Host genetic diversity contributes to disease outcome in Crimean-Congo hemorrhagic fever virus infection

Host genetic diversity contributes to disease outcome in Crimean-Congo hemorrhagic fever virus infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Host genetic diversity contributes to disease outcome in Crimean-Congo hemorrhagic fever virus infection Deepashri Rao, Matthew Lewis, Kimberly Meade-White, Carl Shaia, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4644919/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever (CCHF), a widely distributed disease with significant morbidity and mortality. The virus has high genetic diversity correlated with geographic distribution, but limited temporal evolution within regions. Despite this, cases of CCHF within a region present as a spectrum of disease from often unrecognized asymptomatic infections to a severe, fatal viral hemorrhagic fever, suggesting host factors may play a role in disease outcome. We investigated the effect of host genetic diversity on the outcome of CCHFV infection in the genetically diverse Collaborative Cross mouse model. Infected mice recapitulated the full spectrum of disease recognized in humans and similar to human disease, virus replication, tissue pathology and inflammatory responses were associated with disease severity. Our study demonstrates that host genetics contribute to disease outcome in CCHF infection and establishes the Collaborative Cross mouse resource as a model to understand how host genetic diversity contributes to CCHF outcome. Biological sciences/Microbiology/Virology/Virus host interactions Biological sciences/Microbiology/Virology/Viral pathogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Crimean-Congo hemorrhagic fever (CCHF) is a severe, febrile illness that can progress to a viral hemorrhagic fever (VHF) in humans. The causative agent is the Crimean-Congo hemorrhagic fever virus (CCHFV), a tickborne RNA virus with a widespread geographic distribution 1,2 . Domestic and wild animals serve as amplifying hosts, while humans are incidental hosts. Exposure to the virus in humans primarily occurs through tick bites, as well as during handling of livestock during practices such as farming and butchering, which may lead to contact with infected blood 1,2 . Due to climate change, there is a possibility of an expansion in the geographic range of the tick vector 3 , resulting in a higher risk of more human exposure to the virus. Case fatality rate is variable but can be higher than 30% in endemic regions 4 . Interestingly, although CCHFV can productively infect a wide range of domestic and wild animal species, with studies showing seroconversion in rabbits, cattle and even tortoises 5,6 symptomatic disease has only been reported in humans. Additionally, within humans, infection with CCHFV results in a spectrum of disease, from asymptomatic infection, to severe, sometimes lethal disease 7,8 . The virus is also genetically diverse, with diversity correlating with geography 9,10 . However, within a geographical region, there is often minimal diversity of the virus, with studies showing strong sequence conservation in strains of CCHFV isolated from the same regions decades apart 11,12 . It is therefore likely that within a region, people are infected with similar strains of virus and yet, exhibit different outcomes. A number of factors including viral determinants, virus dose, route of exposure, host immune responses, and access to healthcare resources are likely to contribute to disease outcome and case fatality rates. Several studies have demonstrated the association of polymorphisms in certain innate signaling genes to CCHF disease severity in humans, suggesting that host genetics may also contribute to disease outcome 13–18 . However, the effect of genetic diversity on CCHF disease is poorly understood and study of innate immunity to CCHFV limited by the historical requirement that mice be deficient in type I interferon to develop symptomatic disease. We recently developed a mouse-adapted strain of CCHFV (MA-CCHFV) that infects and results in symptomatic disease in immunocompetent mice 19 . With this model, we also identified sex-linked differences in disease severity, with male mice developing more severe disease than female mice. These sex-linked differences were maintained across several strains of mice tested, including C57BL/6J, C57BL/6NCR, and BALB/c/J mice. Interestingly, both male and female 129S1 mice were largely resistant to disease 19 . These findings suggested that with the MA-CCHFV virus, there exist sex and host strain-specific differences in disease outcome. Since these conventional inbred strains represent only a subset of mouse genetic diversity, here we explored the effect of host genetics on disease progression after infection with the MA-CCHFV using the genetically diverse Collaborative Cross (CC) mouse 20–24 , a genetic reference population. Infection of male and female mice of ten CC strains resulted in a wide spectrum of disease outcomes, from asymptomatic infection to lethal disease. Disease severity in CC mice showed similar correlates to disease severity in human CCHF cases such as viral replication, liver pathology and inflammatory cytokine production. Our findings demonstrate that host genetics contribute to disease outcome in MA-CCHFV infection of mice and establish the CC mouse resource for continued study of how host responses contribute to CCHF disease outcome. Methods Biosafety and Ethics All infectious work with CCHFV was performed in the biosafety level 4 (BSL4) maximum containment laboratory at the Integrated Research Facility, Rocky Mountain Laboratories (RML), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) according to standard operating procedures (SOPs) approved by the RML institutional Biosafety Committee (IBC). All animal studies were approved by the Rocky Mountain Laboratories Institutional Animal Care and Use Committee (IACUC) and performed according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International and the Office of Laboratory Animal Welfare by trained and experienced personnel. Humane endpoint criteria in compliance with IACUC-approved scoring parameters were used to determine when animals should be humanely euthanized. Mice Male and female CC mice, 7–15 weeks of age and bred at the University of North Carolina at Chapel Hill’s Systems Genetics Core Facility, purchased between January and October 2023, were used in the study. Mice from ten different CC strains (CC003/Unc, CC046/Unc, CC080/TauUnc, CC004/TauUnc, CC037/TauUnc, CC002/Unc, CC051/TauUnc, CC037/TauUnc, CC030/GeniUnc, CC042/GeniUnc and CC012/GeniUnc, hereafter referred to without suffixes) were included. Mice were acclimatized to ABSL4 conditions prior to the start of experiments, and were provided with food, water and nesting material ad libitum . Cage changes were performed by animal caretakers every 14 days. Mice were randomly assigned to groups. Only the histology team was blinded to the study. For procedures requiring anesthesia, animals were anesthetized using isoflurane. Following infection, mice were weighed daily up to day 14 or euthanasia timepoint and surviving mouse weights were recorded every 3 days until study endpoint. For survival studies, anesthetized mice were implanted with telemetry transponders (UCT-2112, UID) via subcutaneous implantation to enable monitoring of body temperature and activity levels. Implanted mice were allowed to recover for at least one week prior to CCHFV challenge. Data were recorded continuously with a zone interval of 250 ms, 2 cycles per series and a 1s series delay. Data are reported as mean of readings collected during 6-h intervals corresponding to vivarium light–dark cycles. Following survival studies, the disease was further characterized in a second set of mice of 6 CC strains. Infected mice were euthanized at peak disease (corresponding with highest weight loss in survival studies) for virological, immunological and histopathology analyses. Mice were euthanized using exsanguination under deep anesthesia followed by cervical dislocation or isoflurane overdose followed by cervical dislocation. Virus stock and infections The stock of MA-CCHFV used here is same as described previously 25 . Anesthetized mice were inoculated intraperitoneally with 10 4 TCID 50 of MA-CCHFV in 100µL of Dulbecco’s Modified Eagle Medium (DMEM) or mock infected with DMEM only. qRT-PCR Viral RNA copies in the blood, liver and spleen were quantified by qRT-PCR as previously described 19 . Limit of detection (LoD) of the assay was determined by extrapolating the standard curve to the copy number given by a Ct (crossing of threshold) value of 40. Samples without any amplification were set at the LoD. The last standard to amplify was set as the limit of quantification (LoQ) of the assay. Cytokine analysis The levels of cytokines in the sera were analyzed by the Bio-Plex Pro Mouse Cytokine 23-plex Assay (Bio-Rad Cat #M60009RDPD) according to manufacturer’s instructions. Enzyme-linked immunosorbent assay (ELISA) IgG responses to CCHFV in the sera were evaluated by an in-house ELISA as previously described 26 . Absorbance of negative samples were used to determine limits of detection. Histology and immunohistochemistry Liver and spleen tissues were harvested from mice at peak disease for each strain. Tissues were fixed in 10% Neutral Buffered Formalin x2 changes, for a minimum of 7 days. Fixed tissues were placed in cassettes and processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded series of ethanol, xylene, and PureAffin. Embedded tissues were sectioned at 5µm and dried overnight at 42°C prior to staining. Specific anti-CCHFV immunoreactivity was detected using Rabbit anti-CCHFV N IBT (Bioservices, cat#04–0011) at a 1:2000 dilution. The secondary antibody is the Immpress-VR horse anti-rabbit IgG polymer kit (Vector Laboratories, cat#MP-6401). The tissues were then processed for immunohistochemistry using the Discovery Ultra automated stainer (Ventana Medical Systems) with a ChromoMap DAB kit (Roche Tissue Diagnostics cat#760 − 159). Statistics Statistics was performed on GraphPad Prism 10 using recommended tests. Sample sizes were determined based on previous experience with the MA-CCHFV model, and mice were assigned to groups in a random manner. For survival studies, 5 male and 5 five female mice of each strain were analyzed. However, one female mouse each of strains CC037, CC004 and CC012 did not recover from anesthesia and were excluded from analyses, resulting in N = 4 for these groups for survival studies. One male mouse of strain CC012 was euthanized for non-study related reasons and was also excluded from analyses, resulting in N = 4 for this group for survival studies. For timed necropsy studies, 6 mice per sex per strain were included for MA-CCHFV-infected groups, and 2 mice per sex per strain were included for mock-infected groups. Results Infection with MA-CCHFV results in a spectrum of clinical manifestations in CC mice Since infection with MA-CCHFV results in disease in strains of immunocompetent conventional inbred WT mice with minimal genetic diversity 19 , we hypothesized that infection of the genetically diverse CC mice would result in a range of clinical presentations across these genotypes. Male and female mice of 10 CC strains were infected with MA-CCHFV and monitored for the development of clinical signs. Infection of male CC mice resulted in a range of disease outcomes, from severe, lethal disease to moderate disease and mild and asymptomatic infections, as indicated by weight loss, fluctuations in body temperature, reduced activity levels and reduced survival (Fig. 1 a -d). The infection was uniformly lethal in male mice of two strains, CC003 and CC046, both of which rapidly lost weight and all succumbed to the disease at 5- and 6-days post-infection (dpi) respectively (Fig. 1 a and b). Both strains showed hyperthermia (Fig. 1 c) and reduced activity levels (Fig. 1 d) beginning early after infection followed by hypothermia before succumbing to disease. Male mice of strains CC080 and CC004 showed partial lethality, with 60% mortality in CC080 mice by 6 dpi (3/5 mice succumbed) and 40% in CC004 mice, with mice succumbing on 6–7 dpi (2/5 mice succumbed) (Fig. 1 b). Both strains developed hyperthermia followed by hypothermia corresponding to peak weight loss, along with a decline in activity levels, before reaching euthanasia criteria or recovering (Fig. 1 a, c and d). Strains CC037, CC002 and CC051 showed moderate disease in male mice with a peak weight loss of approximately 10% of their starting body weight and 100% survival (Fig. 1 a and b). Strain CC037 also displayed a biphasic disease pattern, with peak weight loss at day 5, and subsequently gaining weight until day 14, when body weight again dropped slightly before complete recovery (Fig. 1 b). All three strains showed slight elevations in body temperatures and slight reduction in activity levels coinciding with peak weight loss (Fig. 1 c and d). Male mice of strain CC030 displayed mild disease with 10% body weight loss rapidly following infection on day 1, before quickly recovering the lost weight by day 3 and all survived (Fig. 1 a and b). There were no changes in body temperature or activity levels of these mice throughout the course of infection. Two strains, CC042 and CC012 did not show any weight loss, changes in body temperature or otherwise overt clinical disease. Strain CC012 showed mildly reduced activity levels during the first week of infection, but subsequently returned to normal activity levels (Fig. 1 d). Female mice infected with MA-CCHFV also displayed a spectrum ranging from asymptomatic to moderate disease (Fig. 1 e). However, lethal disease was not observed in female mice of any of the 10 strains tested, (Fig. 1 f) suggesting sex-linked differences observed in inbred laboratory strains of mice remain in outbred CC mice. Female mice of strain CC004 showed moderate disease with 20% weight loss at peak disease on day 5. Coinciding with peak weight loss, mice exhibited a decline in body temperature and activity levels before recovery. A majority of the strains tested (7/10) only developed mild disease in female mice. Female mice of strains CC003 and CC046, in which the infection was lethal in male mice, only lost ~ 10% of their body weight at peak disease. Female CC046 exhibited hyper followed by hypothermia (Fig. 1 g and h) and there was a prolonged recovery with mice not returning to starting weight until 21 dpi (Fig. 1 e). However, by temperature and activity, return to baseline was observed by 14 dpi. The remaining 5 strains, CC037, CC030, CC051, CC080 and CC002 only lost 5–8% body weight, and all rapidly recovered from disease. Slightly elevated body temperature early after infection, followed by a drop at peak disease was observed in all strains except CC030, which had stable body temperature and activity. The same two strains that did not show any clinical symptoms of disease in males, CC042 and CC012, were also resistant to disease in females, with little-to-no weight loss or changes in body temperature or activity levels observed. Since the MA-CCHFV strain displays sex-linked differences in disease severity, with male mice developing higher weight loss than female mice in several strains tested 19 , we also compared weight loss between male and female mice of each CC line that showed weight loss (8/10 strains) (Supplementary Fig. 1). The same pattern of higher weight loss in male mice than female mice was observed in 6 of the 8 strains: CC037 (Supplementary Fig. 1a), CC046 (Supplementary Fig. 1b), CC051 (Supplementary Fig. 1c), CC080 (Supplementary Fig. 1d), CC002 (Supplementary Fig. 1e), and CC003 (Supplementary Fig. 1f). The weight loss pattern was similar in male and female mice of strain CC030, both of which had mild weight loss very early post-infection before complete recovery (Supplementary Fig. 1h). Interestingly, CC004 was the only strain in which female mice presented with higher weight loss than male mice (Supplementary Fig. 1g), despite the partial lethality observed in male mice (Fig. 1 b) and 100% survival in female mice (Fig. 1 f). Together, these findings demonstrate that the CC mice recapitulate the full spectrum of CCHF disease observed in humans and that the sex-bias in disease severity with MA-CCHFV infection of in-bred laboratory strains of mice such as C57BL6/J 19 was largely maintained in all CC strains tested. The notable exception was the strain CC004 mice which showed a distinct sex-linked phenotype with greater weight loss in female mice but greater lethality in male mice. MA-CCHFV replicates in all CC strains tested Next, we performed a more in-depth characterization of CCHFV infection in a subset of these strains of CC mice to understand the association of the different disease outcomes with virology, pathology and inflammation. Based on the survival studies, we selected 6 strains for further characterization of CCHFV infection: CC003 and CC046, in which the infection was lethal in male mice, and displayed mild to moderate disease in female mice, CC004 in which partial lethality was observed in male mice but female mice had higher weight loss, CC037 in which both male and female mice had mild disease, and strains CC012 and CC042, in which both male and female mice did not show any clinical signs of disease. We first investigated viral replication as disease severity in humans typically correlates with viral loads and we also hypothesized that resistant CC strains may be resistant to infection. Mice were infected with MA-CCHFV on day 0 and euthanized at peak disease corresponding with highest weight loss for each sex of each strain in the survival study. Since the MA-CCHFV strain replicates to high titers in several organs including the blood, liver and spleen of WT mice 19 , we first examined viral RNA loads in the blood and tissues of infected CC mice strains, to see if they would support infection similar to WT mice. Analysis of viral RNA loads showed that both male (Fig. 2 a) and female (Fig. 2 d) mice of all 6 CC strains tested had detectable viremia at peak disease. Mice of all 6 strains also had viral RNA in the livers (Fig. 2 b and e) and spleens (Fig. 2 c and f). Of particular interest were strains CC012 and CC042, which despite showing no clinical signs of disease, still supported virus replication in the blood, liver, and spleen. We confirmed these results by analyzing serum IgG responses to CCHFV in these two strains at 28 dpi. CCHFV-specific IgG was observed in both sexes of CC012 and CC042 (Supplemental Fig. 2), confirming productive infection of both strains. Overall, these results suggest that resistance to disease upon infection with CCHFV as observed in strains CC012 and CC042, is not due to an inherent inability of these two strains to be infected. Instead, MA-CCHFV replicated in all CC mouse strains tested. MA-CCHFV causes liver pathology associated with disease severity in CC mice Similar to CCHFV infection of humans, the liver is the primary target of MA-CCHFV, which causes significant pathology upon infection of WT mice 19 . Next, we examined histological evidence of liver pathology in the 6 CC strains infected with MA-CCHFV. Pathological findings indicated that male mice of strain CC003 had more necrosis than female mice in the liver (Fig. 3 , Supplemental Fig. 4a & e) and spleen (Supplemental Fig. 3, Supplemental Fig. 4c & g) at peak disease. Consistent with the higher viral loads in the livers and spleens, male mice of strain CC003 also had more CCHFV-specific immunoreactivity than females in the livers (Fig. 3 Supplemental Fig. 4b & f) and spleens (Supplemental Fig. 3, Supplemental Fig. 4d & h). Male mice of strain CC046 also had liver necrosis at peak disease, with immunoreactivity to CCHFV mainly observed in the necrotic foci (Fig. 3 ). Livers of CC046 females displayed high inflammatory foci, which were also the focus of CCHFV-specific immunoreactivity (Fig. 3 ). Spleens of male mice showed occasional immunoreactivity; whereas, almost no immunoreactivity was observed in spleens of female mice (Supplemental Fig. 3, Supplemental Fig. 4d & h). Both male and female mice of strain CC004 had similar lesions in the liver (Fig. 3 , Supplemental Fig. 4a & e). The higher weight loss in female CC004 mice was associated with high CCHFV antigen distribution throughout the liver and the spleen at peak disease. In agreement with the mild disease in both male and female mice of strain CC037, there were only small inflammatory foci in the livers of both sexes. Consistent with the higher weight loss in male mice, there was a slight increase in overall anti-CCHFV immunoreactivity in the livers of the males over the females (Fig. 3 ), but no difference was noted in the spleens (Supplemental Fig. 3). The two strains that were resistant to clinical disease, strains CC042 and CC012, did not demonstrate any major histologic lesions in the liver (Fig. 3 ) or spleen (Supplemental Fig. 3). Mild (CC012) or no (CC042) pathology and occasional (CC012) or extremely rare (CC042) CCHFV-specific immunoreactivity consistent with reduced viral loads was noted in the livers (Fig. 3 ) and spleens (Supplemental Fig. 3) of male and female mice of these two strains. Collectively, these findings demonstrate the MA-CCHFV infection of CC mice results in pathology of the liver and spleen that correlates with disease severity across genetic backgrounds. MA-CCHFV induces an inflammatory cytokine response associated with disease severity in CC mice Disease severity in humans and MA-CCHFV infection of WT mice correlates with systemic production of multiple inflammatory cytokines 19,27–29 . Therefore, we evaluated serum cytokine response in male and female mice of the 6 CC strains that were necropsied at peak disease. Compared to mock infected mice, the 100% lethal disease in male mice of strains CC003 and CC046 was associated with significantly higher levels of multiple cytokines including Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-4, IL-6, IL-9. IL-10, IL-12(p40), IL-13, CXCL1, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα (Supplemental Figs. 5 and 6). Notably, the inflammatory cytokines G-CSF, CXCL1, MCP-1, MIP-1a, MIP-1b and RANTES were increased several fold in the sera at peak disease (day 5 for CC003, day 4 for CC046) compared to mock-infected mice (Fig. 4 , Supplemental Figs. 5 and 6). Female mice of both strains did not have significant elevations in most of the cytokines tested, except IL-5 in CC046, correlating with the mild disease observed in them (Supplemental Figs. 5 and 6). Interestingly, strain CC004, in which the infection was lethal in ~ 40% of male mice, did not have increases in any cytokines over mock mice (Fig. 4 ), and their levels were similar to mock infected mice at 7 dpi at peak disease (Supplemental Fig. 7). In contrast, the higher weight loss in female mice of this strain was associated with significantly higher levels of several inflammatory cytokines in the serum at peak disease (day 5 post infection) compared to mocks (Supplemental Fig. 7). Similarly, the levels of multiple cytokines were significantly higher compared to mock or infected female mice in male mice of strain CC037, correlating with the higher weight loss observed (Supplemental Fig. 8). In contrast, mild disease in female CC037 mice was associated with no changes in the levels of several cytokines compared to mocks (Supplemental Fig. 8). Corresponding to the overall mild course of infection in this strain, the levels of some of these cytokines were decreased in both male and female CC037 mice including IL-3, IL-17A and TNFα (Fig. 4 , Supplemental Fig. 8) and there was no change in the levels of cytokines Eotaxin, IFNγ, IL-10, IL-12(p40), CXCL1 and RANTES in both male and female CC037 mice (Fig. 4 ). However, male and female CC037 mice did have an increase in MCP-1 and MIP-1α, although this was significant only in male mice compared to mocks and female mice. In agreement with no overt clinical disease throughout the course of the infection, male mice of strain CC012 did not have any changes in cytokine levels compared to mock infected mice (Supplemental Fig. 9). On the other hand, the slight decrease in body weight at 2 dpi in female mice was correlated with significantly higher levels of a number of inflammatory cytokines compared to mock and infected male mice (Supplemental Fig. 9). Finally, in agreement with no overt clinical disease throughout the course of the experiment, both male and female mice of strain CC042 had similar levels of cytokines as that observed in mock mice (Supplemental Fig. 10). Overall, mild disease was associated with a significant decrease of IL-3, IL-9, IL-12(p70), IL-17A and TNFα; whereas absence of disease was associated with little change in cytokines. In contrast lethal disease is associated with increased inflammatory cytokines such as Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-4, IL-6, IL-9, IL-10, IL-12(p40), IL-13, CXCL1, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα, several of which have been associated with fatal human cases of CCHF. Taken together, these results suggest that similar to human cases of CCHF, disease severity in CC mice correlates with inflammatory cytokine production. Discussion For many VHFs, host immunopathology contributes significantly to morbidity and mortality but for CCHF it remains largely unclear how host factors contribute to disease outcome. Like other VHFs, CCHFV has multiple hosts, but upon infection, causes disease primarily in humans. In infected humans, a wide range of clinical manifestations are observed, even within regions with minimal virus diversity. This leads to speculation that there could be a strong role for host genetics in determining disease presentation. Although results from some studies indicate that genetic variation in humans may determine severity of CCHF disease 13–18 , this has not been characterized in depth nor modeled in genetically diverse model systems. Thus a mechanistic understanding of the contribution of host genetics to CCHF disease outcome is lacking. This report sheds light on the role of host genetic diversity in CCHFV pathogenesis and disease outcome. We observed the full spectrum of CCHF disease by screening 10 CC strains infected with MA-CCHFV, demonstrating a clear association of host genetic diversity with disease severity as well as the recapitulation of several aspects of each disease phenotype that is observed in humans. Our previous studies with MA-CCHFV infection in several conventional inbred laboratory mouse strains revealed that except for strain 129S1, all strains developed overt clinical disease and the sex-linked bias in disease severity was maintained 19 . In the present study, MA-CCHFV infection of male and female mice of ten CC strains resulted in a range of disease phenotypes, with consistently more severe disease in male than female mice. Sex differences in disease presentations have been described in the CC for other infectious diseases 30,31 . We identified two strains in which the infection in male mice was lethal and which exhibited correlates similar to fatal human cases of CCHF. High viremia is a predictor of fatal disease in humans, with viral titers higher than 10 9 genome copies per milliliter of plasma being indicative of lethal disease 32–34 . Interestingly, recapitulating what is observed in humans, high viremia of approximately 10 10 genome copies per milliliter of serum was observed in most of the mice with lethal disease in our study. Moreover, lethal CCHF disease in male CC mice was also associated with severe involvement of the liver with significant necrosis, also in agreement with lethal human cases. Finally, production of inflammatory cytokines, a hallmark feature of fatal disease in humans 27,28 , was also observed in the two CC strains with lethal disease in male mice. Notably, we observed an increase in MCP-1 and TNFα in both strains that exhibited lethal outcome, in agreement with what has been reported in fatal human CCHF cases 27,28 . We have previously shown that low dose (10 4 TCID 50 ) MA-CCHFV challenge of C57BL6/J mice resulted in elevated MCP-1 but not TNFα 19 ; whereas, high-dose, lethal MA-CCHFV challenge resulted in significant MCP-1 and TNFα production later in infection 25 . These data suggest that in addition to host determinants there could be an impact due to inoculating dose, and that disease outcome correlates with inflammatory cytokine production. Thus, lethal CCHFV infection in two CC mice strains has several correlates also measured in lethal disease in humans suggesting similar disease mechanisms. These strains represent a useful model for identifying host genes that could potentially direct the course of CCHFV infection or more specifically disentangle the relationships between inflammatory cytokines, uncontrolled replication, and resultant disease. Polymorphisms in several key factors involved in the innate immune response to CCHFV including NF-κB, TLRs and IFNα have been shown to be associated with fatal outcome in CCHF cases in humans 13–17 . It will be interesting to examine these genes in our CC lines to see if the same alleles observed in humans associate with lethality in CC strains. The CC mice have proven valuable in the study of other viruses including Ebola in CC and CC recombinant inbred intercrossed (CC-RIX) mice using a mouse-adapted strain of Ebola virus (MA-EBOV) 21,22,35 . More recently, strains CC051 (which had mild disease in both males and females infected with MA-CCHFV) and CC004 (which had partial lethality in the males and moderate disease in the females) were investigated for susceptibility to MA-EBOV infection 22 . Infection of CC051 showed mild to moderate disease, and CC004 was highly susceptible to EBOV infection and showed uniform mortality. Thus, MA-EBOV infection in these two strains was more severe than MA-CCHFV infection, suggesting that genes involved in the susceptibility to diverse VHF viruses may be distinct. The MA-CCHFV strain displays a sex-linked bias in disease severity, with more severe disease in male than female WT mice across a number of mouse genotypes 19 . At a higher dose of the virus, male but not female mice all succumb to the disease 25 . This male versus female phenotype was also maintained in six out of the eight CC strains that showed disease suggesting the mechanism is conserved in the genetically diverse CC model. However, we identified one strain, CC004 in which female mice showed higher weight loss, increased viral loads and inflammatory cytokines at peak disease (day 5) than male mice at peak disease (day 7). In CC003 and CC046 mice, in which males showed uniform lethality, female mice developed only mild disease demonstrating stark sex-linked differences in disease outcome. This finding suggests a role for host genetics in driving sex differences in the immune response to MA-CCHFV infection. Sex differences in immunity have been described for a number of pathogens in both humans and mice, with genetic mediators including sex chromosomes, microRNAs and genetic polymorphisms all shown to play roles in sex-differential effects on immunity 36 . For example, infection with coxsackievirus B3 (CVB3) results in sexual dimorphism in C57BL/6 WT mice, with increased mortality and severe cardiac inflammation in males compared to females 37 . Gonadectomy leads to a flipping of these phenotypes, with reduced CVB3 pathogenicity in male mice and increased pathogenicity in females 37 . Studies have also reported higher CCHFV infection of human males 38–41 , although this may be explained by cultural practices in those regions, where activities such as farming and butchering are more likely carried out by men 42–44 . It is therefore unclear whether a sex-linked bias in human CCHF disease exists. Nevertheless, our results provide an opportunity to investigate host genes associated with sex-linked determinants of outcome of viral infections, an understudied area in the context of viral hemorrhagic fevers. A majority of the CC strains screened presented with mild to moderate disease, and two strains were resistant to clinical disease in both male and female mice. This is consistent with human infections, a majority of which are likely subclinical and underappreciated 45,46 . Our data indicate that asymptomatic or subclinical infections were not due to resistance to infection but instead likely due to host responses that resulted in rapid control of the infection with minimal immunopathology. This data is consistent with serological evidence indicating productive infection of numerous animal species in the absence of clinical disease 5 , suggesting that although CCHFV can infect numerous animal species, unique determinants within the human host enables symptomatic disease. Interestingly, mild disease in male and female CC037 and female CC003 mice was associated with decreased expression of several cytokines such as IL-3, IL-9, IL-12p70, IL-17A and TNFα. In contrast, the absence of clinical disease despite viremia and seroconversion in male and female CC012 and CC042 was associated with largely no cytokine response to the infection. These data suggest that there exist distinct cytokine responses and likely overall host immune responses to the infection in asymptomatic, mild, and severe CCHFV infections. We were also able to identify more subtle disease phenotypes, such as mice with mild weight loss but prolonged recovery (CC051 males) versus mice with rapid severe weight loss but rapid recovery (CC080 males). These models may be useful for studying the long-term sequelae that may follow recovery from acute CCHF, an area in need of further research. The presence of replicating virus and inflammatory cytokines in all ten infected CC lines irrespective of disease severity leads to an important area to explore- host factors limiting disease in mild and resistant strains. Our study has several important limitations. First, the significant difference in disease between MA-CCHFV infected male and female mice has not been reported in human CCHFV infections. Although several studies have reported men are more likely to develop CCHFV or be seropositive for infection 38–41 , this can also be attributed to cultural practices in which men are more likely to be exposed to ticks or livestock 44 . Epidemiological studies that account for these confounding variables are necessary to determine if there are sex-linked differences in disease outcomes in CCHFV-infected humans. Nevertheless, the similar correlates of disease severity in MA-CCHFV infected mice and infected humans along with distinct disease outcomes in a genetically variable population infected with the same strain of virus provides a powerful tool to investigate how host genetics contribute to CCHF outcome. Second, the CCHFV strain used is mouse-adapted and was generated by serial passage in mice on the C57BL6/J background. WT C57/BL6J mice are resistant to disease upon CCHFV infection 19 and therefore we did not test exposure of the CC strains to WT virus. Thus, it is possible that adaptation of MA-CCHFV to C57BL6/J mice may link to host-genes not linked to disease in humans. Notably, using a mouse-adapted EBOV and the CC mice, Price et al. identified key cell types that were associated with tolerance of Ebola virus disease (EVD), and through transcriptomics, generated gene expression signature profiles that were used to successfully predict clinical outcome in patients with EVD in Africa 35 . Further, human quantitative trait loci (QTL) and key genes identified in severe disease for viruses such as Influenza and SARS-CoV-2 also mapped as key genes that drive disease severity in the CC platform 31,47,48 demonstrating the utility of the CC platform in understanding viral pathogenesis. These studies provide encouraging evidence that gene signatures identified in disease phenotypes in CC mice can be accurately linked to disease outcome in humans using mouse-adapted viruses. Third, CCHFV is a genetically diverse virus and case fatality rates can vary by region 46,49–51 . MA-CCHFV is based on CCHFV strain Hoti, isolated from a fatal human case in Eastern Europe. Therefore, we were unable to model the contribution of viral diversity to disease outcome and it is possible that distinct strains of CCHFV may interact with distinct host factors to cause disease, and this concern suggests further studies in mouse strains with discordant disease outcomes. Fourth, we did not perform paired timed necropsies on each strain at all timepoints and thus differences observed in pathology, cytokines or viral loads between sexes and strains may be due to different timepoints analyzed. In this report we intended to establish the MA-CCHFV infection of CC mice as a model to investigate host contributions to disease outcome. Future studies will perform more in-depth temporal analyses of these factors at multiple timepoints. In conclusion, using just ten CC lines, we were able to recapitulate the full spectrum of CCHF manifestations, providing strong evidence that the host genetic background is critical in determining the fate of MA-CCHFV infected mice. Our study demonstrates the utility of the CC model for examining host responses associated with the range of disease phenotypes observed in CCHF infection. Similar correlates of disease severity measured in human CCHF cases such as viral loads, tissue pathology and inflammatory cytokines were also measured in MA-CCHFV infected CC mice. Identifying host genes that can be linked to these distinct phenotypes is the focus of ongoing studies. Using a systems biology approach, the CC can be used to assess the interaction of genetic diversity and host responses leading to disease. These studies will allow for a more thorough understanding of CCHFV pathogenesis and host responses, eventually leading to the identification of novel therapeutic interventions for CCHF. Declarations Competing interests The authors declare that they have no competing interests. Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Funding: This work was supported by the Intramural Research Program of the NIAID, NIH. Author Contribution D.R., D.H., H.F., A.S., M.T. F., and R.B. conceptualized the study. A.S., M.T. F., R.B., and D.H. designed the methodology of the study. D.R., M.L., K.M-W., C.S., A.O., and D.H. performed all the investigations. D.R. analyzed data from all the analyses except histopathology, which was analyzed by C.R. M.F., A.S., R.B., and H.F. provided the resources and D.H. and H.F. supervised the studies. H.F. acquired funding for the study. D.R., D.H., and H.F. wrote the original draft of the manuscript. D.R., M.L., K.M-W., C.S., A.O., A.S., M.T. F., R.B., H.F. and D.H. all reviewed and edited the final manuscript. Acknowledgement Authors wish to acknowledge the support provided by the Rocky Mountain Veterinary Branch, animal caretakers and the BSL-4 facilities staff for the ABSL-4 and BSL-4 studies. We also wish to acknowledge the UNC SGCF, which maintains and distributes the CC lines. Data Availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. References Hawman, D. W. & Feldmann, H. Crimean-Congo haemorrhagic fever virus. Nat Rev Microbiol 21 , 463-477 (2023). https://doi.org/10.1038/s41579-023-00871-9 Hawman, D. W. & Feldmann, H. Recent advances in understanding Crimean-Congo hemorrhagic fever virus. F1000Res 7 (2018). https://doi.org/10.12688/f1000research.16189.1 Grandi, G. et al. 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Am J Trop Med Hyg 57 , 519-525 (1997). https://doi.org/10.4269/ajtmh.1997.57.519 Khurshid, A. et al. CCHF virus variants in Pakistan and Afghanistan: Emerging diversity and epidemiology. J Clin Virol 67 , 25-30 (2015). https://doi.org/10.1016/j.jcv.2015.03.021 Additional Declarations No competing interests reported. Supplementary Files RaoEtAlCCmouseSupplementaryInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Aug, 2024 Reviews received at journal 26 Aug, 2024 Reviews received at journal 12 Aug, 2024 Reviewers agreed at journal 21 Jul, 2024 Reviewers agreed at journal 19 Jul, 2024 Reviewers invited by journal 19 Jul, 2024 Editor assigned by journal 18 Jul, 2024 Submission checks completed at journal 17 Jul, 2024 First submitted to journal 26 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4644919","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":338245075,"identity":"58ae502e-d12b-4092-ab3e-d47d8285b0d4","order_by":0,"name":"Deepashri Rao","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Deepashri","middleName":"","lastName":"Rao","suffix":""},{"id":338245076,"identity":"f5a9d4ee-20e5-494d-9452-123263209ff8","order_by":1,"name":"Matthew Lewis","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Lewis","suffix":""},{"id":338245077,"identity":"3e7ea1dd-8374-47aa-bb54-f9f403449137","order_by":2,"name":"Kimberly Meade-White","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Kimberly","middleName":"","lastName":"Meade-White","suffix":""},{"id":338245078,"identity":"12a38294-8119-425d-8d10-62eaff4f7c2f","order_by":3,"name":"Carl Shaia","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Carl","middleName":"","lastName":"Shaia","suffix":""},{"id":338245079,"identity":"7f9d8d5e-584f-432b-bda3-c031f09327a1","order_by":4,"name":"Atsushi Okumura","email":"","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Okumura","suffix":""},{"id":338245080,"identity":"e3f6a2dd-5bfe-4960-92a1-d6cf3b201388","order_by":5,"name":"Martin T. 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Hawman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYDCCA0DEAyJB4AODRAJpWhhnEKuFAaaFmYeBgbAWvuNnDA+8Ybgjb3Dt8LPHtjss8hj4Fx+TwKdF8kyOwcE5DM8MN9xOMzfOPSNRzCDxLA2vFoMDaQmHeRgOM267nWAmndsmkdggccbYAK+W88/AWuy33U7/Jm1JlJYbyQdAWhK33c4xk2YEaeHvMXyA1y83Hh84OMfgcPL+2zllkr1ALW0SbIl4tfCdT2z+8KbisO3M2enbJH621SX28x8+cACfFqjzkNhsxMQmGuAnwo5RMApGwSgYUQAA8mhTzh4hOLUAAAAASUVORK5CYII=","orcid":"","institution":"Rocky Mountain Laboratories, NIAID/NIH","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"W.","lastName":"Hawman","suffix":""}],"badges":[],"createdAt":"2024-06-26 21:36:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4644919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4644919/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62656074,"identity":"03ae645c-0c73-4628-80c9-5171190122bd","added_by":"auto","created_at":"2024-08-17 01:57:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInfection of CC mice with MA-CCHFV results in a spectrum of disease. \u003c/strong\u003eMale (a – d) or female (e – h) mice of ten CC strains were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of MA-CCHFV intraperitoneally and monitored for clinical signs and symptoms of disease. N= 5 males per group for all strains except CC012, for which N=4 males. N= 5 females per group for all strains except CC0037, CC004 and CC012, for which N= 4 females. Mice were weighed daily, (a \u0026amp; e), monitored sur survival (b \u0026amp; f) and body temperature (c \u0026amp; g) and activity (d \u0026amp; h) were measured by the cage telemetry system. Data shown as mean plus SEM (a \u0026amp; e). For telemetry data (c, d, g, h) data points were smoothed using 4 neighbors to each side and a 2\u003csup\u003end\u003c/sup\u003e order polynomial. Cages were changed on days -1 and 14 relative to infection, indicated by DC (d \u0026amp; h).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4644919/v1/efebe3901976f0fcf460b4b6.jpg"},{"id":62656871,"identity":"e4ed11ce-09e6-4021-9304-a72c42e8d404","added_by":"auto","created_at":"2024-08-17 02:05:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82681,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisease progression in CC mice following MA-CCHFV infection is associated with virus replication. \u003c/strong\u003eGroups of male (a -c) or female (d – f) mice of 6 CC strains were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of MA-CCHFV intraperitoneally and euthanized at peak disease for each sex of each strain. N= 6 mice per sex per strain. Viral loads in the indicated tissues were evaluated by qRT-PCR. Data presented as mean plus standard deviation.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4644919/v1/9aaa239dc92ed9cacec3f24d.jpg"},{"id":62656076,"identity":"e6eacc22-d325-4a5e-a5cd-0cbf212d53de","added_by":"auto","created_at":"2024-08-17 01:57:00","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":166383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCC mice infected with MA-CCHFV develop liver pathology correlating with disease severity. \u003c/strong\u003eGroups of male and female mice of 6 CC strains were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of MA-CCHFV via the intraperitoneal route or mock-infected. N= 6 mice per sex per strain for CCHFV-infected groups. N= 2 mice per sex per strain for mock-infected groups, except strain CC037, for which N= 1 male and 1 female mouse for mock-infected group. At peak disease, mice were euthanized, and tissues were fixed in formalin and paraffin embedded sections stained with H and E or an antibody against the CCHFV NP to identify viral antigen (IHC). Representative images of liver are shown. Images shown at 200 magnification and scale bar indicates 100 mm. Study performed once.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4644919/v1/1bee51c0f1dd5c64ec8e7f33.jpg"},{"id":62656078,"identity":"72884215-c770-4bb3-ba82-6f84e5969110","added_by":"auto","created_at":"2024-08-17 01:57:00","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":86495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAn inflammatory immune response associated with disease severity develops in MA-CCHFV infected CC mice. \u003c/strong\u003eGroups of male and female CC mice were infected with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50 \u003c/sub\u003eof MA-CCHFV via the intraperitoneal route or mock-infected. N= 6 mice per sex per strain for CCHFV-infected groups. N= 2 mice per sex per strain for mock-infected groups, except strain CC037, for which N= 1 male and 1 female mouse for mock-infected group. At peak disease, cytokine levels in the sera were measured by 23-plex cytokine assay. Data shown as fold change in the levels of each cytokine in the MA-CCHFV infected mice over mocks. Study performed once. Statistics were calculated with a one-way ANOVA with Tukey’s multiple comparison test. Asterisks indicate significantly higher (in black), or lower (in white) levels of cytokines in male and female MA-CCHFV infected mice compared to mocks- *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4644919/v1/903f6b15afeebcba5ca3e705.jpg"},{"id":62656872,"identity":"cce89b09-05b6-42aa-b84b-6e6cefe95ed5","added_by":"auto","created_at":"2024-08-17 02:05:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1123699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4644919/v1/c4add937-211b-4d50-8e5b-502164401ff0.pdf"},{"id":62656077,"identity":"609464e3-4136-4e37-ad27-9f883ff69d7b","added_by":"auto","created_at":"2024-08-17 01:57:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4330780,"visible":true,"origin":"","legend":"","description":"","filename":"RaoEtAlCCmouseSupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4644919/v1/af6363b2b70fae23ea54c0d8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Host genetic diversity contributes to disease outcome in Crimean-Congo hemorrhagic fever virus infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCrimean-Congo hemorrhagic fever (CCHF) is a severe, febrile illness that can progress to a viral hemorrhagic fever (VHF) in humans. The causative agent is the Crimean-Congo hemorrhagic fever virus (CCHFV), a tickborne RNA virus with a widespread geographic distribution \u003csup\u003e1,2\u003c/sup\u003e. Domestic and wild animals serve as amplifying hosts, while humans are incidental hosts. Exposure to the virus in humans primarily occurs through tick bites, as well as during handling of livestock during practices such as farming and butchering, which may lead to contact with infected blood \u003csup\u003e1,2\u003c/sup\u003e. Due to climate change, there is a possibility of an expansion in the geographic range of the tick vector \u003csup\u003e3\u003c/sup\u003e, resulting in a higher risk of more human exposure to the virus. Case fatality rate is variable but can be higher than 30% in endemic regions \u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, although CCHFV can productively infect a wide range of domestic and wild animal species, with studies showing seroconversion in rabbits, cattle and even tortoises \u003csup\u003e5,6\u003c/sup\u003e symptomatic disease has only been reported in humans. Additionally, within humans, infection with CCHFV results in a spectrum of disease, from asymptomatic infection, to severe, sometimes lethal disease \u003csup\u003e7,8\u003c/sup\u003e. The virus is also genetically diverse, with diversity correlating with geography \u003csup\u003e9,10\u003c/sup\u003e. However, within a geographical region, there is often minimal diversity of the virus, with studies showing strong sequence conservation in strains of CCHFV isolated from the same regions decades apart \u003csup\u003e11,12\u003c/sup\u003e. It is therefore likely that within a region, people are infected with similar strains of virus and yet, exhibit different outcomes. A number of factors including viral determinants, virus dose, route of exposure, host immune responses, and access to healthcare resources are likely to contribute to disease outcome and case fatality rates. Several studies have demonstrated the association of polymorphisms in certain innate signaling genes to CCHF disease severity in humans, suggesting that host genetics may also contribute to disease outcome \u003csup\u003e13\u0026ndash;18\u003c/sup\u003e. However, the effect of genetic diversity on CCHF disease is poorly understood and study of innate immunity to CCHFV limited by the historical requirement that mice be deficient in type I interferon to develop symptomatic disease.\u003c/p\u003e \u003cp\u003eWe recently developed a mouse-adapted strain of CCHFV (MA-CCHFV) that infects and results in symptomatic disease in immunocompetent mice \u003csup\u003e19\u003c/sup\u003e. With this model, we also identified sex-linked differences in disease severity, with male mice developing more severe disease than female mice. These sex-linked differences were maintained across several strains of mice tested, including C57BL/6J, C57BL/6NCR, and BALB/c/J mice. Interestingly, both male and female 129S1 mice were largely resistant to disease \u003csup\u003e19\u003c/sup\u003e. These findings suggested that with the MA-CCHFV virus, there exist sex and host strain-specific differences in disease outcome.\u003c/p\u003e \u003cp\u003eSince these conventional inbred strains represent only a subset of mouse genetic diversity, here we explored the effect of host genetics on disease progression after infection with the MA-CCHFV using the genetically diverse Collaborative Cross (CC) mouse \u003csup\u003e20\u0026ndash;24\u003c/sup\u003e, a genetic reference population. Infection of male and female mice of ten CC strains resulted in a wide spectrum of disease outcomes, from asymptomatic infection to lethal disease. Disease severity in CC mice showed similar correlates to disease severity in human CCHF cases such as viral replication, liver pathology and inflammatory cytokine production. Our findings demonstrate that host genetics contribute to disease outcome in MA-CCHFV infection of mice and establish the CC mouse resource for continued study of how host responses contribute to CCHF disease outcome.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBiosafety and Ethics\u003c/h2\u003e \u003cp\u003e All infectious work with CCHFV was performed in the biosafety level 4 (BSL4) maximum containment laboratory at the Integrated Research Facility, Rocky Mountain Laboratories (RML), Division of Intramural Research (DIR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) according to standard operating procedures (SOPs) approved by the RML institutional Biosafety Committee (IBC). All animal studies were approved by the Rocky Mountain Laboratories Institutional Animal Care and Use Committee (IACUC) and performed according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International and the Office of Laboratory Animal Welfare by trained and experienced personnel. Humane endpoint criteria in compliance with IACUC-approved scoring parameters were used to determine when animals should be humanely euthanized.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eMale and female CC mice, 7\u0026ndash;15 weeks of age and bred at the University of North Carolina at Chapel Hill\u0026rsquo;s Systems Genetics Core Facility, purchased between January and October 2023, were used in the study. Mice from ten different CC strains (CC003/Unc, CC046/Unc, CC080/TauUnc, CC004/TauUnc, CC037/TauUnc, CC002/Unc, CC051/TauUnc, CC037/TauUnc, CC030/GeniUnc, CC042/GeniUnc and CC012/GeniUnc, hereafter referred to without suffixes) were included. Mice were acclimatized to ABSL4 conditions prior to the start of experiments, and were provided with food, water and nesting material \u003cem\u003ead libitum\u003c/em\u003e. Cage changes were performed by animal caretakers every 14 days. Mice were randomly assigned to groups. Only the histology team was blinded to the study. For procedures requiring anesthesia, animals were anesthetized using isoflurane. Following infection, mice were weighed daily up to day 14 or euthanasia timepoint and surviving mouse weights were recorded every 3 days until study endpoint. For survival studies, anesthetized mice were implanted with telemetry transponders (UCT-2112, UID) via subcutaneous implantation to enable monitoring of body temperature and activity levels. Implanted mice were allowed to recover for at least one week prior to CCHFV challenge. Data were recorded continuously with a zone interval of 250 ms, 2 cycles per series and a 1s series delay. Data are reported as mean of readings collected during 6-h intervals corresponding to vivarium light\u0026ndash;dark cycles. Following survival studies, the disease was further characterized in a second set of mice of 6 CC strains. Infected mice were euthanized at peak disease (corresponding with highest weight loss in survival studies) for virological, immunological and histopathology analyses. Mice were euthanized using exsanguination under deep anesthesia followed by cervical dislocation or isoflurane overdose followed by cervical dislocation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eVirus stock and infections\u003c/h2\u003e \u003cp\u003eThe stock of MA-CCHFV used here is same as described previously \u003csup\u003e25\u003c/sup\u003e. Anesthetized mice were inoculated intraperitoneally with 10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of MA-CCHFV in 100\u0026micro;L of Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) or mock infected with DMEM only.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eqRT-PCR\u003c/h3\u003e\n\u003cp\u003eViral RNA copies in the blood, liver and spleen were quantified by qRT-PCR as previously described \u003csup\u003e19\u003c/sup\u003e. Limit of detection (LoD) of the assay was determined by extrapolating the standard curve to the copy number given by a Ct (crossing of threshold) value of 40. Samples without any amplification were set at the LoD. The last standard to amplify was set as the limit of quantification (LoQ) of the assay.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCytokine analysis\u003c/h2\u003e \u003cp\u003e The levels of cytokines in the sera were analyzed by the Bio-Plex Pro Mouse Cytokine 23-plex Assay (Bio-Rad Cat #M60009RDPD) according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eIgG responses to CCHFV in the sera were evaluated by an in-house ELISA as previously described \u003csup\u003e26\u003c/sup\u003e. Absorbance of negative samples were used to determine limits of detection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eLiver and spleen tissues were harvested from mice at peak disease for each strain. Tissues were fixed in 10% Neutral Buffered Formalin x2 changes, for a minimum of 7 days. Fixed tissues were placed in cassettes and processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded series of ethanol, xylene, and PureAffin. Embedded tissues were sectioned at 5\u0026micro;m and dried overnight at 42\u0026deg;C prior to staining. Specific anti-CCHFV immunoreactivity was detected using Rabbit anti-CCHFV N IBT (Bioservices, cat#04\u0026ndash;0011) at a 1:2000 dilution. The secondary antibody is the Immpress-VR horse anti-rabbit IgG polymer kit (Vector Laboratories, cat#MP-6401). The tissues were then processed for immunohistochemistry using the Discovery Ultra automated stainer (Ventana Medical Systems) with a ChromoMap DAB kit (Roche Tissue Diagnostics cat#760\u0026thinsp;\u0026minus;\u0026thinsp;159).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistics was performed on GraphPad Prism 10 using recommended tests. Sample sizes were determined based on previous experience with the MA-CCHFV model, and mice were assigned to groups in a random manner. For survival studies, 5 male and 5 five female mice of each strain were analyzed. However, one female mouse each of strains CC037, CC004 and CC012 did not recover from anesthesia and were excluded from analyses, resulting in N\u0026thinsp;=\u0026thinsp;4 for these groups for survival studies. One male mouse of strain CC012 was euthanized for non-study related reasons and was also excluded from analyses, resulting in N\u0026thinsp;=\u0026thinsp;4 for this group for survival studies. For timed necropsy studies, 6 mice per sex per strain were included for MA-CCHFV-infected groups, and 2 mice per sex per strain were included for mock-infected groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInfection with MA-CCHFV results in a spectrum of clinical manifestations in CC mice\u003c/h2\u003e \u003cp\u003eSince infection with MA-CCHFV results in disease in strains of immunocompetent conventional inbred WT mice with minimal genetic diversity \u003csup\u003e19\u003c/sup\u003e, we hypothesized that infection of the genetically diverse CC mice would result in a range of clinical presentations across these genotypes. Male and female mice of 10 CC strains were infected with MA-CCHFV and monitored for the development of clinical signs. Infection of male CC mice resulted in a range of disease outcomes, from severe, lethal disease to moderate disease and mild and asymptomatic infections, as indicated by weight loss, fluctuations in body temperature, reduced activity levels and reduced survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea -d). The infection was uniformly lethal in male mice of two strains, CC003 and CC046, both of which rapidly lost weight and all succumbed to the disease at 5- and 6-days post-infection (dpi) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). Both strains showed hyperthermia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) and reduced activity levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) beginning early after infection followed by hypothermia before succumbing to disease. Male mice of strains CC080 and CC004 showed partial lethality, with 60% mortality in CC080 mice by 6 dpi (3/5 mice succumbed) and 40% in CC004 mice, with mice succumbing on 6\u0026ndash;7 dpi (2/5 mice succumbed) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Both strains developed hyperthermia followed by hypothermia corresponding to peak weight loss, along with a decline in activity levels, before reaching euthanasia criteria or recovering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, c and d). Strains CC037, CC002 and CC051 showed moderate disease in male mice with a peak weight loss of approximately 10% of their starting body weight and 100% survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). Strain CC037 also displayed a biphasic disease pattern, with peak weight loss at day 5, and subsequently gaining weight until day 14, when body weight again dropped slightly before complete recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). All three strains showed slight elevations in body temperatures and slight reduction in activity levels coinciding with peak weight loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d). Male mice of strain CC030 displayed mild disease with 10% body weight loss rapidly following infection on day 1, before quickly recovering the lost weight by day 3 and all survived (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). There were no changes in body temperature or activity levels of these mice throughout the course of infection. Two strains, CC042 and CC012 did not show any weight loss, changes in body temperature or otherwise overt clinical disease. Strain CC012 showed mildly reduced activity levels during the first week of infection, but subsequently returned to normal activity levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFemale mice infected with MA-CCHFV also displayed a spectrum ranging from asymptomatic to moderate disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). However, lethal disease was not observed in female mice of any of the 10 strains tested, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) suggesting sex-linked differences observed in inbred laboratory strains of mice remain in outbred CC mice. Female mice of strain CC004 showed moderate disease with 20% weight loss at peak disease on day 5. Coinciding with peak weight loss, mice exhibited a decline in body temperature and activity levels before recovery. A majority of the strains tested (7/10) only developed mild disease in female mice. Female mice of strains CC003 and CC046, in which the infection was lethal in male mice, only lost\u0026thinsp;~\u0026thinsp;10% of their body weight at peak disease. Female CC046 exhibited hyper followed by hypothermia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg and h) and there was a prolonged recovery with mice not returning to starting weight until 21 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). However, by temperature and activity, return to baseline was observed by 14 dpi. The remaining 5 strains, CC037, CC030, CC051, CC080 and CC002 only lost 5\u0026ndash;8% body weight, and all rapidly recovered from disease. Slightly elevated body temperature early after infection, followed by a drop at peak disease was observed in all strains except CC030, which had stable body temperature and activity. The same two strains that did not show any clinical symptoms of disease in males, CC042 and CC012, were also resistant to disease in females, with little-to-no weight loss or changes in body temperature or activity levels observed.\u003c/p\u003e \u003cp\u003eSince the MA-CCHFV strain displays sex-linked differences in disease severity, with male mice developing higher weight loss than female mice in several strains tested \u003csup\u003e19\u003c/sup\u003e, we also compared weight loss between male and female mice of each CC line that showed weight loss (8/10 strains) (Supplementary Fig.\u0026nbsp;1). The same pattern of higher weight loss in male mice than female mice was observed in 6 of the 8 strains: CC037 (Supplementary Fig.\u0026nbsp;1a), CC046 (Supplementary Fig.\u0026nbsp;1b), CC051 (Supplementary Fig.\u0026nbsp;1c), CC080 (Supplementary Fig.\u0026nbsp;1d), CC002 (Supplementary Fig.\u0026nbsp;1e), and CC003 (Supplementary Fig.\u0026nbsp;1f). The weight loss pattern was similar in male and female mice of strain CC030, both of which had mild weight loss very early post-infection before complete recovery (Supplementary Fig.\u0026nbsp;1h). Interestingly, CC004 was the only strain in which female mice presented with higher weight loss than male mice (Supplementary Fig.\u0026nbsp;1g), despite the partial lethality observed in male mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) and 100% survival in female mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eTogether, these findings demonstrate that the CC mice recapitulate the full spectrum of CCHF disease observed in humans and that the sex-bias in disease severity with MA-CCHFV infection of in-bred laboratory strains of mice such as C57BL6/J \u003csup\u003e19\u003c/sup\u003e was largely maintained in all CC strains tested. The notable exception was the strain CC004 mice which showed a distinct sex-linked phenotype with greater weight loss in female mice but greater lethality in male mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMA-CCHFV replicates in all CC strains tested\u003c/h2\u003e \u003cp\u003eNext, we performed a more in-depth characterization of CCHFV infection in a subset of these strains of CC mice to understand the association of the different disease outcomes with virology, pathology and inflammation. Based on the survival studies, we selected 6 strains for further characterization of CCHFV infection: CC003 and CC046, in which the infection was lethal in male mice, and displayed mild to moderate disease in female mice, CC004 in which partial lethality was observed in male mice but female mice had higher weight loss, CC037 in which both male and female mice had mild disease, and strains CC012 and CC042, in which both male and female mice did not show any clinical signs of disease.\u003c/p\u003e \u003cp\u003eWe first investigated viral replication as disease severity in humans typically correlates with viral loads and we also hypothesized that resistant CC strains may be resistant to infection. Mice were infected with MA-CCHFV on day 0 and euthanized at peak disease corresponding with highest weight loss for each sex of each strain in the survival study. Since the MA-CCHFV strain replicates to high titers in several organs including the blood, liver and spleen of WT mice \u003csup\u003e19\u003c/sup\u003e, we first examined viral RNA loads in the blood and tissues of infected CC mice strains, to see if they would support infection similar to WT mice. Analysis of viral RNA loads showed that both male (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and female (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) mice of all 6 CC strains tested had detectable viremia at peak disease. Mice of all 6 strains also had viral RNA in the livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and e) and spleens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and f). Of particular interest were strains CC012 and CC042, which despite showing no clinical signs of disease, still supported virus replication in the blood, liver, and spleen. We confirmed these results by analyzing serum IgG responses to CCHFV in these two strains at 28 dpi. CCHFV-specific IgG was observed in both sexes of CC012 and CC042 (Supplemental Fig.\u0026nbsp;2), confirming productive infection of both strains. Overall, these results suggest that resistance to disease upon infection with CCHFV as observed in strains CC012 and CC042, is not due to an inherent inability of these two strains to be infected. Instead, MA-CCHFV replicated in all CC mouse strains tested.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMA-CCHFV causes liver pathology associated with disease severity in CC mice\u003c/h2\u003e \u003cp\u003eSimilar to CCHFV infection of humans, the liver is the primary target of MA-CCHFV, which causes significant pathology upon infection of WT mice \u003csup\u003e19\u003c/sup\u003e. Next, we examined histological evidence of liver pathology in the 6 CC strains infected with MA-CCHFV. Pathological findings indicated that male mice of strain CC003 had more necrosis than female mice in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplemental Fig.\u0026nbsp;4a \u0026amp; e) and spleen (Supplemental Fig.\u0026nbsp;3, Supplemental Fig.\u0026nbsp;4c \u0026amp; g) at peak disease. Consistent with the higher viral loads in the livers and spleens, male mice of strain CC003 also had more CCHFV-specific immunoreactivity than females in the livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e Supplemental Fig.\u0026nbsp;4b \u0026amp; f) and spleens (Supplemental Fig.\u0026nbsp;3, Supplemental Fig.\u0026nbsp;4d \u0026amp; h). Male mice of strain CC046 also had liver necrosis at peak disease, with immunoreactivity to CCHFV mainly observed in the necrotic foci (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Livers of CC046 females displayed high inflammatory foci, which were also the focus of CCHFV-specific immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Spleens of male mice showed occasional immunoreactivity; whereas, almost no immunoreactivity was observed in spleens of female mice (Supplemental Fig.\u0026nbsp;3, Supplemental Fig.\u0026nbsp;4d \u0026amp; h). Both male and female mice of strain CC004 had similar lesions in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplemental Fig.\u0026nbsp;4a \u0026amp; e). The higher weight loss in female CC004 mice was associated with high CCHFV antigen distribution throughout the liver and the spleen at peak disease. In agreement with the mild disease in both male and female mice of strain CC037, there were only small inflammatory foci in the livers of both sexes. Consistent with the higher weight loss in male mice, there was a slight increase in overall anti-CCHFV immunoreactivity in the livers of the males over the females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), but no difference was noted in the spleens (Supplemental Fig.\u0026nbsp;3). The two strains that were resistant to clinical disease, strains CC042 and CC012, did not demonstrate any major histologic lesions in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) or spleen (Supplemental Fig.\u0026nbsp;3). Mild (CC012) or no (CC042) pathology and occasional (CC012) or extremely rare (CC042) CCHFV-specific immunoreactivity consistent with reduced viral loads was noted in the livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and spleens (Supplemental Fig.\u0026nbsp;3) of male and female mice of these two strains. Collectively, these findings demonstrate the MA-CCHFV infection of CC mice results in pathology of the liver and spleen that correlates with disease severity across genetic backgrounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMA-CCHFV induces an inflammatory cytokine response associated with disease severity in CC mice\u003c/h2\u003e \u003cp\u003eDisease severity in humans and MA-CCHFV infection of WT mice correlates with systemic production of multiple inflammatory cytokines \u003csup\u003e19,27\u0026ndash;29\u003c/sup\u003e. Therefore, we evaluated serum cytokine response in male and female mice of the 6 CC strains that were necropsied at peak disease. Compared to mock infected mice, the 100% lethal disease in male mice of strains CC003 and CC046 was associated with significantly higher levels of multiple cytokines including Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-4, IL-6, IL-9. IL-10, IL-12(p40), IL-13, CXCL1, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα (Supplemental Figs.\u0026nbsp;5 and 6). Notably, the inflammatory cytokines G-CSF, CXCL1, MCP-1, MIP-1a, MIP-1b and RANTES were increased several fold in the sera at peak disease (day 5 for CC003, day 4 for CC046) compared to mock-infected mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplemental Figs.\u0026nbsp;5 and 6). Female mice of both strains did not have significant elevations in most of the cytokines tested, except IL-5 in CC046, correlating with the mild disease observed in them (Supplemental Figs.\u0026nbsp;5 and 6). Interestingly, strain CC004, in which the infection was lethal in ~\u0026thinsp;40% of male mice, did not have increases in any cytokines over mock mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and their levels were similar to mock infected mice at 7 dpi at peak disease (Supplemental Fig.\u0026nbsp;7). In contrast, the higher weight loss in female mice of this strain was associated with significantly higher levels of several inflammatory cytokines in the serum at peak disease (day 5 post infection) compared to mocks (Supplemental Fig.\u0026nbsp;7). Similarly, the levels of multiple cytokines were significantly higher compared to mock or infected female mice in male mice of strain CC037, correlating with the higher weight loss observed (Supplemental Fig.\u0026nbsp;8). In contrast, mild disease in female CC037 mice was associated with no changes in the levels of several cytokines compared to mocks (Supplemental Fig.\u0026nbsp;8). Corresponding to the overall mild course of infection in this strain, the levels of some of these cytokines were decreased in both male and female CC037 mice including IL-3, IL-17A and TNFα (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplemental Fig.\u0026nbsp;8) and there was no change in the levels of cytokines Eotaxin, IFNγ, IL-10, IL-12(p40), CXCL1 and RANTES in both male and female CC037 mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, male and female CC037 mice did have an increase in MCP-1 and MIP-1α, although this was significant only in male mice compared to mocks and female mice. In agreement with no overt clinical disease throughout the course of the infection, male mice of strain CC012 did not have any changes in cytokine levels compared to mock infected mice (Supplemental Fig.\u0026nbsp;9). On the other hand, the slight decrease in body weight at 2 dpi in female mice was correlated with significantly higher levels of a number of inflammatory cytokines compared to mock and infected male mice (Supplemental Fig.\u0026nbsp;9). Finally, in agreement with no overt clinical disease throughout the course of the experiment, both male and female mice of strain CC042 had similar levels of cytokines as that observed in mock mice (Supplemental Fig.\u0026nbsp;10). Overall, mild disease was associated with a significant decrease of IL-3, IL-9, IL-12(p70), IL-17A and TNFα; whereas absence of disease was associated with little change in cytokines. In contrast lethal disease is associated with increased inflammatory cytokines such as Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1α, IL-4, IL-6, IL-9, IL-10, IL-12(p40), IL-13, CXCL1, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα, several of which have been associated with fatal human cases of CCHF. Taken together, these results suggest that similar to human cases of CCHF, disease severity in CC mice correlates with inflammatory cytokine production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor many VHFs, host immunopathology contributes significantly to morbidity and mortality but for CCHF it remains largely unclear how host factors contribute to disease outcome. Like other VHFs, CCHFV has multiple hosts, but upon infection, causes disease primarily in humans. In infected humans, a wide range of clinical manifestations are observed, even within regions with minimal virus diversity. This leads to speculation that there could be a strong role for host genetics in determining disease presentation. Although results from some studies indicate that genetic variation in humans may determine severity of CCHF disease \u003csup\u003e13\u0026ndash;18\u003c/sup\u003e, this has not been characterized in depth nor modeled in genetically diverse model systems. Thus a mechanistic understanding of the contribution of host genetics to CCHF disease outcome is lacking. This report sheds light on the role of host genetic diversity in CCHFV pathogenesis and disease outcome. We observed the full spectrum of CCHF disease by screening 10 CC strains infected with MA-CCHFV, demonstrating a clear association of host genetic diversity with disease severity as well as the recapitulation of several aspects of each disease phenotype that is observed in humans.\u003c/p\u003e \u003cp\u003eOur previous studies with MA-CCHFV infection in several conventional inbred laboratory mouse strains revealed that except for strain 129S1, all strains developed overt clinical disease and the sex-linked bias in disease severity was maintained \u003csup\u003e19\u003c/sup\u003e. In the present study, MA-CCHFV infection of male and female mice of ten CC strains resulted in a range of disease phenotypes, with consistently more severe disease in male than female mice. Sex differences in disease presentations have been described in the CC for other infectious diseases \u003csup\u003e30,31\u003c/sup\u003e. We identified two strains in which the infection in male mice was lethal and which exhibited correlates similar to fatal human cases of CCHF. High viremia is a predictor of fatal disease in humans, with viral titers higher than 10\u003csup\u003e9\u003c/sup\u003e genome copies per milliliter of plasma being indicative of lethal disease \u003csup\u003e32\u0026ndash;34\u003c/sup\u003e. Interestingly, recapitulating what is observed in humans, high viremia of approximately 10\u003csup\u003e10\u003c/sup\u003e genome copies per milliliter of serum was observed in most of the mice with lethal disease in our study. Moreover, lethal CCHF disease in male CC mice was also associated with severe involvement of the liver with significant necrosis, also in agreement with lethal human cases. Finally, production of inflammatory cytokines, a hallmark feature of fatal disease in humans \u003csup\u003e27,28\u003c/sup\u003e, was also observed in the two CC strains with lethal disease in male mice. Notably, we observed an increase in MCP-1 and TNFα in both strains that exhibited lethal outcome, in agreement with what has been reported in fatal human CCHF cases \u003csup\u003e27,28\u003c/sup\u003e. We have previously shown that low dose (10\u003csup\u003e4\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e) MA-CCHFV challenge of C57BL6/J mice resulted in elevated MCP-1 but not TNFα \u003csup\u003e19\u003c/sup\u003e; whereas, high-dose, lethal MA-CCHFV challenge resulted in significant MCP-1 and TNFα production later in infection \u003csup\u003e25\u003c/sup\u003e. These data suggest that in addition to host determinants there could be an impact due to inoculating dose, and that disease outcome correlates with inflammatory cytokine production. Thus, lethal CCHFV infection in two CC mice strains has several correlates also measured in lethal disease in humans suggesting similar disease mechanisms. These strains represent a useful model for identifying host genes that could potentially direct the course of CCHFV infection or more specifically disentangle the relationships between inflammatory cytokines, uncontrolled replication, and resultant disease. Polymorphisms in several key factors involved in the innate immune response to CCHFV including NF-κB, TLRs and IFNα have been shown to be associated with fatal outcome in CCHF cases in humans \u003csup\u003e13\u0026ndash;17\u003c/sup\u003e. It will be interesting to examine these genes in our CC lines to see if the same alleles observed in humans associate with lethality in CC strains. The CC mice have proven valuable in the study of other viruses including Ebola in CC and CC recombinant inbred intercrossed (CC-RIX) mice using a mouse-adapted strain of Ebola virus (MA-EBOV) \u003csup\u003e21,22,35\u003c/sup\u003e. More recently, strains CC051 (which had mild disease in both males and females infected with MA-CCHFV) and CC004 (which had partial lethality in the males and moderate disease in the females) were investigated for susceptibility to MA-EBOV infection \u003csup\u003e22\u003c/sup\u003e. Infection of CC051 showed mild to moderate disease, and CC004 was highly susceptible to EBOV infection and showed uniform mortality. Thus, MA-EBOV infection in these two strains was more severe than MA-CCHFV infection, suggesting that genes involved in the susceptibility to diverse VHF viruses may be distinct.\u003c/p\u003e \u003cp\u003eThe MA-CCHFV strain displays a sex-linked bias in disease severity, with more severe disease in male than female WT mice across a number of mouse genotypes \u003csup\u003e19\u003c/sup\u003e. At a higher dose of the virus, male but not female mice all succumb to the disease \u003csup\u003e25\u003c/sup\u003e. This male versus female phenotype was also maintained in six out of the eight CC strains that showed disease suggesting the mechanism is conserved in the genetically diverse CC model. However, we identified one strain, CC004 in which female mice showed higher weight loss, increased viral loads and inflammatory cytokines at peak disease (day 5) than male mice at peak disease (day 7). In CC003 and CC046 mice, in which males showed uniform lethality, female mice developed only mild disease demonstrating stark sex-linked differences in disease outcome. This finding suggests a role for host genetics in driving sex differences in the immune response to MA-CCHFV infection. Sex differences in immunity have been described for a number of pathogens in both humans and mice, with genetic mediators including sex chromosomes, microRNAs and genetic polymorphisms all shown to play roles in sex-differential effects on immunity \u003csup\u003e36\u003c/sup\u003e. For example, infection with coxsackievirus B3 (CVB3) results in sexual dimorphism in C57BL/6 WT mice, with increased mortality and severe cardiac inflammation in males compared to females \u003csup\u003e37\u003c/sup\u003e. Gonadectomy leads to a flipping of these phenotypes, with reduced CVB3 pathogenicity in male mice and increased pathogenicity in females \u003csup\u003e37\u003c/sup\u003e. Studies have also reported higher CCHFV infection of human males \u003csup\u003e38\u0026ndash;41\u003c/sup\u003e, although this may be explained by cultural practices in those regions, where activities such as farming and butchering are more likely carried out by men \u003csup\u003e42\u0026ndash;44\u003c/sup\u003e. It is therefore unclear whether a sex-linked bias in human CCHF disease exists. Nevertheless, our results provide an opportunity to investigate host genes associated with sex-linked determinants of outcome of viral infections, an understudied area in the context of viral hemorrhagic fevers.\u003c/p\u003e \u003cp\u003eA majority of the CC strains screened presented with mild to moderate disease, and two strains were resistant to clinical disease in both male and female mice. This is consistent with human infections, a majority of which are likely subclinical and underappreciated \u003csup\u003e45,46\u003c/sup\u003e. Our data indicate that asymptomatic or subclinical infections were not due to resistance to infection but instead likely due to host responses that resulted in rapid control of the infection with minimal immunopathology. This data is consistent with serological evidence indicating productive infection of numerous animal species in the absence of clinical disease \u003csup\u003e5\u003c/sup\u003e, suggesting that although CCHFV can infect numerous animal species, unique determinants within the human host enables symptomatic disease. Interestingly, mild disease in male and female CC037 and female CC003 mice was associated with decreased expression of several cytokines such as IL-3, IL-9, IL-12p70, IL-17A and TNFα. In contrast, the absence of clinical disease despite viremia and seroconversion in male and female CC012 and CC042 was associated with largely no cytokine response to the infection. These data suggest that there exist distinct cytokine responses and likely overall host immune responses to the infection in asymptomatic, mild, and severe CCHFV infections. We were also able to identify more subtle disease phenotypes, such as mice with mild weight loss but prolonged recovery (CC051 males) versus mice with rapid severe weight loss but rapid recovery (CC080 males). These models may be useful for studying the long-term sequelae that may follow recovery from acute CCHF, an area in need of further research. The presence of replicating virus and inflammatory cytokines in all ten infected CC lines irrespective of disease severity leads to an important area to explore- host factors limiting disease in mild and resistant strains.\u003c/p\u003e \u003cp\u003eOur study has several important limitations. First, the significant difference in disease between MA-CCHFV infected male and female mice has not been reported in human CCHFV infections. Although several studies have reported men are more likely to develop CCHFV or be seropositive for infection \u003csup\u003e38\u0026ndash;41\u003c/sup\u003e, this can also be attributed to cultural practices in which men are more likely to be exposed to ticks or livestock \u003csup\u003e44\u003c/sup\u003e. Epidemiological studies that account for these confounding variables are necessary to determine if there are sex-linked differences in disease outcomes in CCHFV-infected humans. Nevertheless, the similar correlates of disease severity in MA-CCHFV infected mice and infected humans along with distinct disease outcomes in a genetically variable population infected with the same strain of virus provides a powerful tool to investigate how host genetics contribute to CCHF outcome. Second, the CCHFV strain used is mouse-adapted and was generated by serial passage in mice on the C57BL6/J background. WT C57/BL6J mice are resistant to disease upon CCHFV infection \u003csup\u003e19\u003c/sup\u003e and therefore we did not test exposure of the CC strains to WT virus. Thus, it is possible that adaptation of MA-CCHFV to C57BL6/J mice may link to host-genes not linked to disease in humans. Notably, using a mouse-adapted EBOV and the CC mice, Price et al. identified key cell types that were associated with tolerance of Ebola virus disease (EVD), and through transcriptomics, generated gene expression signature profiles that were used to successfully predict clinical outcome in patients with EVD in Africa \u003csup\u003e35\u003c/sup\u003e. Further, human quantitative trait loci (QTL) and key genes identified in severe disease for viruses such as Influenza and SARS-CoV-2 also mapped as key genes that drive disease severity in the CC platform \u003csup\u003e31,47,48\u003c/sup\u003e demonstrating the utility of the CC platform in understanding viral pathogenesis. These studies provide encouraging evidence that gene signatures identified in disease phenotypes in CC mice can be accurately linked to disease outcome in humans using mouse-adapted viruses. Third, CCHFV is a genetically diverse virus and case fatality rates can vary by region \u003csup\u003e46,49\u0026ndash;51\u003c/sup\u003e. MA-CCHFV is based on CCHFV strain Hoti, isolated from a fatal human case in Eastern Europe. Therefore, we were unable to model the contribution of viral diversity to disease outcome and it is possible that distinct strains of CCHFV may interact with distinct host factors to cause disease, and this concern suggests further studies in mouse strains with discordant disease outcomes.\u003c/p\u003e \u003cp\u003eFourth, we did not perform paired timed necropsies on each strain at all timepoints and thus differences observed in pathology, cytokines or viral loads between sexes and strains may be due to different timepoints analyzed. In this report we intended to establish the MA-CCHFV infection of CC mice as a model to investigate host contributions to disease outcome. Future studies will perform more in-depth temporal analyses of these factors at multiple timepoints.\u003c/p\u003e \u003cp\u003eIn conclusion, using just ten CC lines, we were able to recapitulate the full spectrum of CCHF manifestations, providing strong evidence that the host genetic background is critical in determining the fate of MA-CCHFV infected mice. Our study demonstrates the utility of the CC model for examining host responses associated with the range of disease phenotypes observed in CCHF infection. Similar correlates of disease severity measured in human CCHF cases such as viral loads, tissue pathology and inflammatory cytokines were also measured in MA-CCHFV infected CC mice. Identifying host genes that can be linked to these distinct phenotypes is the focus of ongoing studies. Using a systems biology approach, the CC can be used to assess the interaction of genetic diversity and host responses leading to disease. These studies will allow for a more thorough understanding of CCHFV pathogenesis and host responses, eventually leading to the identification of novel therapeutic interventions for CCHF.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eData and materials availability\u003c/h2\u003e \u003cp\u003e All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by the Intramural Research Program of the NIAID, NIH.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eD.R., D.H., H.F., A.S., M.T. F., and R.B. conceptualized the study. A.S., M.T. F., R.B., and D.H. designed the methodology of the study. D.R., M.L., K.M-W., C.S., A.O., and D.H. performed all the investigations. D.R. analyzed data from all the analyses except histopathology, which was analyzed by C.R. M.F., A.S., R.B., and H.F. provided the resources and D.H. and H.F. supervised the studies. H.F. acquired funding for the study. D.R., D.H., and H.F. wrote the original draft of the manuscript. D.R., M.L., K.M-W., C.S., A.O., A.S., M.T. F., R.B., H.F. and D.H. all reviewed and edited the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAuthors wish to acknowledge the support provided by the Rocky Mountain Veterinary Branch, animal caretakers and the BSL-4 facilities staff for the ABSL-4 and BSL-4 studies. We also wish to acknowledge the UNC SGCF, which maintains and distributes the CC lines.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHawman, D. W. \u0026amp; Feldmann, H. Crimean-Congo haemorrhagic fever virus. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 463-477 (2023). https://doi.org/10.1038/s41579-023-00871-9\u003c/li\u003e\n\u003cli\u003eHawman, D. W. \u0026amp; Feldmann, H. 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S.\u003cem\u003e et al.\u003c/em\u003e An outbreak of Crimean-Congo hemorrhagic fever in the United Arab Emirates, 1994-1995. \u003cem\u003eAm J Trop Med Hyg\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 519-525 (1997). https://doi.org/10.4269/ajtmh.1997.57.519\u003c/li\u003e\n\u003cli\u003eKhurshid, A.\u003cem\u003e et al.\u003c/em\u003e CCHF virus variants in Pakistan and Afghanistan: Emerging diversity and epidemiology. \u003cem\u003eJ Clin Virol\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 25-30 (2015). https://doi.org/10.1016/j.jcv.2015.03.021\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4644919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4644919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Crimean-Congo hemorrhagic fever virus (CCHFV) causes Crimean-Congo hemorrhagic fever (CCHF), a widely distributed disease with significant morbidity and mortality. The virus has high genetic diversity correlated with geographic distribution, but limited temporal evolution within regions. Despite this, cases of CCHF within a region present as a spectrum of disease from often unrecognized asymptomatic infections to a severe, fatal viral hemorrhagic fever, suggesting host factors may play a role in disease outcome. We investigated the effect of host genetic diversity on the outcome of CCHFV infection in the genetically diverse Collaborative Cross mouse model. Infected mice recapitulated the full spectrum of disease recognized in humans and similar to human disease, virus replication, tissue pathology and inflammatory responses were associated with disease severity. Our study demonstrates that host genetics contribute to disease outcome in CCHF infection and establishes the Collaborative Cross mouse resource as a model to understand how host genetic diversity contributes to CCHF outcome.\u003c/p\u003e","manuscriptTitle":"Host genetic diversity contributes to disease outcome in Crimean-Congo hemorrhagic fever virus infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-17 01:56:55","doi":"10.21203/rs.3.rs-4644919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-27T19:38:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-26T04:40:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-12T15:48:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320161270116921974330400165008652611195","date":"2024-07-21T15:34:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81559603463551667754650302636993591744","date":"2024-07-19T21:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-19T19:37:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-18T04:51:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-18T03:42:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Viruses","date":"2024-06-26T21:32:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-viruses","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Viruses](https://www.nature.com/npjviruses)","snPcode":"44298","submissionUrl":"https://submission.springernature.com/new-submission/44298/3","title":"npj Viruses","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"05ea5fb1-04ee-45cb-89de-d7585c749bdf","owner":[],"postedDate":"August 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":35837273,"name":"Biological sciences/Microbiology/Virology/Virus host interactions"},{"id":35837274,"name":"Biological sciences/Microbiology/Virology/Viral pathogenesis"}],"tags":[],"updatedAt":"2025-02-13T16:23:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-17 01:56:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4644919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4644919","identity":"rs-4644919","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}