Discussion
Swine coronavirus (SCoV) are cause effect important diseases in pigs.
PDCoV is the most resent SCoV discovered during 2012 in China. Later, it
spread around the world causing serious economic losses. It has been
suggested that PDCoV spread from the United States to Mexico due to the
commercial swine exchange and the geographical proximity of these two
countries (He et al., 2020; Perez-Rivera et al., 2019). Identification
and diagnosis in early stages are necessary to prevent and control the
diseases. Therefore, the goal in this study was to develop a recombinant
PDCoV protein M based on a consensus sequence from 138 available
sequences from China, Laos, Vietnam, Thailand, and USA (Table 1).
We found a highly conserve M
protein among the 138 sequences in the phylogenetic analysis (Fig 1).
The similarity among the sequences was >94%. The high
frequency of M in the virus envelope (molar ratio S:N:M of 1:8:16 and
1:6:15) (de Haan & Rottier, 2005) and its role in the assembly process
like a “building block (Wang et al., 2020), are probably the cause of
the highly conserve property of M protein.
Moreover, at a structural level (Fig 2a), the consensus sequences folds
in three hydrophobic domains
alternating with short hydrophilic regions in the N-terminal half of the
protein (Fig 2b). This tertiary structure was observed in all sequences
compared but not in the TGE Miller M6 sequence. TGE M proteins has a
cleavage N-terminal, but it is still not clear the function of this
(Kapke et al., 1988). However, this incision could give an alternative
configuration like the observed in figure 2a. Similarly, the topological
tertiary structure comparison between the PDCoV sequences and the
consensus sequence shows a significant TM-Score of 0.8 (Table 2).
Overall, the tertiary structure observed for consensus M protein
correlates with the reported in the literature (Neuman et al., 2011).
It has been observed that PDCoV is rare among enteric SCoV infections
(Perez-Rivera et al., 2019), due to PDCoV is an emergent disease. During
the present study, it was not possible to obtain and isolate a viral
sample. Therefore, we decided to construct a synthetic gene. The
integrity and weight of the synthetic gene was evaluated (Fig. 3a). We
successfully expressed and purified a recombinant M protein
(r M-PDCoV) from E. coli . The r M-PDCoV was evaluated
by SDSPAGE and identified by western blotting with ~37.7
kDa expected molecular weight. Using the r M-PDCoV, we identify
eight positive sera by western blot among 17 from “El Bajio” pig
farms. Thus, the r M-PDCoV developed in this study, was able to
detect antibodies against PDCoV. It has been mentioned that co-infection
with PDCoV and PED (19.6%) are likely to occurred (Song et al., 2015).
However, the amino acid identity (Stothard, 2000) of r M-PDCoV and
the CV777 PEDV M protein (GenBank: AAK38659) is 23.5%, for TGE Miller
M6 (GenBank: ABG89300) is 18.89%. This percentage of identity was
consistent with the observed by Thachil and cols. These amino acid
identity values suggest that the PDCoV consensus protein M used in this
study is unlikely to cross-react with PED or TGE (Thachil et al., 2015).
Also, this result is in accordance with reported that antibodies
cross-reactivity of PDCoV with PED has not been observed (Jung et al.,
2016; Ma et al., 2015).
On the other hand, according to the TM-Score value > 0.8
(table 2) the r M-PDCoV protein structure is similar to other
reported PDCoV M protein sequences. It has been reported that that
structures with a score higher than 0.5 assume roughly the same fold
(Zhang & Skolnick, 2004, 2005). Therefore, it is presumed that a
vaccine based on r M-PDCoV protein may stimulate the immunity
against the disease. To validate the above, three groups of eight mice
each was used to determine the r M-PDCoV capability to produce
antibodies. Two doses were applied to each mouse subcutaneously at day
one and at day 14. The sera were collected through the caudal vein at
days 7, 14, 21, and 28. The antibody response induced in BALB/c mice by
the r M-PDCoV protein was significantly higher (P <
0.001) using immunostimulating complex (Iscom®) (Fig 5). These results
are in agreement with previously reported (Sun et al., 2009), who
described that vaccines enhanced with Iscom® have been shown to be
highly immunogenic, inducing both antibody and cellular immune responses
(Sanders et al., 2005) .
Additionally, in figure 5, the line corresponding to r M-PDCoV
show an increased antibodies (P < 0.01) production along the
weeks (Fig 5) In this context, we found nine antigenic sites along the
consensus amino acid sequence and from these, five were in the
N-terminal region (Fig 2c). Also, the nine antigenic sites lie in
hydrophilic regions ready to be recognized by the immune system. (Fig
2d). Furthermore, it has been reported that viral structural proteins,
like M protein, possess much higher immunogenicity for T cell responses
than the nonstructural proteins (Li et al., 2008). For instance, it has
been observed that the M protein N-terminal region play a role as a
dominant immunogen for cellular immune response (Liu et al., 2010).
Similarly, in alpha, beta, and gamma-coronaviruses, the N-terminal
region has an interferogenic activity to produce monoclonal antibodies
(Baudoux et al., 1998; Laude et al., 1992).
These results indicates that the r M-PDCoV developed in this study
has significant potential to be used as antigen in vaccine or in
immune-detection systems. In this scenario, diagnostic system based on
recombinant proteins has been frequently developed. Specifically, there
has been an interest to develop a recombinant protein (Balamurugan et
al., 2010). For example, several immune assays based on recombinant
proteins (S, M, N) from PEDV have been developed using E. coli as
an expression system (Cao et al., 2013; Pan et al., 2015; Ren et al.,
2011; Shenyang et al., 2007). Also, a recombinant TGEV N protein was
developed with high sensitivity and specificity (Liu et al., 2001).
Additionally, a recombinant S, M, and N protein have been developed to
antibodies detection for PDCoV (Luo et al., 2017; Su et al., 2016).
In Mexico, despite PDCoV in an emergent coronavirus, there is no a
developed recombinant systems to prevent and control de diseases. Also,
in Mexico there is not enough information about the current prevalence
of PDCoV. Moreover, recent findings about PDCoV shows an evolutionary
change and adaptation leading to human infections by coronaviruses
outside of the previously recognized human-associated coronavirus groups
(Lednicky et al., 2021), indicating the important risk that PDCoV
represents.