4 Discussion
Overall, Mammalia, Aves, and Reptilia formed a distinct cluster,
indicating the TMPRSS2 was more likely to be used for recognizing and
processing S-protein of SARS-CoV-2 in Aves and Reptilia rather than
Amphibia and fish. Human, pangolin, cat, dog and hamster are proved to
be be infected by SARS-CoV-2 in previous
studies[5,6,7,8,9,10,11],
especially for human, the TMPRSS2 was shown to be used for priming
S-protein on ACE2 to help SARS-CoV-2 to enter into
cells[13]. We then focused on these
species to predict the utilizing capability of TMPRSS2 for S-protein
priming. Primates closely clustered together with human (Fig S1, Fig
S2), indicating the TMPRSS2 could be effectively used in primates for
S-protein priming. Cat, dog clustered with bat (Fig 2), which was
inconsistent with the phylogenetic result of
ACE2[14], and also showed the high
possibility of TMPRSS2 utilizing capability. The brown bear was placed
with pangolin, bat and dog forming a distinct clade (Fig S1), showing
yet another possible wild animal of high probability to use TMPRSS2 to
cleave S-protein of SARS-CoV-2. Considering the lack of direct contact
with the human, brown bear was less likely to be the intermediate host
to transfer the virus to human. Interestingly, the TMPRSS2 of horse
closely clustered with hamster, but not with its much closer relatives
(Fig S1), showing a potentially effective function of TMPRSS2 in horse
to process the S-protein for priming. When focused on animals that have
a close relationship with humans; the pig, sheep, cattle and pangolin
formed a distinct clade (Fig 2). Although the incubation test showed the
pig was not susceptible with SARS-CoV-2
infection[7], our result indicated the
probable availability of TMPRSS2 for S-protein priming in the pig. A
previous study showed that the SARS-CoV-2 was not able to use ACE2 to
enter into the mouse cell[4]. In our
study, however, rat and mouse TMPRSS2 were closely clustered with human
and hamster TMPRSS2, showing that the TMPRSS2 in mouse and rat should be
able to activate the S-protein of SARS-CoV-2.
ACE2 can tolerate a variety of amino acid changes among animals, making
the low species barrier of
SARS-CoV-2[20]. We expected the same
situation for TMPRSS2, so we further used the PolyPhen-2 to evaluate the
possible impact on the function of TMPRSS2 at single amino level, but
not at the whole sequence level, because many amino acid changes will
not cause change in function. By scoring the TMPRSS2 of all 164 species,
we found that the prediction result was highly similar to prediction
from the phylogenetic tree, with Mammalia having the highest scores, and
then Aves, Reptilia, Amphibia and fish. However, the most important
three amines (His296, Ser441 and
Asp435)[21] located at the catalytic
triad of TMPRSS2 were ultra-conserved (Fig 1, Fig S4). Only four out of
164 species were found to have mutations at these three amines,
indicating a conserved utilizing capability of TMPRSS2 across species,
although other amino acid changes may also influence the efficiency of
TMPRSS2 to cleave the S-protein of SARS-CoV-2. Animals that have been
proven to be affected by the SARS-CoV-2, like pangolin, cat, dog, and
hamster, exhibited very high scores (Table 1), which showed the
rationality of this scoring method. Both high score and less damaging
amino acid changes were found in rat, mouse and pig than pangolin and
cat, which in turn supported the prediction of the phylogenetic tree,
showing the effective utilizing capability of TMPRSS2 in rat, mouse and
pig. In addition, both the phylogenetic tree and PolyPhen-2 scores
showed relatively low TMPRSS2 capability in poultry.
COVID19 is still progressing and SARS-COV-2 strains are constantly
evolving. It needs to be emphasized again that we need to pay more
attention to mammals, especially pigs and rats, to prevent these animals
from becoming intermediate hosts of a future pandemic, considering the
more effective utilizing capability of TMPRSS2 in mammals than in other
animals.