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Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19), with effective versus dysregulated responses influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the diverse genetic backgrounds of the Collaborative Cross founder strains crossed to K18-hACE2 transgenic mice that confers high susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 F1 progeny resulted in a spectrum of weight loss, survival, viral replication kinetics, histopathology, and cytokine profiles, some of which were sex-specific. Importantly, survival was associated with early type I interferon (IFN) expression and a phased proinflammatory response distinct from mice with severe disease. Thus, dynamics of inflammatory responses observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding antiviral immunity and evaluating countermeasures. ONE SENTENCE SUMMARY: Genetically diverse mice display a broad spectrum of clinically relevant responses to SARS-CoV-2 infection, reflecting variability in COVID-19 disease.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.One Sentence SummaryGenetically diverse mice model a spectrum of clinically relevant innate immune responses to SARS-CoV-2 infection.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, particularly with respect to immune responses, we utilized the rhesus macaque model of SARS-CoV-2 infection. Two cohorts of eight older (16-23 years) and eight younger (3-5 years) rhesus macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at pre-defined timepoints in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between age cohorts. Transcriptional signatures of inflammation-associated genes in cells isolated from bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both younger and older animals. These findings suggested that age did not substantially skew major facets of acute disease in this model. However, age-specific divergence of immune responses emerged during the post-acute phase of infection (7-21 dpi). Older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, in contrast to persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling in SARS-CoV-2-infected rhesus macaques suggests that age may delay or impair the induction of anti-viral cellular immune responses and delay efficient return to immune homeostasis following acute infection.

Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens, Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV NS5 antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplified the production of pro-inflammatory chemokines through PKR sensing of mitochondrial RNA. In Ajuba−/− mice, ZIKV induced early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy, and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

Single-cell RNA sequencing of lungs of SARS-CoV-2–infected African green monkeys reveals virus replication and host response dynamics in vivo., A dynamic response to SARS-CoV-2 While a number of animal models for SARS-CoV-2 have been developed, nonhuman primates are among the best at recapitulating human COVID-19. Here, Speranza et al. used single-cell RNA sequencing to demonstrate that SARS-CoV-2 replicates in the lung of African green monkeys and that immune cell populations in the lung change over the course of infection. Sequencing data revealed that pneumocytes are the site of viral replication in the lung, and infiltrating macrophages are responsible for clearing infected cells and cellular debris early in infection. Together, these findings further our understanding of the dynamics of SARS-CoV-2 infection and host immune responses in vivo., Detailed knowledge about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is important for uncovering the viral and host factors that contribute to coronavirus disease 2019 (COVID-19) pathogenesis. Old-World nonhuman primates recapitulate mild to moderate cases of COVID-19, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with infectious SARS-CoV-2 or irradiated, inactivated virus to study the dynamics of virus replication throughout the respiratory tract. Genomic RNA from the animals inoculated with the irradiated virus was found to be highly stable, whereas subgenomic RNA, an indicator of viral replication, was found to degrade quickly. We combined this information with single-cell RNA sequencing of cells isolated from the lung and lung-draining mediastinal lymph nodes and developed new analysis methods for unbiased targeting of important cells in the host response to SARS-CoV-2 infection. Through detection of reads to the viral genome, we were able to determine that replication of the virus in the lungs appeared to occur mainly in pneumocytes, whereas macrophages drove the inflammatory response. Monocyte-derived macrophages recruited to the lungs, rather than tissue-resident alveolar macrophages, were most likely to be responsible for phagocytosis of infected cells and cellular debris early in infection, with their roles switching during clearance of infection. Together, our dataset provides a detailed view of the dynamics of virus replication and host responses over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.

Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens, Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV NS5 antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplified the production of pro-inflammatory chemokines through PKR sensing of mitochondrial RNA. In Ajuba -/- mice, ZIKV induced early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy, and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

Chlamydia trachomatis infection causes severe inflammatory disease resulting in blindness and infertility. The pathophysiology of these diseases remains elusive but myeloid cell-associated inflammation has been implicated. Here we show NLRP3 inflammasome activation is essential for driving a macrophage-associated endometritis resulting in infertility by using a female mouse genital tract chlamydial infection model. We find the chlamydial parasitophorous vacuole protein CT135 triggers NLRP3 inflammasome activation via TLR2/MyD88 signaling as a pathogenic strategy to evade neutrophil host defense. Paradoxically, a consequence of CT135 mediated neutrophil killing results in a submucosal macrophage-associated endometritis driven by ATP/P2X7R induced NLRP3 inflammasome activation. Importantly, macrophage-associated immunopathology occurs independent of macrophage infection. We show chlamydial infection of neutrophils and epithelial cells produce elevated levels of extracellular ATP. We propose this source of ATP serves as a DAMP to activate submucosal macrophage NLRP3 inflammasome that drive damaging immunopathology. These findings offer a paradigm of sterile inflammation in infectious disease pathogenesis., Myeloid cells are implicated in the innate immune and inflammatory response during infection with Chlamydia trachomatis. Here the authors show the evasion of the neutrophil response to infection and concomitant induction of sterile immunity via the purinergic P2X7 receptor.

Detailed knowledge about the dynamics of SARS-CoV-2 infection is important for unraveling the viral and host factors that contribute to COVID-19 pathogenesis. Old-World nonhuman primates recapitulate mild-moderate COVID-19 cases, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with SARS-CoV-2 or inactivated virus to study the dynamics of virus replication throughout the respiratory tract. RNA sequencing of single cells from the lungs and mediastinal lymph nodes allowed a high-resolution analysis of virus replication and host responses over time. Viral replication was mainly localized to the lower respiratory tract, with evidence of replication in the pneumocytes. Macrophages were found to play a role in initiating a pro-inflammatory state in the lungs, while also interacting with infected pneumocytes. Our dataset provides a detailed view of changes in host and virus replication dynamics over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets. Funding: This work is supported by the Intramural Research Program of NIAID, NIH. Conflict of Interest: The authors have no competing interests to declare. Ethical Approval: All animal experiments were approved by the Institutional Animal Care and Use Committee of Rocky Mountain Laboratories, NIH and carried out by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International accredited facility, according to the institution’s guidelines for animal use, following the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures., Author summary Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic disease virus (AHFV) are tick-borne flaviviruses that cause viral hemorrhagic fevers in India and the Arabian Peninsula, respectively. Bonnet macaques and black-faced langurs are susceptible to KFDV infection, but these animals do not experience hemorrhagic signs as seen in human cases with KFDV. This work characterizes for the first time experimental infection of KFDV and AHFV in pigtailed macaques (PTMs). Infected PTMs can develop moderate to severe disease that models multiple features of human disease, including some hemorrhagic signs. Together these data describe the PTM model for KFDV and AHFV as a valuable tool for future work to study viral pathogenesis and for assessing the efficacy of vaccines and antivirals.

Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality-control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV nonstructural protein 5 (NS5) antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplifies the production of pro-inflammatory chemokines through protein kinase R (PKR) sensing of mitochondrial RNA. In Ajuba-/- mice, ZIKV induces early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

SUMMARY Tripartite motif-containing protein 5α (TRIM5α) is a cellular antiviral restriction factor that prevents early events in retrovirus replication. The activity of TRIM5α is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. In contrast to this current understanding, we show that both human and rhesus macaque TRIM5α suppress replication of specific flaviviruses. Multiple viruses in the tick-borne encephalitis complex are sensitive to TRIM5α-dependent restriction, but mosquito-borne flaviviruses, including yellow fever, dengue, and Zika viruses, are resistant. TRIM5α suppresses replication by binding to the viral protease NS2B/3 to promote its K48-linked ubiquitination and proteasomal degradation. Importantly, TRIM5α contributes to the antiviral function of IFN-I against sensitive flaviviruses in human cells. Thus, TRIM5α possesses remarkable plasticity in the recognition of diverse virus families, with the potential to influence human susceptibility to emerging flaviviruses of global concern., Graphical Abstract, In Brief The antiviral activity of TRIM5α is thought to be limited to retroviruses as a result of highly specific interactions with capsid lattices. Here, Chiramel et al. demonstrate that TRIM5α restricts replication of specific flaviviruses by binding and degrading the viral protease.

Summary The unprecedented 2013–2016 outbreak of Ebola virus (EBOV) resulted in over 11,300 human deaths. Host resistance to RNA viruses requires RIG-I-like receptor (RLR) signaling through the adaptor protein, mitochondrial antiviral signaling protein (MAVS), but the role of RLR-MAVS in orchestrating anti-EBOV responses in vivo is not known. Here we apply a systems approach to MAVS −/− mice infected with either wild-type or mouse-adapted EBOV. MAVS controlled EBOV replication through the expression of IFNα, regulation of inflammatory responses in the spleen, and prevention of cell death in the liver, with macrophages implicated as a major cell type influencing host resistance. A dominant role for RLR signaling in macrophages was confirmed following conditional MAVS deletion in LysM+ myeloid cells. These findings reveal tissue-specific MAVS-dependent transcriptional pathways associated with resistance to EBOV, and they demonstrate that EBOV adaptation to cause disease in mice involves changes in two distinct events, RLR-MAVS antagonism and suppression of RLR-independent IFN-I responses.

Zika virus (ZIKV) belongs to the family Flaviviridae, and is related to other viruses that cause human diseases. Unlike other flaviviruses, ZIKV infection can cause congenital neurological disorders and replicates efficiently in reproductive tissues1-3. Here we show that the envelope protein (E) of ZIKV is polyubiquitinated by the E3 ubiquitin ligase TRIM7 through Lys63 (K63)-linked polyubiquitination. Accordingly, ZIKV replicates less efficiently in the brain and reproductive tissues of Trim7-/- mice. Ubiquitinated E is present on infectious virions of ZIKV when they are released from specific cell types, and enhances virus attachment and entry into cells. Specifically, K63-linked polyubiquitin chains directly interact with the TIM1 (also known as HAVCR1) receptor of host cells, which enhances virus entry in cells as well as in brain tissue in vivo. Recombinant ZIKV mutants that lack ubiquitination are attenuated in human cells and in wild-type mice, but not in live mosquitoes. Monoclonal antibodies against K63-linked polyubiquitin specifically neutralize ZIKV and reduce viraemia in mice. Our results demonstrate that the ubiquitination of ZIKV E is an important determinant of virus entry, tropism and pathogenesis.

The Chlamydia trachomatis plasmid and inclusion membrane protein CT135 are virulence factors in the pathogenesis of murine female genital tract infection. To determine if these virulence factors play a similar role in female nonhuman primates, we infected pig-tailed macaques with the same C. trachomatis strains shown to be important in the murine model. Wild-type C. trachomatis and its isogenic mutant strain deficient in both plasmid and CT135 were used to infect macaques. Macaques were given primary and repeated cervicovaginal challenges with the wild-type and mutant strains. The infection rate, infection duration, and antibody response were similar among macaques infected with both strains. Unexpectedly, colposcopy, laparoscopy, and histologic analysis revealed no substantial genital tract pathology following either primary or repeated cervicovaginal challenges. Cytokine analysis of cervicovaginal secretions from both challenged groups revealed low concentrations of interleukin 1β (IL-1β) and elevated levels of the interleukin 1 receptor agonist (IL-1RA). We propose that an imbalance of IL-1β and IL-1RA in macaques is the reason for the mild inflammatory responses observed in infected urogenital tissues. Thus, understanding the pathobiology of chlamydial infection requires a better understanding of host epigenetic and chlamydial genetic factors. Our findings also have implications for understanding the high frequency of asymptomatic infections in humans.

We studied infection and immunity of hysterectomized mice infected with Chlamydia muridarum and Chlamydia trachomatis to determine if there were differences between these species in their ability to infect vaginal squamous epithelial cells in vivo independently of proximal upper genital tract tissues. We found that C. muridarum readily colonized and infected vaginal squamous epithelial cells, whereas C. trachomatis did not. Primary infection of the vaginal epithelium with C. muridarum produced infections of a duration longer than that reported for normal mice. Infection resulted in an inflammatory response in the vagina characterized by neutrophils and infiltrating submucosal plasma cells consisting primarily of T cells. Despite the delayed clearance, rechallenged C. muridarum -infected mice were highly immune. Mice vaginally infected with C. muridarum produced serum and vaginal wash antibodies and an antigen-specific gamma interferon-dominated Th1-biased T cell response. By comparison, mice vaginally infected with C. trachomatis exhibited transient low-burden infections, produced no detectable tissue inflammatory response, and failed to seroconvert. We discuss how these marked differences in the biology of vaginal infection between these otherwise genetically similar species are possibly linked to pathogen-specific virulence genes and how they may influence pathology and immunity in the upper genital tract.

Chlamydia trachomatis is an obligate intracellular pathogen characterized by a unique biphasic developmental cycle that alternates between infectious and non-infectious organisms. Chlamydial ChxR is a transcriptional activator that has been implicated in the regulation of the development cycle. We used a reverse genetics approach to generate three chxR null mutants. All three mutants grew normally in cultured mammalian cells. Whole genome sequencing identified SNPs in other genes; however, none of the mutated genes were common to all three ChxR null mutants arguing against a genetic compensatory mechanism that would explain the non-essential in vitro growth phenotype. Comparative proteomics identified five proteins, CT005, CT214, CT565, CT694 and CT695, that were significantly downregulated in all ChxR null mutants. This group includes established inclusion membrane and type III secreted proteins. ChxR transcriptional regulation of these genes was confirmed by qRT-PCR. Importantly, while ChxR null mutants exhibited no growth deficiencies in in vitro, they did show significant differences in in vivo growth using a mouse genital tract model. Collectively, our findings demonstrated that ChxR is a transcriptional activator that regulates the expression of virulence genes whose functions are restricted to in vivo infection.

Introduction Previous studies demonstrated that a Chlamydia trachomatis plasmid negative ocular strain (A/P-) resulted in attenuation of infection and pathology, paired with immune stimulation that produced protective immunity in a monkey ocular model. Expanding upon these findings, we assessed a plasmid negative genital strain (D/P-) in the pigtailed macaque model of chlamydial reproductive tract infection. Infection and pathology were compared to a plasmid positive genital strain (D/P+). Methods Groups of six macaques were cervically challenged with C. trachomatis D/P+ or D/P-. All animals were followed for infection, circulating antibody, local immunity, and tissue inflammation. Upon spontaneous clearance of cervical infection, each animal underwent repeated challenges with matched strains to drive upper reproductive tract disease. The same strains were similarly compared in the macaque ocular infection model. Results Similar rates and duration of chlamydial genital infection were documented in both the P+ and P- challenged macaques. Likewise serum and local antibodies were similar. Tissue inflammation graded by gross observation during laparoscopic procedures and by tissue histology yielded no discernible patterns to disease pathology between P+ and P- strains. Because A/P+ and A/P- ocular strains in macaques exhibited dramatic differences in infectivity and pathology in the eye, we ocularly challenged animals with D/P+ and D/P-. Unexpectedly, no differences in infectivity or pathology were observed between the D/P+ and D/P- strains; each produced similar infection kinetics with ocular disease characterised by conjunctival hyperemia and follicle formation. Conclusions Unlike the ocular strain, the plasmid negative genital strain is not attenuated in either genital or ocular macaque infection models. This suggests that genetic determinants unrelated to the plasmid play a dominant role in the pathogenesis of urogenital strains. Disclosure of interest statement This research was supported by NIH Contract Numbers HHSN266200700013C and HHSN272201400016C, and by the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health through Grant Number P51 OD010425 Washington National Primate Research Centre. No pharmaceutical grants were received in the development of this study.

Chlamydia trachomatis is an important human pathogen causing both ocular and sexually transmitted disease. Recently, we identified CT135 as an important virulence determinant in a mouse infection model. Results from CEL 1 digestion assays and sequencing analyses indicated that CT135 was much more polymorphic in high in vitro passage reference serovars than it was in clinical strains that had undergone limited passaging. Herein, we used targeted next-generation sequencing of the CT134-135 locus, from reference strains and clinical isolates, enabling accurate discovery of single nucleotide polymorphisms and other population genetic variations. Our results indicate that CT134 is stable in all C. trachomatis serovars examined. In contrast, CT135 is highly polymorphic in high-passaged reference ocular and non-LGV genital serovars, with the majority of the mutations resulting in gene disruption. In low-passaged ocular clinical isolates, CT135 was frequently disrupted, whereas in genital clinical isolates CT135 was intact in almost all instances. When a serovar K isolate, with an intact CT134 and CT135, was subjected to serial passage in vitro CT134 remained invariable, while numerous gene interrupting mutations rapidly accumulated in CT135. Collectively, our data indicate that, for genital serovars, CT135 is under strong positive selection in vivo, and negative selection in vitro.