在一名COVID-19死亡病例中，SARS-CoV-2刺突蛋白的组织趋向性及其功能适应性

《Journal of Virology》：Tissue tropism and functional adaptation of the SARS-CoV-2 spike protein in a fatal case of COVID-19

研究分析了一例严重免疫缺陷患者尸检中27种不同组织中的SARS-CoV-2基因组，发现存在多个基因型，尤其在受体结合域（RBD）有11处突变。蛋白模拟显示这些突变提高了病毒与ACE2受体的结合稳定性和亲和力，部分突变与Delta和Omicron变异株相关。组织聚类分析表明，病毒在免疫相关组织（如肺、心脏）和非免疫相关组织（如脑、眼）中存在分区传播，且变异程度与组织类型相关。

　　SARS-CoV-2, the virus responsible for the global pandemic of COVID-19, has been observed to spread beyond the respiratory system into various extrapulmonary tissues, including the brain, heart, and kidneys. These observations have raised critical questions about the virus's ability to adapt to different tissue environments and its potential to evolve new mutations that could influence its persistence, transmission, and pathogenicity. This study presents a detailed analysis of SARS-CoV-2 genetic diversity across 27 tissue sites from an autopsy case of a patient with a primary immune deficiency. The findings suggest that such individuals may serve as a unique environment for the virus to diversify and evolve, potentially leading to the emergence of new variants with altered functional properties.

The patient, a 57-year-old male, had a history of chronic granulomatous disease (CGD), a condition that impairs the immune system's ability to fight off certain infections. He had undergone a hematopoietic stem cell transplant (HSCT) in January 2021 and was receiving daily immunosuppressive therapy with sirolimus, which further weakened his immune response. His initial symptoms of fever, lethargy, shortness of breath, and cough were reported on 29 September 2021, approximately 7 days after his first dose of a SARS-CoV-2 vaccine. He was treated with monoclonal antibodies and remdesivir, and while his symptoms initially improved, he was readmitted in late October with recurrent respiratory symptoms and a negative SARS-CoV-2 test. He eventually died from respiratory failure in early November 2021. The study was conducted using tissues collected during an autopsy on 2 December 2021, which provided a unique opportunity to examine the genetic diversity of the virus across multiple organ systems.

The analysis of the collected tissues revealed that SARS-CoV-2 RNA was present in 61 of the 73 tissue sites examined, with a wide range of viral loads. These findings highlight the potential for the virus to persist in non-respiratory tissues, even after the resolution of acute symptoms. A key aspect of the study was the identification of multiple SARS-CoV-2 genotypes across different tissues, with some mutations observed in only specific locations. This tissue-specific genetic diversity suggests that the virus may undergo distinct evolutionary trajectories in different parts of the body, potentially due to differences in the local microenvironment, immune response, and cellular interactions.

One of the most notable findings was the presence of mutations primarily in the receptor-binding domain (RBD) of the spike protein, which is a critical region for viral entry into host cells. These mutations, such as K417R, G476S, S477N, and Q493K, were found to be associated with increased structural stability and stronger binding to the ACE2 receptor. The spike protein's RBD is known to interact directly with ACE2, and mutations in this region can significantly affect the virus's ability to bind and enter cells. The study used protein simulations and binding assays to evaluate the functional impact of these mutations. These simulations indicated that the presence of these mutations could enhance the virus's ability to persist in the host and potentially evade immune responses, which is a crucial factor in the prolonged infection and the development of long-term symptoms, such as those seen in long-COVID.

The patient's condition of immunosuppression is believed to have contributed to the high genetic diversity of SARS-CoV-2 observed in this study. Patients with weakened immune responses may not effectively clear the virus, allowing it to replicate and evolve within various tissue compartments. This is supported by the observation that the patient's tissues contained a significant number of minor variants, which were not typically found in other individuals. The study also noted that certain mutations, such as G446V and Y453F, are associated with reduced neutralization by monoclonal antibodies, which may have implications for the effectiveness of current therapies.

In addition to identifying mutations in the spike protein, the study also examined the broader genetic landscape of the virus across the genome. The findings revealed that while the majority of mutations were nonsynonymous, some were synonymous, meaning they did not change the amino acid sequence of the virus. However, even synonymous mutations can have functional implications, especially when they occur in regions that are structurally important for viral stability. The analysis of the viral genome suggested that the observed mutations may have played a role in the virus's ability to adapt to the host's immune environment and persist in non-respiratory tissues.

The study further explored the possibility of viral recombination, which could lead to the emergence of new variants. By isolating infectious virus from different tissue sites, the researchers were able to demonstrate that multiple genotypes can coexist within the same tissue, potentially increasing the likelihood of recombination events. This is particularly relevant in the context of immunocompromised individuals, who may have prolonged viral replication and a higher chance of harboring diverse viral populations. The presence of these multiple genotypes may have implications for the transmission of the virus to new hosts, as it could lead to the emergence of more virulent or transmissible variants.

The findings of this study also have implications for the understanding of long-COVID, a condition where individuals continue to experience symptoms for weeks or even months after the initial infection. The persistence of SARS-CoV-2 in non-respiratory tissues, such as the brain and gastrointestinal tract, may contribute to the ongoing symptoms observed in some patients. The study's results suggest that the virus may not be completely cleared from the body, even in the absence of detectable viral loads in the respiratory tract, and that this persistence could be due to the virus's ability to adapt to different tissue environments.

The analysis of the viral genome also highlighted the importance of considering the role of tissue-specific factors in viral evolution. The study found that different tissues exhibited varying levels of genetic diversity, with some tissues showing a higher degree of mutation accumulation than others. This variation may be due to differences in the expression of ACE2 and TMPRSS2, the primary receptors and proteases involved in viral entry, as well as the presence of immune-privileged sites that may provide a more permissive environment for viral replication. The presence of immune-privileged sites, such as the brain and eyes, is particularly interesting, as these tissues are generally considered to be less accessible to the immune system.

The study also examined the impact of different viral strains on the host's immune response. The patient's tissues contained a variety of SARS-CoV-2 genotypes, including those associated with the Omicron BA.1 lineage and Delta variants. The presence of these variants in the patient's tissues, despite the fact that the majority of circulating SARS-CoV-2 was of the Omicron lineage, suggests that the virus may have undergone significant adaptation to the host's immune environment. The researchers also noted that the patient's immune status, characterized by a suppressed adaptive immune response, may have allowed for the accumulation of multiple viral variants, some of which may have conferred an advantage in terms of viral replication and persistence.

In terms of functional implications, the study found that certain spike mutations, such as Q493K, could significantly impact the virus's ability to bind to the ACE2 receptor. This mutation, which changes a polar amino acid to a positively charged one, may enhance the virus's binding affinity and contribute to its persistence in the host. The study also demonstrated that the presence of these mutations could influence the virus's ability to infect different cell types, as evidenced by the differences in binding and internalization efficiencies observed in the experimental assays.

The findings of this study underscore the importance of understanding the genetic diversity of SARS-CoV-2 in immunocompromised individuals. These patients may represent a unique population in which the virus can evolve and persist, potentially leading to the emergence of new variants that could have implications for public health. The study's results also highlight the need for more comprehensive surveillance of SARS-CoV-2 in non-respiratory tissues, as these sites may serve as reservoirs for the virus.

In conclusion, this study provides valuable insights into the genetic and functional diversity of SARS-CoV-2 in an immunocompromised individual. The findings suggest that the virus can persist in non-respiratory tissues and evolve new mutations that may enhance its ability to replicate and transmit. The study's results also highlight the importance of considering the role of tissue-specific factors in viral evolution and the potential impact of immune suppression on the virus's ability to diversify. These findings have important implications for the development of more effective therapies and the prevention of long-term complications associated with SARS-CoV-2 infections.