PART Ⅱ: SARS-CoV-2 Infection in The Syrian Hamster Model Causes Inflammation As Well As Type I Interferon Dysregulation in Both Respiratory And Non-respiratory Tissues Including The Heart And Kidney

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Magen Ellen Francis, Una Goncin & et al.

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Abstract

COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection is a disease affecting several organ systems. A model that captures all clinical symptoms of COVID-19, as well as long-hauler disease, is needed. We investigated the host responses associated with infection in several major organ systems including the respiratory tract, the heart, and the kidneys after SARS-CoV-2 infection in Syrian hamsters. We found significant increases in inflammatory cytokines (IL-6, IL-1beta, and TNF) and type II interferons whereas type I interferons were inhibited. Examination of extrapulmonary tissue indicated inflammation in the kidney, liver, and heart which also lacked type I interferon upregulation. Histologically, the heart had evidence of myocarditis and microthrombi while the kidney had tubular inflammation. These results give insight into the multiorgan disease experienced by people with COVID-19 and possibly the prolonged disease in people with post-acute sequelae of SARS-CoV-2 (PASC). coronavirus 2)infection can affect several organ systems including the respiratory tract, urinary tract, neurological system, and cardiovascular system. To appropriately study this disease and evaluate therapeutics, a model that captures all clinical symptoms of COVID-19 is required. It is known that damage that occurs after viral infection is often due to the host's own immune responses. Therefore, here we investigated how the immune host responses are regulated in several major organ systems including the respiratory tract, the heart, and the kidneys after SARS-CoV-2 infection in Syrian hamsters. We found significant increases in inflammatory cytokines(IL-6, IL-1beta, and TNF), as well as antiviral cytokines, called type I interferons. Interestingly the type I interferons of the antiviral response was inhibited in the respiratory tract. Examination of extrapulmonary tissue indicated inflammation in the kidney, liver, and heart which also lacked type I interferon upregulation. Histologically, the heart had evidence of myocarditis and microthrombi while the kidney had tubular inflammation. These results give insight into the multiorgan disease experienced by people with COVID-19 and possibly the prolonged disease in people with post-acute sequelae of SARS-CoV-2(PASC).

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SARS-CoV-2 infection results in significant inflammation or immune regulation in the heart, lymph node, and spleen as well as eosinophil signatures in the large intestine

Immune markers of inflammation, adaptive immunity, and antiviral responses were also assessed in non-respiratory tissues alongside histopathology of the tissues. We investigated the mediastinal lymph node, heart, kidney, spleen, liver, and large intestine for regulation of immune targets categorized as above by type I IFN, type II/General IFN Response, and Innate/ Adaptive Mediators (Fig 6) as well as for immunopathology (Figs 7 and S4). If the initial assessment of histopathology suggested the involvement of specific immune mechanisms (S4 Fig), high-resolution scanning was performed to acquire a greater depth of information (Fig 7). Mediastinal lymph nodes were not able to be recovered on day 15. Corresponding significances for the gene expression can be found in the S2 Table. The type I and type II IFN responses in all the extrapulmonary organs except the liver had similar regulation profiles as those of the lungs and nasal turbinates (Fig 6). Gene regulation was characterized by decreased type I responses and increases in type II responses. STAT2 was significantly decreased in the mediastinal lymph node, kidney, spleen, and large intestine. The heart had significant and sustained decreases in IRF1 while the large intestine had decreases in IFN-beta (Fig 6). Conversely, marked increases in IFN-gamma were observed in the mediastinal lymph node and kidney and to a lower extent in the heart and spleen. CXCL10, the T cell chemoattractant, was increased to high levels in the spleen, liver, and mediastinal lymph node where the regulation was biphasic in the lymph node (Fig 6). Regulation of the innate/adaptive mediator genes in the spleen and lymph node was seen for all immune targets examined with the exception of TGF-beta, IL-5R, and CCR3 which were not effectively regulated. Specifically, significant regulation of CD3, CD4, CD8A, IL-2, IL-21, IL-10, IL-4, IL-5, IL-13, and IL-12 was noted in these secondary immune organs. The regulation of the innate/adaptive immune mediators was particularly interesting in the heart where IL-2, IL-10, IL-4, and IL-5 were increased 100 times over baseline. The highest upregulation of inflammatory cytokines was detected in the heart followed by the kidney, liver, and mediastinal lymph node. In the heart, IL-12 was significantly increased early and, along with TNF, remained elevated at day 15 pi. IL-6 and IL-1beta were also transiently induced in the heart tissue on days 5 and 8 (Fig 6). Significant changes in the heart were observed by histological analysis (S4 Fig). High-resolution imaging found evidence of microthrombi (Fig 7A, white arrows) as well as myocarditis (Fig 7B, white arrows) in the heart. CD14, IL-1beta, and TNF were upregulated early in the kidney but decreased by day 8 to 12 pi (Fig 6) which coincided with infiltration of inflammatory cells into the kidney and possibly acute kidney injury or tubular injury (indicated by the white arrows, Fig 7C). IL-6 expression was sustained in the liver where IL-1beta was also increased at late time points. The mediastinal lymph node had a biphasic increase in IL-12 with transient increases in IL-6 and TNF to fold levels of 12, 8, and 11, respectively. The large intestine had significant regulation of IL-5, IL-5R, and CCR3 with infiltration of eosinophils (Fig 7D), but minimal regulation of any other innate/adaptive mediators. To accompany the analysis of the extra-pulmonary organs, we analyzed the blood chemistry of the animals over the time course to assess organ dysfunction (S5 Fig). We analyzed glucose, albumin, alkaline phosphatase, alanine transferase, blood urea nitrogen, total bilirubin, and total protein as a sign of liver function[27]. Calcium, potassium, albumin, amylase, blood urea nitrogen, and total protein were analyzed to assess the kidney[27]. Gastrointestinal health was evaluated through sodium, potassium, and alkaline phosphatase[27]. Cardiac function was measured using sodium, potassium, and alanine transferase[27]. Globulin upregulation was assessed as a sign of antigenic stimulation[27]. Of the 14 molecules investigated, only amylase and glucose were significantly increased in infected animals when compared to controls, peaking at 1700 U/L and 280 mg/dL, respectively, on day 8 pi. Additionally, potassium concentration ([K+ ]) changes were the most notable and appeared mostly to be time-related over the period of the study as most individuals had an increasing [K+] with the largest change seen at day 15. The increase in albumin was also notable but mild in many of the animals. Total protein concentration increased slightly in most subjects, but the change is relatively mild and likely clinically insignificant. Other liver markers peaked later in the time course, including glucose (280 mg/dL) on day 8, alkaline phosphatase (ALP) on day 12 (3 U/L), and blood urea nitrogen (BUN) (22 mg/dl) on day 15. Alanine transferase (ALT), although peaking on day 2, was high on day 12 at just under 100 U/L suggesting possible liver damage. Kidney markers also peaked later, on day 8 for amylase (1700 U/L), and day 15 for potassium and BUN (12 and 22 mg/dl, respectively). BUN with few exceptions had a possible time-dependent increase in concentration. Calcium, in contrast, peaked on day 2 at 2.6 mg/dl but fell into reference ranges. Markers of gastrointestinal and cardiac health such as sodium, showed minimal regulation, while [K+] increased relatively linearly throughout the infectious time course, peaking at day 15 (12 mg/dl). ALP (gastrointestinal marker) was high on days 5 and 8 at 3u/L and ALT (cardiac marker) was increased in infected hamsters relative to non-infected hamsters on days 2, 8, and 12 at 75–100 U/L but the differences were not significant. Lastly, globulin peaked on day 2 at 0.6 g/dL and decreased relatively linearly over the time course. Taken together, blood analysis indicates some dysregulation in extrapulmonary organs, as well as antigenic stimulation in response to SARS-CoV-2.

Fig 6. SARS-Cov-2 infection in hamsters leads to increases in inflammatory and immune mediator related genes in select extrapulmonary organs possibly indicating myocarditis, adaptive immune evolution, and eosinophil infiltration into the large intestines. qRT-PCR was performed on RNA extracted from mediastinal (M) lymph node, heart, kidney, spleen, liver, and large intestine from SARS-CoV-2 inoculated hamsters. Samples were assessed for type I interferon response-related (IFN-beta, STAT2, IRF1, IRF3, TLR3), type II interferon response-related (IFN-gamma, IRF2, STAT1, CXCL10), and innate/adaptive mediator (CD3, CD4, CD8, CD19, IL-2, TGF-beta, IL-21, IL-10, IL-4, IL-5, IL-5R, IL-13, IL-12, CD14, CCR3, PKR, CCL20, CCL22, IL-1beta, IL-6, TNF) genes leveraging PCR primers specific for hamster sequences (Table 1). Fold-change was calculated via ΔΔCt against baseline (Day 0) with BACT as the housekeeping gene. N is 3 for all timepoints.

Discussion

Originally SARS-CoV-2 infection was thought to mainly cause respiratory disease, but it is now clear that COVID-19 is a disease of many faces affecting several organ systems[25,26]. To gain a better understanding of the origin of disease and the myriad of clinical symptoms experienced in humans, we investigated the immunopathology across the respiratory tract as well as non-respiratory organs during SARS-CoV-2 infection in Syrian hamsters. Infection with 105 TCID50 of SARS-CoV-2 resulted in mild to moderate clinical illness. Infectious virus was mainly contained in the respiratory tract although viral RNA was present in all tissues evaluated. Host response profiling in the respiratory tract identified inhibition of the type I IFN response with a concomitant increase in type II IFNs, inflammatory cytokines, innate and adaptive cytokines. Investigation of the extra-pulmonary organs also found a similar profile for type I and II IFN signaling; however, tissue-specific immune regulation was also identified. Specifically, significant expression of innate and adaptive mediators was identified in the heart and spleen. Since antiviral treatment during SARS-CoV-2 infection is only effective during a short window[28], severe COVID-19 is thought to be a consequence of a dysregulated immune response[29]. Understanding immune pathogenesis is essential for identifying therapeutic targets and treating acute non-respiratory symptoms associated with COVID-19 including heart palpitations or migraines and longer-lasting conditions in the recovery phase such as Post Acute Sequelae of SARS-CoV-2 infection (PASC), also referred to as long-COVID[30].

Fig 7. Select extrapulmonary organs have distinct pathology after SARS-CoV-2 infection with evidence of microthrombi and myocarditis in the heart, tubular damaging in the kidney, and eosinophil infiltration in the large intestine. The heart, kidney, and large intestine were collected from each animal at necropsy and fixed in 10% formalin prior to paraffin block embedding. Tissues were H&E stained. Heart tissues exhibited microthrombi (white arrows) over multiple days (A), as well as myocarditis on day 8 (white arrow) (B). The kidney had acute injury/tubular injury (white arrows) (C). The large intestine had eosinophil infiltration (white arrows) (D). Stained tissues were visualized and imaged using the Aperio ScanScope XT slide scanner. The images shown are representative of 3 animals per group, per time point.

The primary site of infection influencing transmission for SARS-CoV-2 in people is the respiratory tract. Therefore, the initial focus of our investigation was the respiratory tract, including associated immune responses and immunopathology. Here we have demonstrated both upper and lower respiratory infection and associated disease following SARS-CoV-2 infection in Syrian hamsters. Viral infection in the lung led to severe pathology characterized by infiltration of inflammatory cells resulting in bronchopneumonia by day 5 pi and hemorrhage by day 8. These findings were consistent with SARS-CoV-2 infection in prior Syrian hamster studies[19,20,31,32] and larger animal models including rhesus macaques[33] as well as severe pneumonia identified in some human COVID-19 patients[34-36]. The consistency of the data supports the use of the hamster to model SARS-CoV-2 infection human respiratory disease and the associated immunopathology. We also noted that some hamsters experienced larger weight loss which was correlated to higher viral load. Differential weight may have been due to the natural instinct for hamsters to hide food within their cheeks, inefficient inoculation, or that there are inherent host properties that may lead to differential weight loss and viral replication in this animal stock. Since all animals were checked for food storage in their mouths and all animals were positive for the SARS-CoV-2 virus, we do not feel this result is a reflection of either weighing errors or poor inoculation. More work is required to determine if there are genetic differences within Syrian hamsters from Charles River. Understanding the purity of the animal stock will provide important information for the use of these animals in SARS-CoV-2 therapeutic studies and if it is possible to leverage this knowledge for the development of a more severe hamster SARS-CoV-2 model. In our study, we also identified the upregulation of key inflammatory genes, such as IL-6 and TNF in the lungs and nasal turbinates confirming previous immune modeling in hamsters[32,37]. These findings are important as these inflammatory markers have been shown to be predictors of severe disease via assessment of blood in humans[38-42]. More in-depth analysis of the host response to SARS-CoV-2 in the respiratory tissues of hamsters has been done using transcriptome sequencing analysis[43,44]. Hoagland and colleagues assessed the lungs of SARS-CoV-2 infected hamsters for host immune gene regulation and found a prominent upregulation of IL-6, TNF, and IL-1beta[43] consistent with our PCR results. In another study single-cell sequencing analysis revealed a prominent T cell response including upregulation of IFN-gamma and IL-2[44], two markers we also noted to be highly expressed in the lungs. Additionally, our analyses of the lungs revealed upregulation of T cell markers CD3, CD4, and CD8A. Importantly, the qRT-PCR analysis we reported in our present study is in agreement with these other studies thereby validating the use of qRT-PCR as a method to quickly assess the effectiveness of vaccines and therapeutics being evaluated in the hamster SARS-CoV-2 model. Our qRT-PCR pro-files provide targeted biomarkers that can easily be employed. Our study and interpretation were limited by only having the presence of pathogenesis analysis. Future studies should utilize our panel of immune targets for use in vaccine and therapeutic studies.

The antiviral response is essential for the initial control of a virus by active destruction of viral replication and immune stimulation to limit the spread to other cells. After identifying the presence of a virus or viral antigens, the induction of interferons is central to the establishment of an antimicrobial state within cells where the balance between the interferons and other innate cytokines is critical to effective action[45]. Recently, inhibition of type I interferons(IFN-alpha and IFN-beta) has been associated with severe disease in COVID-19 suggesting the virus is able to block antiviral immunity leading to increased pathogenesis [14,46,47].In our study, we found that hamsters infected with SARS-CoV-2 have a prototypical type II IFN response. In contrast, the type IIFN response is downregulated, as evidenced by the decrease in IFN-beta gene expression and increase in the IRF2 gene expression, a type I antagonist. Numerous studies have demonstrated impaired type I IFN responses following SARS-CoV-2 infection in vitro[14,48-51]. The IFN dysregulation we report here has not yet been analyzed in the respiratory tissue of humans with severe COVID-19, but systemic immune analyses of blood have indicated similar results[14,46,47,52]. Although the mechanisms inhibiting type I interferon responses have not been fully elucidated, viral and host factors have been implicated in this dysregulation[53,54].

Dysregulation of the interferon response is a strategy employed by viruses to evade host immunity. Previous investigations of the host response to SARS-CoV(severe acute respiratory syndrome coronavirus 1)and MERS-CoV(Middle East respiratory syndrome coronavirus)infection suggest that multiple coronaviruses employ this strategy[55-59]. The 2020 study by Xia et al. determined this dysregulation utilized by the highly pathogenic coronaviruses extends to SARS-CoV-2 by demonstrating that all three of these beta-coronaviruses downregulate the type I interferon pathway. Specifically, NSP1, NSP6 and NSP13[50] interfere with IFN-beta translation, either by blocking host ribosome in the case of NSP1[60] or targeting upstream proteins necessary for IFN-beta induction in the case of NSP13[61]. Other SARS-CoV-2 virus proteins have also been implicated in interferon downregulation[62]. ORF3b and ORF9b both specifically target a mitochondrial antiviral-signaling protein complex in order to inhibit type I IFN signaling [63,64]. Host factors affecting SARS-CoV-2 infection outcomes include age and sex; furthermore, comorbidities have also been implicated in interferon dysre-gulation[65]. A 2020 study by Trouillet-Assent et al.examined COVID-19 patient outcomes in a cohort including individuals systemically expressing type I IFN and individuals who had significantly lower levels of both IFN-alpha and beta. They found patients lacking type I interferon expression were older, and more likely to be male compared to those who did express type I IFNs[66. In a similar study, significantly higher systemic levels of IFN-alpha2 were identified in females compared to levels in males|67]. In our recent study, we found that older adult male ferrets had a delayed-type I interferon response when compared to adult females, with males lacking induction of essential antiviral genes including OASL, MIX1, and IDG15 which coincided with increased viral burden[68].In our present study using the hamster model, all of our animals were male suggesting that investigation into sex biases is needed to gain a full understanding of our observed type I interferon dysregulation. At this time, it is not known why or how host factors influence interferon responses to SARS-CoV-2. A study by Bastard et al.identified the presence of autoantibodies against type I interferons in males with severe COVID-19[69]. This offers a possible explanation of the immune mechanisms involved in decreased type I interferon responses in a subset of human patients. The mechanism for reduced interferon responses in our present study, where all hamsters were adult male animals, awaits further investigation. Moreover, taken together, the immune profile we have identified together with the respiratory tract pathology suggests hamsters be an ideal model to explore these mechanisms and test therapeutics such as type Iinterferons[48,49,70] or interferon lambda [Z1, Z2], both of which have demonstrated effectiveness in treating COVID-19 patients [Z3-75] but more research is needed.

COVID-19 is a disease that involves the pathogenesis of both respiratory and non-respiratory organs[25,26, however little is understood about the host response outside of the respiratory tract. In our study, we identified vRNA in several tissues outside of the respiratory tract including the mediastinal lymph node, heart, kidney, liver, spleen, and large intestine. Viral RNA presence can be an indicator of live virus replication and tissue infection but may also be present without a live virus. We did not find significant live virus outside of the respiratory tract suggesting minimal if any infection of extrapulmonary organs is occurring. However, the presence of viral RNA may be significant to clinical disease and associated mechanisms. The presence of viral RNA could be the result of a virus in the blood which is transported systemically or viral antigen that was transported by antigen-presenting cells to immunologically important tissues, such as the spleen and mediastinal lymph node. The presence of viral RNA can play a role in immune responses and associated pathogenesis within these tissues. Therefore, we went on to investigate immune-related host responses in the tissues that were positive for viral RNA. Although we found upregulation of inflammatory and type II interferon genes in all non-pulmonary tissue, the most striking response was the increase of immune-modulatory cytokines in the heart. Within the heart, our analysis identified the increase of cytokines including IL-10, IL-2, IL-4, IL-5, IL-5R, IL-12, CD14, and CCR3 where some cytokine mRNA transcripts were increased 100-fold. Although select cytokines here can be produced by different T cell subsets, the absence of TGF-beta suggests the presence of T-regulatory cells is less likely. Additionally, the absence of IL-13 decreases the likelihood of a TH2 response. Apart from T-lymphocytes, the increase in gene expression of IL-10, TNF, and IL-6 could be attributed to monocytes or macrophages[76]. This is further supported by the increase in CD14[76]. The expression of IL-5, IL-5R, IL-4, and CCR3 also suggests the presence of eosinophils[77]. A large number of clinical studies have now detected cardiac involvement in COVID-19[78-84]. In fact, it is estimated that approximately 30% of people who have recovered from COVID-19 have some type of cardiac injury85]. A German study of 100 COVID-19 patients found that myocardial inflammation was common even months after recovery from SARS-CoV-2 infec-tion[84]. Within the tested cohort, 78% of patients experienced cardiac-related symptoms regardless of their degree of COVID-19 severity[84]. Monocytes are among cell types known to infiltrate the heart in myocardial inflammation, producing IL-10, IL-1beta, IL-6, and TNF [86], as well as IL-12[87,88], all of which were upregulated in the heart post-SARS-CoV-2 inoculation in our present study. In addition, another subset of cardiac issues related to COVID-19 has been attributed to eosinophils resulting in eosinophilic myocarditis[89]. Other viruses are known to cause eosinophilic myocarditis after infection[90-93]. This pathology typically results from the virus infecting the tissues of the heart for instance with Coxsackievirus B3 infection[94]. Eosinophilic myocarditis has been diagnosed in COVID-19 patients, whereupon autopsy the heart tissue exhibited infiltration of inflammatory cells including lymphocytes, macrophages, and prominently, eosinophils[89]. In this case, the increase in eosinophils and overall inflammation in the heart was found despite the lack of live virus in the heart at the time of death[95]. We found evidence in our study of eosinophilic myocarditis in the histopathology and also the cytokine signature found in the heart. It is possible in our study that eosinophils lead to inflammatory mediated cardiomyopathy via upregulation of IL-4, which was increased in terms of gene expression in the hearts of hamsters on day8 post-SARS-CoV-2 inoculation. An increase of potassium and slight increases in alanine transferase in the plasma of infected hamsters also may have supported cardiac dysregulation. With this in mind, the mechanisms regulating cardiac injury related to COVID-19 are still not known. Considering the prevalence of cardiac involvement in COVID-19 patients, more research is needed to understand the mechanisms of disease. Here we have provided evidence that hamsters may be useful for understanding the defining mechanisms of cardiac injury during SARS-CoV-2 infection and evaluating therapeutics for this pathology.

Immunologically, our analyses in the spleen and mediastinal lymph nodes suggest the induction of adaptive immunity. The mediastinal lymph node identified a notable biphasic regulation in T cell-related genes, with upregulation in the gene expression of CD3, CD4, IL-2, IL-21, I-10, IL-4, IL-5, and IL-13 on days2 and 15. This profile suggests early antigen presentation within the draining lymph node shortly after lung infection by dendritic cells, which would be supported by the day 2 upregulation of IL-12 production in the lymph node [96,97]. Dendritic cell migration leading toT cell activation eventually culminates in IL-2 stimulating T cell growth and proliferation[98]. Globulin levels in the blood on day 2 post-infection in infected hamsters also support early antigenic stimulation. The upregulation of IL-4, IL-5, and IL-13 suggests a TH2 skewed response[99]. Early studies have demonstrated that asymptomatic or mild COVID-19 cases with an immune skewing towards a systemic TH2 characterized by increases in IL-4 and IL-10[100].IL-10, a regulatory cytokine, was also upregulated on day 2in our study. Our data suggest that activated T cells are recruited to tissues with a live viral infection such as the nasal turbinates, where we observed an increase in T cell-related genes on day 5 post-inoculation. The secondary upregulation of genes in the lymph node on day 15 also suggests an expansion of T cell subsets. In the absence of an infectious viruses at this time, this could be an increase in T cell subsets that directly contribute to the development of a memory response. As central memory T cells also produce IL-2[101] our data showing increased IL-2 on day 15 post-inoculation may be indicative of T cell memory. IL-21 was the most upregulated T cell-related gene on day 15.IL-21 is primarily produced by T-follicular helper cells, a subset that aids germinal center B cells[102], particularly skewing B cells toward affinity maturation and memory development. B Cell marker CD19 was also increased in the lymph node, with peak gene expression occurring on day 15.A 2021 study by Lakshmanappa et al.assessed SARS-CoV-2 infection in rhesus macaques, revealing a robust T cell response in the mediastinal lymph node with specific increases in CD4, as well as T follicular helper cell markers CD28 and CXCR5[103]. It is possible the increased T cell gene expression observed in our study is also indicative of a germinal center response. An elevated systemic T cell response has also been observed in patients with SARS-CoV-2 who recover and develop long-term memory [97,104]. Deeper analysis with increased markers of germinal center reactions for lymph node assessment would be beneficial for vaccine studies.

The elevated lymph node activity could also be indicative of pathogenesis. The lymph node was the only non-respiratory organ where the live virus was detected. Live viral infection of the lymph node, as well as the spleen, has been observed in humans who succumbed to SARS-CoV-2[105].In a study by Kaneko et al., human lymph node and spleen samples from deceased COVID-19 patients lacked BCL6+ B cells as well as entire germinal centers in general. The authors hypothesized this was the cause of poor humoral response to SARS-CoV-2 [106]. Since we identified a robust neutralizing antibody response that was detected by day 8 post-infection which coincided with a decrease in viral shedding, it is more likely that the live virus found in the lymph nodes of our study was indicative of functional B cell responses and germinal center reactions.

The spleen also showed prominent immune responses characterized by increases in gene expression linearly from day 8 to day 15. This late upregulation correlates with typical splenic involvement with the immune response to pathogens which would occur after activation in local draining nodes[107]. The spleen, a lymphoid organ, does also activates T and B cells in response to viral antigen.High gene expression of CD3, CD4, IL-2, and I-21 is suggestive of T-follicular cell as well as germinal center dependant B cell activity, as we saw in the lymph node[108]. Additionally, the response in the spleen may be a consequence of viremia, where plasmacytoid dendritic cells(pDCs) are involved in viral antigen recognition via TLR7 and TLR9 for CD8+ T cell and Natural Killer T(NKT)cell activation. Both of these cell types function in the clearing of virally infected cells after activation in the spleen by IFN-beta and IL-12 [109], which were increased in our model, along with CD8.IL-12, IL-10, and CD14 were upregulated in the spleen of infected hamsters, potentially pointing to macrophage phagocytic activity[110,111]. Also, innate lymphoid cells(ILCs)in the spleen support tissue repair in response to injury and produce IL-5 and IL-13[112]. Taken together, this later response in the spleen points to the recycling function of the spleen, as well as potential lymphocyte activation for long-term memory responses.

The large intestine showed minimal regulation of the assessed cytokines, with the notable exception of I-5, IL-5R, and CCR3. Expression of all three of these genes increased significantly on days 2 through 15 with a peak fold-change of 74 for IL-5, relative to baseline on day 8. IL-5 is predominantly secreted by type 2 T-helper cells and eosinophils[113]. Given the lower gene expression levels of other Th2 related genes, such as IL-4 and IL-13, and the increase in known eosinophil markers IL-5R and CCR3, it is likely IL-5 is being produced primarily by eosinophils which can be seen in our histological analysis of the large intestine. Eosinophils play a direct role in antiviral defense, as evidenced by RNases in their granules and have been associated with the clearance of other respiratory viruses[114,115]. Large numbers of eosinophils are also found in the gastrointestinal tract[116].In our study, the large intestine of infected hamsters did not exhibit live viruses. The large intestine was, however, positive for SARS-CoV-2 viral RNA on days 2 and5post inoculation, a finding consistent with Sia et al., who also observed vRNA in the colon in the absence of an inflammatory response[20]. Up to 20% of COVID-19 patients report gastrointestinal symptoms such as diarrhea[39,117]with viruses being shed in the feces[118]. This could be explained by the decrease in eosinophils that have been reported in cases of severe COVID-19[119-121]. The increase in IL-5 gene expression by eosinophils could contribute to the antiviral state of the large intestine, thereby preventing infection. Further investigation is needed to confirm the exact cell type leading to this heightened IL-5 response.

In our study we also investigated the blood chemistry of SARS-CoV-2 infected Syrian hamsters. We determined a reference range using uninfected hamsters from our animal source as well as compared the readings to published reference ranges for hamsters, specifically Syrian hamsters[122]. Some preanalytical factors that may affect the blood chemistry of our animals prior to infection that must be considered for analysis include the level of stress the animals may be undergoing as well as the isoflurane anesthetic used when blood is being drawn which may alter the blood chemistry. It has been shown that glucose, ALT, ALP, and inorganic phosphorus levels were significantly higher in rats under isoflurane and other anesthetics not indicated here[123].In our study of SARS-CoV-2 infected hamsters, one of the most notable changes after infection included the potassium concentrations which increased over time. Potassium, which is a base, acts as a buffer to exchange for Hydrogen(H')(acid) when a patient is in an acidic state. Since blood acidosis can be caused by changes in the respiratory function it is not surprising that the hamsters which showed signs of pneumonia/respiratory dis-tress (suggesting an increase in Carbon Dioxide(CO2)being retained (or expelled less) and less Oxygen (O2)being taken due to decreased alveolar function)would be entering an acid state leading to potassium increases in the blood. In regard to the lower albumin levels in comparison to our reference range, it would have been expected that albumin would increase in response to inflammatory cytokine expression. It is possible that the lower levels of albumin were due to the use of isoflurane which has been shown previously[122-124]. In respect to the glucose over time, most of the infected animals had an increase in glucose concentration suggesting cortisol-induced stress, epinephrine-induced excitement, or dysregulation in insulin production or secretion/receptor interaction. In summary, the most interesting finding of this analysis was the suggestion of decreased oxygen levels associated with increased potassium levels. These results suggest potassium monitoring may be used to identify pneumonia during the in-life portion of COVID-19 animal studies. Moving forward, this work can also be used to build reference values for the Syrian hamster SARS-CoV-2 infection model and be used for in-life screening of therapeutics.

Our study offers important insights into SARS-CoV-2 infection and COVID-19 disease regulation. Preclinical models are the cornerstone of biomedical research offering the ability to experimentally isolate disease mechanisms and also evaluate potential medical countermeasures including immunomodulatory drugs, antivirals, and vaccines. As SARS-CoV-2 first emerged, we had limited understanding of the mechanisms of pathogenesis, no identified therapeutics or prophylactics, and no preclinical model to leverage for medical countermeasure discovery. Since the time of viral emergence, huge leaps have been made especially in preclinical model development, but our understanding of COVID-19 and its many manifestations is still limited. Our study confirmed the viral tropism and respiratory pathology previously identified in our SARS-COV-2 hamster studies but was also able to extend our understanding of immune regulation and pathogenesis within the respiratory tract and beyond.

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