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.