Bioscience Methods 2024, Vol.15, No.4, 162-172 http://bioscipublisher.com/index.php/bm 1 67 scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163, which also conferred complete resistance to PRRSV infection without impairing the biological functions of the protein (Burkard et al., 2018). 4.2 Strategies for enhancing resistance to bacterial pathogens While the focus on genetic modifications has predominantly been on viral pathogens, there are emerging strategies aimed at enhancing resistance to bacterial infections in pigs. These strategies often involve the manipulation of genes associated with the immune response to bacterial pathogens. For example, the IRF8-miR-10a-SRP14 regulatory pathway has been identified as a critical mechanism by which host cells resist PRRSV infection. This pathway could potentially be targeted to enhance resistance to bacterial pathogens as well (Zheng et al., 2022). 4.3 Case studies and experimental data on pathogen resistance Several case studies and experimental data highlight the effectiveness of genetic modifications in conferring resistance to pathogens in pigs. In one study, pigs with a modified CD163 gene showed a substantial decrease in viral load and relief from PRRSV-induced symptoms, with a significant number of modified pigs surviving the infection compared to wild-type controls (Figure 3) (Chen et al., 2019). Another study demonstrated that CD163 knockout pigs were completely resistant to highly pathogenic PRRSV, showing no signs of infection or viremia (Yang et al., 2018). Additionally, pigs lacking the SRCR5 domain of CD163 were resistant to multiple subtypes of PRRSV, further validating the effectiveness of this genetic modification (Burkard et al., 2018). Moreover, research on the IRF8-miR-10a-SRP14 pathway has provided insights into the molecular mechanisms of host resistance to PRRSV, suggesting potential new antiviral strategies that could be applied to bacterial infections as well (Zheng et al., 2022). These studies collectively underscore the potential of genetic modifications in enhancing the biosafety of transplantable pig organs by conferring resistance to both viral and bacterial pathogens. Chen et al. (2019) investigated the resistance of CD163-mutated porcine alveolar macrophages (PAMs) to infection by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). The study found that CD163-mutated PAMs exhibited significantly reduced susceptibility to viral infection, evidenced by lower viral titers and RNA expression levels. This resistance was consistently observed across different infection doses and time points. Further protein and immunofluorescence analyses revealed a significant reduction in viral protein expression in these mutated cells. The results suggest that the CD163 mutation restricts the replication and spread of HP-PRRSV in PAMs, thereby inhibiting the efficiency of viral infection. These findings provide new insights and potential molecular targets for developing antiviral strategies against PRRSV infection. 5 Comprehensive Biosafety Strategies 5.1 Combining genetic modifications with other biosafety measures Combining genetic modifications with other biosafety measures forms a robust framework for enhancing the safety of transplantable pig organs. Genetic modifications, such as the alteration of the CD163 gene to confer resistance to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), provide a fundamental line of defense against specific pathogens (Wiater et al., 2020). However, the complexity of pathogen dynamics and the risk of new infections necessitate additional biosafety measures. Biosecure facilities play a crucial role by implementing stringent controls that prevent pathogen entry and spread, including controlled access, quarantine protocols, and regular sanitization. Alongside, continuous monitoring through advanced diagnostic tools and routine health checks allows for the early detection and management of infections, thereby complementing the genetic defenses. Integrating vaccination programs with these measures adds another layer of protection, further reducing the risk of infection and enhancing overall biosafety. This comprehensive approach ensures that genetic modifications are supported by a well-rounded system that addresses multiple aspects of pathogen control, ultimately enhancing the safety of pig organs intended for transplantation (Sykes and Sachs, 2019). 5.2 Role of antimicrobial peptides and other genetic enhancements Antimicrobial peptides (AMPs) and other genetic enhancements represent promising advancements in the effort to eliminate porcine pathogens. AMPs, such as porcine β-defensins, have broad-spectrum antimicrobial properties that can effectively target and neutralize various pathogens, including bacteria, viruses, and fungi. By introducing
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