Deciphering bat influenza H18N11 infection dynamics in male Jamaican fruit bats on a single-cell level.

Jamaican fruit bats (Artibeus jamaicensis) naturally harbor a wide range of viruses of human relevance. These infections are typically mild in bats, suggesting unique features of their immune system. To better understand the immune response to viral infections in bats, we infected male Jamaican fruit bats with the bat-derived influenza A virus (IAV) H18N11. Using comparative single-cell RNA sequencing, we generated single-cell atlases of the Jamaican fruit bat intestine and mesentery. Gene expression profiling showed that H18N11 infection resulted in a moderate induction of interferon-stimulated genes and transcriptional activation of immune cells. H18N11 infection was predominant in various leukocytes, including macrophages, B cells, and NK/T cells. Confirming these findings, human leukocytes, particularly macrophages, were also susceptible to H18N11, highlighting the zoonotic potential of this bat-derived IAV. Our study provides insight into a natural virus-host relationship and thus serves as a fundamental resource for future in-depth characterization of bat immunology.

Integrative single-cell characterization of a frugivorous and an insectivorous bat kidney and pancreas.

Frugivory evolved multiple times in mammals, including bats. However, the cellular and molecular components driving it remain largely unknown. Here, we use integrative single-cell sequencing (scRNA-seq and scATAC-seq) on insectivorous (Eptesicus fuscus; big brown bat) and frugivorous (Artibeus jamaicensis; Jamaican fruit bat) bat kidneys and pancreases and identify key cell population, gene expression and regulatory differences associated with the Jamaican fruit bat that also relate to human disease, particularly diabetes. We find a decrease in loop of Henle and an increase in collecting duct cells, and differentially active genes and regulatory elements involved in fluid and electrolyte balance in the Jamaican fruit bat kidney. The Jamaican fruit bat pancreas shows an increase in endocrine and a decrease in exocrine cells, and differences in genes and regulatory elements involved in insulin regulation. We also find that these frugivorous bats share several molecular characteristics with human diabetes. Combined, our work provides insights from a frugivorous mammal that could be leveraged for therapeutic purposes.

Regulatory T cell-like response to SARS-CoV-2 in Jamaican fruit bats (Artibeus jamaicensis) transduced with human ACE2.

Insectivorous Old World horseshoe bats (Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats (Rousettus aegyptiacus) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats (Eptesicus fuscus) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats (Artibeus jamaicensis) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyer's patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4+ helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-ß, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptible to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease.

Regulatory T Cell-like Response to SARS-CoV-2 in Jamaican Fruit Bats ( Artibeus jamaicensis ) Transduced with Human ACE2.

Insectivorous Old World horseshoe bats ( Rhinolophus spp.) are the likely source of the ancestral SARS-CoV-2 prior to its spillover into humans and causing the COVID-19 pandemic. Natural coronavirus infections of bats appear to be principally confined to the intestines, suggesting fecal-oral transmission; however, little is known about the biology of SARS-related coronaviruses in bats. Previous experimental challenges of Egyptian fruit bats ( Rousettus aegyptiacus ) resulted in limited infection restricted to the respiratory tract, whereas insectivorous North American big brown bats ( Eptesicus fuscus ) showed no evidence of infection. In the present study, we challenged Jamaican fruit bats ( Artibeus jamaicensis ) with SARS-CoV-2 to determine their susceptibility. Infection was confined to the intestine for only a few days with prominent viral nucleocapsid antigen in epithelial cells, and mononuclear cells of the lamina propria and Peyer's patches, but with no evidence of infection of other tissues; none of the bats showed visible signs of disease or seroconverted. Expression levels of ACE2 were low in the lungs, which may account for the lack of pulmonary infection. Bats were then intranasally inoculated with a replication-defective adenovirus encoding human ACE2 and 5 days later challenged with SARS-CoV-2. Viral antigen was prominent in lungs for up to 14 days, with loss of pulmonary cellularity during this time; however, the bats did not exhibit weight loss or visible signs of disease. From day 7, bats had low to moderate IgG antibody titers to spike protein by ELISA, and one bat on day 10 had low-titer neutralizing antibodies. CD4 + helper T cells became activated upon ex vivo recall stimulation with SARS-CoV-2 nucleocapsid peptide library and exhibited elevated mRNA expression of the regulatory T cell cytokines interleukin-10 and transforming growth factor-ß, which may have limited inflammatory pathology. Collectively, these data show that Jamaican fruit bats are poorly susceptibility to SARS-CoV-2 but that expression of human ACE2 in their lungs leads to robust infection and an adaptive immune response with low-titer antibodies and a regulatory T cell-like response that may explain the lack of prominent inflammation in the lungs. This model will allow for insight of how SARS-CoV-2 infects bats and how bat innate and adaptive immune responses engage the virus without overt clinical disease. Author Summary: Bats are reservoir hosts of many viruses that infect humans, yet little is known about how they host these viruses, principally because of a lack of relevant and susceptible bat experimental infection models. Although SARS-CoV-2 originated in bats, no robust infection models of bats have been established. We determined that Jamaican fruit bats are poorly susceptible to SARS-CoV-2; however, their lungs can be transduced with human ACE2, which renders them susceptible to SARS-CoV-2. Despite robust infection of the lungs and diminishment of pulmonary cellularity, the bats showed no overt signs of disease and cleared the infection after two weeks. Despite clearance of infection, only low-titer antibody responses occurred and only a single bat made neutralizing antibody. Assessment of the CD4 + helper T cell response showed that activated cells expressed the regulatory T cell cytokines IL-10 and TGFß that may have tempered pulmonary inflammation.

Involvement of Pro-Inflammatory Macrophages in Liver Pathology of Pirital Virus-Infected Syrian Hamsters.

New World arenaviruses cause fatal hemorrhagic disease in South America. Pirital virus (PIRV), a mammarenavirus hosted by Alston’s cotton rat (Sigmodon alstoni), causes a disease in Syrian golden hamsters (Mesocricetus auratus) (biosafety level-3, BSL-3) that has many pathologic similarities to the South American hemorrhagic fevers (BSL-4) and, thus, is considered among the best small-animal models for human arenavirus disease. Here, we extend in greater detail previously described clinical and pathological findings in Syrian hamsters and provide evidence for a pro-inflammatory macrophage response during PIRV infection. The liver was the principal target organ of the disease, and signs of Kupffer cell involvement were identified in mortally infected hamster histopathology data. Differential expression analysis of liver mRNA revealed signatures of the pro-inflammatory response, hematologic dysregulation, interferon pathway and other host response pathways, including 17 key transcripts that were also reported in two non-human primate (NHP) arenavirus liver-infection models, representing both Old and New World mammarenavirus infections. Although antigen presentation may differ among rodent and NHP species, key hemostatic and innate immune-response components showed expression parallels. Signatures of pro-inflammatory macrophage involvement in PIRV-infected livers included enrichment of Ifng, Nfkb2, Stat1, Irf1, Klf6, Il1b, Cxcl10, and Cxcl11 transcripts. Together, these data indicate that pro-inflammatory macrophage M1 responses likely contribute to the pathogenesis of acute PIRV infection.

Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis).

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) highlights the zoonotic potential of Betacoronaviruses. Investigations into the origin of MERS-CoV have focused on two potential reservoirs: bats and camels. Here, we investigated the role of bats as a potential reservoir for MERS-CoV. In vitro, the MERS-CoV spike glycoprotein interacted with Jamaican fruit bat (Artibeus jamaicensis) dipeptidyl peptidase 4 (DPP4) receptor and MERS-CoV replicated efficiently in Jamaican fruit bat cells, suggesting there is no restriction at the receptor or cellular level for MERS-CoV. To shed light on the intrinsic host-virus relationship, we inoculated 10 Jamaican fruit bats with MERS-CoV. Although all bats showed evidence of infection, none of the bats showed clinical signs of disease. Virus shedding was detected in the respiratory and intestinal tract for up to 9 days. MERS-CoV replicated transiently in the respiratory and, to a lesser extent, the intestinal tracts and internal organs; with limited histopathological changes observed only in the lungs. Analysis of the innate gene expression in the lungs showed a moderate, transient induction of expression. Our results indicate that MERS-CoV maintains the ability to replicate in bats without clinical signs of disease, supporting the general hypothesis of bats as ancestral reservoirs for MERS-CoV.

Cloning and sequence analysis of Peromyscus yucatanicus (Rodentia) Th1 (IL-12p35, IFN-γ and TNF) and Th2 (IL-4, IL-10 and TGF-ß) cytokines.

The Yucatan deer mouse, Peromyscus yucatanicus (order Rodentia), is the principal reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico. Experimental infection results in clinical and histopathological features similar to those observed in humans with cutaneous leishmaniasis (CL) as well as peritoneal macrophage production of nitric oxide. These results support the possible use of P. yucatanicus as a novel experimental model to study CL caused by L. (L.) mexicana. However, immunological studies in these rodents have been limited by the lack of specific reagents. To address this issue, we cloned and analyzed cytokine sequences of P. yucatanicus as part of an effort to develop this species as a CL model. We cloned P. yucatanicus interleukin 4 (IL-4), IL-10, IL-12p35, gamma interferon, transforming growth factor beta and tumor necrosis factor partial cDNAs. Most of the P. yucatanicus sequences were highly conserved with orthologs of other mammalian species and the identity of all sequences were confirmed by the presence of conserved amino acids with possible biological functions in each putative polypeptide. The availability of these sequences is a first step which will allow us to carry out studies characterizing the immune response during pathogenic and nonpathogenic L. (L.) mexicana infections in P. yucatanicus.

The two suborders of chiropterans have the canonical heavy-chain immunoglobulin (Ig) gene repertoire of eutherian mammals.

Bats comprise 20% of all mammals, yet little is known about their immune system and virtually nothing about their immunoglobulin genes. We show that four different bat species transcribe genes encoding IgM, IgE, IgA and IgG subclasses, the latter which have diversified after speciation; the canonical pattern for eutherian mammals. IgD transcripts were only recovered from insectivorous bats and were comprised of CH1, CH3 and two hinge exons; the second hinge exon was fused to CH3. IgA in all species resembles human IgA2 with the putative cysteine forming the bridge to the light chain found at position 77. Sequence comparisons yielded no evidence for a diphyletic origin of the suborders. Bats show no close similarity to another mammalian order; the strongest association was with carnivores. Data reveal that CH diversity and VDJ and CDR3 organization are similar to other eutherian mammals, although the expressed VH3 family repertoire was unusually diverse.

Genomic organization and phylogenetic utility of deer mouse (Peromyscus maniculatus) lymphotoxin-alpha and lymphotoxin-beta.

BACKGROUND: Deer mice (Peromyscus maniculatus) are among the most common mammals in North America and are important reservoirs of several human pathogens, including Sin Nombre hantavirus (SNV). SNV can establish a life-long apathogenic infection in deer mice, which can shed virus in excrement for transmission to humans. Patients that die from hantavirus cardiopulmonary syndrome (HCPS) have been found to express several proinflammatory cytokines, including lymphotoxin (LT), in the lungs. It is thought that these cytokines contribute to the pathogenesis of HCPS. LT is not expressed by virus-specific CD4+ T cells from infected deer mice, suggesting a limited role for this pathway in reservoir responses to hantaviruses. RESULTS: We have cloned the genes encoding deer mouse LTalpha and LTbeta and have found them to be highly similar to orthologous rodent sequences but with some differences in promoters elements. The phylogenetic analyses performed on the LTalpha, LTbeta, and combined data sets yielded a strongly-supported sister-group relationship between the two murines (the house mouse and the rat). The deer mouse, a sigmodontine, appeared as the sister group to the murine clade in all of the analyses. High bootstrap values characterized the grouping of murids. CONCLUSION: No conspicuous differences compared to other species are present in the predicted amino acid sequences of LTalpha or LTbeta; however, some promoter differences were noted in LTbeta. Although more extensive taxonomic sampling is required to confirm the results of our analyses, the preliminary findings indicate that both genes (analyzed both separately and in combination) hold potential for resolving relationships among rodents and other mammals at the subfamily level.

Rapid field immunoassay for detecting antibody to Sin Nombre virus in deer mice.

We developed a 1-hour field enzyme immunoassay (EIA) for detecting antibody to Sin Nombre virus in deer mice (Peromyscus maniculatus). The assay specificity and sensitivity were comparable to those of a standard EIA. This test will permit identification of rodents with antibody to this and perhaps other hantaviruses.

Evi3, a zinc-finger protein related to EBFAZ, regulates EBF activity in B-cell leukemia.

Retroviral insertions that activate proto-oncogenes are a primary cause of tumors in certain strains of mice. The AKXD recombinant inbred mice are predisposed to a variety of leukemias and lymphomas as a result of viral integration. One common insertion site, the ecotropic viral insertion site 3 (Evi3), has been implicated in most B-cell tumors in the AKXD-27 strain. The Evi3 gene encodes a zinc-finger protein with sequence similarity to the Early B-cell Factor-Associated Zinc-finger gene (EBFAZ). We show that the Evi3 gene is overexpressed in several tumors with viral insertions at Evi3, which results in the upregulation of Early B-cell Factor (EBF)-target gene expression, suggesting that Evi3 modulates EBF activity. Reconstitution of primary leukemia cells showed that these tumors express high densities of the B-cell surface proteins CD19 and CD38, which are EBF targets. Using a transactivation assay, we show that the terminal six zinc-fingers of Evi3 are required for modification of EBF activity. This is the first evidence that Evi3 expression in tumors alters the level of EBF target genes, and the first characterization of the Evi3 protein domains required for modulation of EBF activity. Further, these data imply that Evi3 misexpression initiates tumorigenesis by perturbing B-cell development via an interaction with EBF.

Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus).

BACKGROUND: Human infections with Sin Nombre virus (SNV) and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS), a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus) are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. RESULTS: To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC) from deer mouse bone marrow using commercially-available house mouse (Mus musculus) granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. CONCLUSIONS: The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

Cloning and characterization of deer mouse (Peromyscus maniculatus) cytokine and chemokine cDNAs.

BACKGROUND: Sin Nombre virus (SNV) establishes a persistent infection in the deer mouse, Peromyscus maniculatus. A strong antibody response occurs in response to SNV infection, but the role of the innate immune response is unclear. To address this issue, we have initiated an effort to identify and characterize deer mouse cytokine and chemokine genes. Such cytokines and chemokines are involved in various aspects of immunity, including the transition from innate to adaptive responses, type I and type II responses, recruitment of leukocytes to sites of infection, and production of mature cells from bone marrow progenitors. RESULTS: We established a colony of SNV antibody-negative deer mice and cloned 11 cytokine and chemokine partial cDNA sequences using directed PCR. Most of the deer mouse sequences were highly conserved with orthologous sequences from other rodent species and functional domains were identified in each putative polypeptide. CONCLUSIONS: The availability of these sequences will allow the examination of the role of these cytokines in deer mouse responses to infection with Sin Nombre virus.

Sequence and expression analysis of deer mouse interferon-gamma, interleukin-10, tumor necrosis factor, and lymphotoxin-alpha.

Deer mice (Peromyscus maniculatus) are the principal host species of Sin Nombre (SN) virus, the primary etiologic agent of hantavirus cardiopulmonary syndrome in North America. The disease is a cytokine-mediated immunopathology characterized by pulmonary mononuclear infiltrates without discernible viral pathology. Infected deer mice remain life-long carriers and virus is found in many organs, including the lungs, but without pathology. It is unclear how deer mice respond to SN virus because no tools exist to examine the immune response in infected animals. As an initial step in examining host responses to SN virus, we have cloned partial cDNAs of deer mouse interferon-gamma (IFN-gamma), interleukin-10 (IL-10), tumor necrosis factor (TNF) and lymphotoxin-alpha (LTalpha). IL-10, TNF and LTalpha sequences are highly conserved compared to orthologs of other mammalian species, while IFN-gamma is substantially less conserved. Phylogenetic analyses indicate that the amino acid sequences of IFN-gamma and TNF may be useful in resolving relationships at the subfamily level within the rodent family Muridae. While all four sets of analyses were able to reconstruct clade Rodentia, they were not able to resolve the relationships among the mammalian orders represented in this study. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of concanavalin A-stimulated splenocytes determined that maximal IFN-gamma and TNF expression occurred rapidly while IL-10 and LTalpha expression was maximal at 24 h.