat-derived MERSr-CoV isolates designated PML − PHE1/RSA/KC869678, 5038/RSA/MF593268, and PDF − 2180/UG/KX574227 had the highest (> 91%) nucleotide sequence identity with human and camel MERS-CoVs (Table 1). Isolates PML − PHE1/RSA/KC869678 and 5038/RSA/MF593268 were detected in Neoromicia capensis bats, members of the Vespertilionidae family, from South Africa. Isolate PDF − 2180/UG/KX574227 was detected in Pipistrellus hesperidus bats, which are also members of the Vespertilionidae family, from Uganda (Fig. 1). Isolate PDF − 2180/UG/KX574227 and its closest MERS-CoVs from humans and camels shared nearly 92% nucleotide sequence identity (Table 1). It also shared 91.93% nucleotide sequence identity with isolate 5038/RSA/MF593268 and 91.79% with isolate PML − PHE1/RSA/KC869678. Both isolates 5038/RSA/MF593268 and PML − PHE1/RSA/KC869678 shared > 93% nucleotide sequence identity with their closest MERS-CoVs from camels and humans (Table 1). Other MERSr-CoV isolates, including HKU5 − 1/CN/EF065509, HKU4 − 1/CN/EF065505, GX2012/CN/KJ473822, JPDB144/CN/KU182965, and GD2013/CN/KJ473820 shared < 83% nucleotide sequence identity with MERS-CoVs from camels and humans (Table 1). MERSr- CoV isolates PML − PHE1/RSA/KC869678, 5038/RSA/MF593268, NL13845/CN/MG021451, HKU25/CN/KX442565, SC2013/CN/KJ473821 206645 − 40/IT/MG596802, and 206645 − 63/IT/MG596803 shared > 85% nucleotide sequence identity with their closest camel and human MERS-CoVs (Table 1), As shown in Fig. 1, the host bats of isolates HKU5 − 1/CN/EF065509, HKU4 − 1/CN/EF065505, GX2012/CN/KJ473822, JPDB144/CN/KU182965, and GD2013/CN/KJ473820 are distantly related to N. capensis bats (Fig. 1). Isolates 206645 − 40/IT/MG596802 and 206645 − 63/IT/MG596803 detected in Hypsugo savii and P. kuhli bats, respectively (Fig. 1), from Italy shared 99.46% nucleotide sequence identity with each other.
Table 1
RdRp nucleotide sequence identity between MERSr-CoVs and camel and human MERS-CoVs.
Bat MERSr- CoVs | Host | Closest camel MERS-CoVs | | Cloest human MERS-CoVs | | Close bat species* |
CoV name | identity | | CoV name | identity | |
5038/RSA/MF593268 | Neoromicia capensis | B78/UAE/MG598668 | 93.77% | | 2285/OMA/KT156560 | 93.77% | | Neo cap |
PML-PHE1/RSA/KC869678 | Neoromicia capensis | T157/KSA/KT368890 | 93.49% | | C20860/KSA/KT806055 | 93.49% | | Neo cap |
PDF-2180/UG/KX574227 | Pipistrellus hesperidus | B25/UAE/MF598617 | 91.99% | | 016/KSA/MN120513 | 91.95% | | Pip hes |
206645-63/CN/MG596803 | Pipistrellus kuhlii | 047(b)/KSA/KT368852 | 86.20% | | 016/KSA/MN120513 | 86.13% | | Pip hes |
NL13845/CN/MG021451 | Ia io | 415915/UAE/MF598718 | 86.16% | | 11958/KSA/KX154687 | 86.20% | | Neo cap |
206645-40/IT/MG596802 | Hypsugo savii | 047(b)/KSA/KTKT368852 | 86.10% | | 016/KSA/MN120513 | 86.03% | | Neo cap |
HKU25/CN/KX442565 | Hypsugo pulveratus | HKU44/ETH/MG923468 | 85.80% | | 10717/KSA/MH310911 | 85.72% | | Neo cap |
SC2013/CN/KJ473821 | Vespertilio sinensis | 415915/UAE/MF598718 | 85.72% | | Bisha/KSA/KF600620 | 85.72% | | Neo cap |
GX2012/CN/KJ473822 | Tylonycteris pachypus | HKU213/MON/MF598718 | 81.70% | | 2362/KSA/MH371127 | 81.92% | | Pip hes |
HKU5-1/CN/EF065509 | Pipistrellus abramus | T157/KSA/KT368890 | 82.99% | | 016/KSA/MN120513 | 83.06% | | Pip hes |
GD2013/CN/KJ473820 | Pipistrellus abramus | T157/KSA/KT368890 | 82.88% | | 18012493/KSA/MK462249 | 82.92% | | Pip hes |
JPDB144/CN/KU182965 | Myotis daubentonii | 047(b)/KSA/KTKT368852 | 81.93% | | 20570/KSA/MG011354 | 81.97% | | Pip hes |
HKU4-1/CN/EF065505 | Tylonycteris pachypus | 047(b)/KSA/KTKT368852 | 81.81% | | 20570/KSA/MG011354 | 81.84% | | Pip hes |
*Neo cap = Neoromicia capenses; Pip hes = Pipistrellus hesperidus |
Rhinolophus sinicus and R. ferrumequinum bats were most commonly found to harbor SARS related coronaviruses (SARSr-CoVs) (Fig. 1). R. sinicus bats are distributed in Asia, and R. ferrumequinum are present in Africa, Asia, and Europe (Fig. 1). Rs3367/CN/KC881006 and YN2013/CN/KJ473816 shared the highest nucleotide identity with SARS-CoV (Table 2) they were dectected from R. sinicus. The SARSr-CoV isolate designated 16BO133/Korea/KY938558 was detected in R. ferrumequinum from South Korea. This isolate was found to be evolutionarily close to SARSr-CoV isolates JL2012/KJ473811 and JTMC15/KU182964 found in the same host species, R. ferrumequinum, from Jilin, China (Fig. 1), which is located north of South Korea. Isolate 16BO133/Korea/KY938558 shared 99.71% nucleotide sequence identity with isolate JL2012/KJ473811 and 99.68% with isolate JTMC15/KU182964, JL2012/CN/KJ473811 and JTMC15/CN/KU182964 shared 99.96% to each other (Table 2).
Table 2
RdRp nucleotide sequence identity between SARSr-CoVs from various species of bats and their closest bat CoVs and human SARS-CoVs.
Bat SARSr- CoVs | Host | Cloest bat CoVs | | Cloest human SARS-CoVs |
CoV name | Identity | | CoV name | identity |
JL2012/CN/KJ473811 | Rhinolophus ferrumequinum | JTMC15/CN/KU182964 | 99.96% | | Sin847/SIN/AY559095 | 92.42% |
JTMC15/CN/KU182964 | Rhinolophus ferrumequinum | JL2012/CN/KJ473811 | 99.96% | | Sin847/SIN/AY559095 | 92.38% |
16BO133/ROK/KY938558 | Rhinolophus ferrumequinum | JL2012/CN/KJ473811 | 99.71% | | Sin847/SIN/AY559095 | 92.42% |
Rf1/CN/DQ412042 | Rhinolophus ferrumequinum | 273/CN/DQ648856 | 99.64% | | icSARS-C7-MA/USA/ MK062184 | 92.45% |
SX2013/CN/KJ473813 | Rhinolophus ferrumequinum | HeB2013/CN/KJ473812 | 99.71% | | icSARS-C7-MA/USA/ MK062184 | 92.95% |
HeB2013/CN/KJ473812 | Rhinolophus ferrumequinum | SX2013/CN/KJ473813 | 99.71% | | icSARS-C7-MA/USA/ MK062184 | 93.13% |
Shaanxi2011/CN/JX993987 | Rhinolophus pusillus | HeB2013/CN/KJ473812 | 95.60% | | icSARS-C7-MA/USA/ MK062184 | 93.13% |
HKU3 − 1/CN/DQ022305 | Rhinolophus sinicus | HKU3 − 1/CN/DQ084200 | 100.00% | | Sin846/SIN/AY559094 | 91.73% |
HuB2013/CN/KJ473814 | Rhinolophus sinicus | Rm1/CN/DQ412043 | 95.13% | | HZS2-E/CN/AY394990 | 93.24% |
Rm1/CN/DQ412043 | Rhinolophus macrotis | 279/CN/DQ648857 | 99.79% | | JMD/CN/AY394988 | 93.56% |
Yunnan2011/CN/JX993988 | Chaerephon plicatus | MLHJC35/CN/KU182963 | 97.03% | | Sin847/SIN/AY559095 | 94.96% |
Anlong − 103/CN/KY770858 | Rhinolophus sinicus | Anlong − 112/CN/KY770859 | 100.00% | | JMD/CN/AY394988 | 98.39% |
GX2013/CN/KJ473815 | Rhinolophus sinicus | Anlong − 112/CN/KY770859 | 98.35% | | HZS2-E/CN/AY394990 | 97.64% |
Rs3367/CN/KC881006 | Rhinolophus sinicus | WIV1/CN/KF367457 | 99.93% | | JMD/CN/AY394988 | 98.39% |
YN2013/CN/KJ473816 | Rhinolophus sinicus | YN2018B/CN/MK211376 | 99.57% | | JMD/CN/AY394988 | 98.39% |
As6526/CN/KY417142 | Aselliscus stoliczkanus | YN2018B/CN/MK211376 | 99.89% | | JMD/CN/AY394988 | 98.28% |
Rp3/CN/DQ071615 | Rhinolophus pearsonii | Anlong-29/CN/KF294439 | 99.03% | | JMD/CN/AY394988 | 97.96% |
LYRa11/CN/KF569996 | Rhinolophus affinis | Rf4092/CN/KY417145 | 98.06% | | JMD/CN/AY394988 | 96.89% |
BM48 − 31/BGR/GU190215 | Rhinolophus blasii | Rs4231/CN/KY417146 | 88.42% | | HZS2-E/CN/AY394990 | 88.18% |
RaTG13/CN/MN996532 | Rhinolophus affinis | SNU01/ROK/MT039890 | 97.85% | | SZ16/HK/AY304488 | 88.26% |
SARSr-CoVs were also detected in bats outside the Rhinolophidae family. The isolate As6526/CN/KY417142 from Aselliscus stoliczkanus bats (family: Hipposideridae), which are evolutionarily close to Rhinolophidae bats, shared 99.89% nucleotide sequence identity with isolates YN2018B/CN/MK211376 found in R. affinis bats from Yunnan, China (Table 2). Isolate Yunnan2011/CN/JX993988 found in Chaerephon plicatus bats, which are relatively distant from Rhinolophus bats (Fig. 1), also shared 97.03% nucleotide sequence identity with isolate MLHJC35/CN/ KU182963 from R. sinicus bats (Table 2). A Betacoronavirus isolate designated BM48 − 31/BGR/GU190215 was detected in R. blasii bats from Bulgaria. These bats are normally found in Africa, southern Europe, and western Asia. This isolate was evolutionarily placed between isolates YRa11/CN/KF569996 and Zhejiang2013/CN/KF636752 on the phylogenetic tree (Fig. 1). It shared 88.42% nucleotide sequence identity with the SARSr-CoV isolate Rs4231/CN/KY417146 from R. sinicus (Table 2). The BatCoV RaTG13/CN/MN996532 was found in R. affinis bats. It is evolutionarily closed to SARSr-CoVs (Fig. 1), and shared 97.83% with COVID-19 strain (SNU01/ROK/MT039890), 88.26 with human SARS-CoV strain (SZ16/HK/AY304488) (Table 2).
Isolates CMR66/CMR/MG693170, HKU9 − 1/CN/EF065513, and GCCDC1 − 356/CN/KU762338 were clustered together on the phylogenetic tree (Fig. 1). These isolates shared 78.03% − 96.24% nucleotide sequence identity with each other, and their hosts were all bats from the Pteropodidae family (flying fox).
In Alphacoronavirus, isolates 1B/CN/EU420137, AH2011/CN/KJ473795, 1A/CN/EU420138, HKU7 − 1/CN/DQ249226, and HKU8/CN/EU420139 were clusterd together on the phylogenetic tree (Fig. 1) and shared 78.38% − 98.24% nucleotide sequence identity with each other. Their hosts were all Miniopterus bats. Isolates AT1A − F1/GHA/KT25327 and KW2E − F151/GHA/KT253269 shared 95.76% nucleotide sequence identity. Their hosts were Hipposideros bats from Africa (Fig. 1).
Bat Paramyxovirus. Results of ParaFit and PACo analyses of nucleotide sequences of bat cyt b gene and paramyxovirus RNA polymerase large (L) gene indicated that paramyxoviruses and their bat hosts had a significant coevolutionary relationship (ParaFitGlobal = 874.11, P = 0.049; m2 global value = 15.49537, P = 0.015). Seven of the 36 individual host-parasite links were significant with a ParaFit1 or Parafit2 value of P ≤ 0.05.
Paramyxoviruses were divided into two major branches on the phylogenetic tree. As most of them were unclassified (Fig. 2), they were divided into 4 groups (PG1-PG4) according to certain characteristics of their hosts. Isolates GB59 − 59/GHA/HQ660162, GB09670/GAB/HQ660156, GB59 − 30/GHA/HQ660161, GH19 − 140/GHA/HQ660153, GD2012/CN/KJ64165, and GB09682/GAB/HQ660157 (paramyxovirus group 1, PG1) were detected in Hipposideros bats (family: Hipposideridae) that are mainly distributed in Africa and Asia (Fig. 2). Isolates RCA − P18/RCA/HQ660152, CD273/DRC/HQ660122, GB1386/GAB/HQ660137, GB1237/GAB/HQ660140, and GH6/GHA/FJ971938 (paramyxovirus group 2, PG2) were detected in bats of the Pteropodidae family from Africa; these bats were not phylogenetically clustered together with the bats in this family from Asia, Oceania, and Australia (Fig. 2). Isolates KCR245H/CRC/JF828297, BR21/BRA/HQ660187, BR310/BRA/HQ660194, BR310/BRA/HQ660194, and BR190/BRA/HQ660190 (paramyxovirus group 3, PG3) were closely related. Their hosts are distributed in South and North America. The host of KCR245H/CRC/JF828297 was Pteronotus parnellii (family: Mormoopidae), and the hosts of the other four isolates were bats of the Pteropodidae family (Fig. 2). Seven closely related isolates GH36/GHA/FJ609192, 3 − 320/BGR/HQ660163, N78 − 14/GER/HQ660166, 6 − 43/BGR/HQ660164, NMS09 − 48/GER/HQ660165, Md − LN2012/CN/KJ641656, and NM98 − 46/GER/HQ660170 (paramyxovirus group 4, PG4) from Europe and Asia (Fig. 2) shared 70.13% − 97.32% nucleotide sequence identity with each other Among them, isolate 6 − 43/BGR/HQ660164 was detected in Myotis capaccinii bats (family: Vespertilionidae) from Bulguria, and isolate NMS09 − 48/GER/HQ660165 was found in M. daubentoni bats (family: Vespertilionidae) from Germany. These two isolates shared the highest nucleotide sequence identity.
Teviot virus (TeV), Tioman virus (TiV), and Menangle virus (MENV) were phylogenetically clustered in the same clade. These viruses are members of the genus Pararubulavirus, and their bat hosts were members of the Pteropodidae family. These bats are distributed in regions ranging from southeast Asia to northwest Oceania (Fig. 2). HeV, NiV, and several unclassified paramyxoviruses were clustered in one clade (Fig. 2). These results indicated that closely related bats carried closely related paramyxoviruses. Some bat hosts of HeV and NiV were found to be also infected by TiV and MENV (Fig. 2).