Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity

Bats are known to harbor a number of emerging and re-emerging zoonotic viruses, many of which are highly pathogenic in other mammals but result in no clinical symptoms in bats. The ability of bats to coexist with viruses may be the result of rapid control of viral replication early in the immune response. IFNs provide the first line of defense against viral infection in vertebrates. Type III IFNs (IFN-λs) are a recently identified IFN family that share similar antiviral activities with type I IFNs. To our knowledge, we demonstrate the first functional analysis of type III IFNs from any species of bat, with the investigation of two IFN-λ genes from the pteropid bat, Pteropus alecto. Our results demonstrate that bat type III IFN has similar antiviral activity to type I and III IFNs from other mammals. In addition, the two bat type III IFNs are differentially induced relative to each other and to type I IFNs after treatment or transfection with synthetic dsRNA. Infection with the bat paramyxovirus, Tioman virus, resulted in no upregulation of type I IFN production in bat splenocytes but was capable of inducing a type III IFN response in three of the four bats tested. To our knowledge, this is the first report to describe the simultaneous suppression of type I IFN and induction of type III IFN after virus infection. These results may have important implications for the role of type III IFNs in the ability of bats to coexist with viruses.     

Interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines

Bats are natural reservoirs for a spectrum of infectious zoonotic diseases including the recently emerged henipaviruses (Hendra and Nipah viruses). Henipaviruses have been observed both naturally and experimentally to cause serious and often fatal disease in many different mammal species, including humans. Interestingly, infection of the flying fox with henipaviruses occurs in the absence of clinical disease. The extreme variation in the disease pattern between humans and bats has led to an investigation into the effects of henipavirus infection on the innate immune response in bat cell lines. We report that henipavirus infection does not result in the induction of interferon expression, and the viruses also inhibit interferon signaling. We also confirm that the interferon production and signaling block in bat cells is not due to differing viral protein expression levels between human and bat hosts. This information, in addition to the known lack of clinical signs in bats following henipavirus infection, suggests that bats control henipavirus infection by an as yet unidentified mechanism, not via the interferon response. This is the first report of henipavirus infection in bat cells specifically investigating aspects of the innate immune system.     

Henipavirus neutralising antibodies in an isolated island population of African fruit bats

Isolated islands provide valuable opportunities to study the persistence of viruses in wildlife populations, including population size thresholds such as the critical community size. The straw-coloured fruit bat, Eidolon helvum, has been identified as a reservoir for henipaviruses (serological evidence) and Lagos bat virus (LBV; virus isolation and serological evidence) in continental Africa. Here, we sampled from a remote population of E. helvum annobonensis fruit bats on Annobón island in the Gulf of Guinea to investigate whether antibodies to these viruses also exist in this isolated subspecies. Henipavirus serological analyses (Luminex multiplexed binding and inhibition assays, virus neutralisation tests and western blots) and lyssavirus serological analyses (LBV: modified Fluorescent Antibody Virus Neutralisation test, LBV and Mokola virus: lentivirus pseudovirus neutralisation assay) were undertaken on 73 and 70 samples respectively. Given the isolation of fruit bats on Annobón and their lack of connectivity with other populations, it was expected that the population size on the island would be too small to allow persistence of viruses that are thought to cause acute and immunising infections. However, the presence of antibodies against henipaviruses was detected using the Luminex binding assay and confirmed using alternative assays. Neutralising antibodies to LBV were detected in one bat using both assays. We demonstrate clear evidence for exposure of multiple individuals to henipaviruses in this remote population of E. helvum annobonensis fruit bats on Annobón island. The situation is less clear for LBV. Seroprevalences to henipaviruses and LBV in Annobón are notably different to those in E. helvum in continental locations studied using the same sampling techniques and assays. Whilst cross-sectional serological studies in wildlife populations cannot provide details on viral dynamics within populations, valuable information on the presence or absence of viruses may be obtained and utilised for informing future studies.     

Induction of alpha interferon by membrane interaction between viral surface and peripheral blood mononuclear cells

Cells infected with viruses and fixed when viral antigens appeared at the cell membrane induced much higher alpha interferon (IFN-alpha) levels in human peripheral blood mononuclear cells (PBMC) than free virions. Relatively few inducer cells were sufficient for triggering IFN production. Optimal IFN yields depended on inducer/producer cell ratio. The response was peculiar to PBMC as it was not found in other cells in which IFN can normally be induced by free virions. IFN inducing activity was also exerted by live virus-infected PBMC, showing that this type of induction may have physiological importance. These findings confirm that viral induction of IFN-alpha is activated by some interaction between viral components presented at the cell surface and PBMC membrane. Thus induction of IFN by circulating cells infected by viruses and presenting viral antigens at the surface may be an efficient host defense mechanism. Since IFN yields close to 10(6) international units per milliliter are obtained, this system has potential for large scale production of native IFN-alpha.     

Disruption of Type I Interferon Induction by HIV Infection of T Cells

Our main objective of this study was to determine how Human Immunodeficiency Virus (HIV) avoids induction of the antiviral Type I Interferon (IFN) system. To limit viral infection, the innate immune system produces important antiviral cytokines such as the IFN. IFN set up a critical roadblock to virus infection by limiting further replication of a virus. Usually, IFN production is induced by the recognition of viral nucleic acids by innate immune receptors and subsequent downstream signaling. However, the importance of IFN in the defense against viruses has lead most pathogenic viruses to evolve strategies to inhibit host IFN induction or responses allowing for increased pathogenicity and persistence of the virus. While the adaptive immune responses to HIV infection have been extensively studied, less is known about the balance between induction and inhibition of innate immune defenses, including the antiviral IFN response, by HIV infection. Here we show that HIV infection of T cells does not induce significant IFN production even IFN I Interferon production. To explain this paradox, we screened HIV proteins and found that two HIV encoded proteins, Vpu and Nef, strongly antagonize IFN induction, with expression of these proteins leading to loss of expression of the innate immune viral RNA sensing adaptor protein, IPS-1 (IFN-β promoter stimulator-1). We hypothesize that with lower levels of IPS-1 present, infected cells are defective in mounting antiviral responses allowing HIV to replicate without the normal antiviral actions of the host IFN response. Using cell lines as well as primary human derived cells, we show that HIV targeting of IPS-1 is key to limiting IFN induction. These findings describe how HIV infection modulates IFN induction providing insight into the mechanisms by which HIV establishes infection and persistence in a host.     

Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba''s short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells.     

Polyinosinic-polycytidylic acid treatment of Friend retrovirus-infected mice improves functional properties of virus-specific T cells and prevents virus-induced disease

The induction of type I IFN is the most immediate host response to viral infections. Type I IFN has a direct antiviral activity mediated by antiviral enzymes, but it also modulates the function of cells of the adaptive immune system. Many viruses can suppress type I IFN production, and in retroviral infections, the initial type I IFN is weak. Thus, one strategy of immunotherapy in viral infection is the exogenous induction of type I IFN during acute viral infection by TLR ligands. Along these lines, the TLR3/MDA5 ligand polyinosinic-polycytidylic acid [poly(I:C)] has already been used to treat viral infections. However, the immunological mechanisms underlying this successful therapy have not been defined until now. In this study, the Friend retrovirus (FV) mouse model was used to investigate the mode of action of poly(I:C) in antiretroviral immunotherapy. Postexposure, poly(I:C) treatment of FV-infected mice resulted in a significant reduction in viral loads and protection from virus-induced leukemia. This effect was IFN dependent because type I IFN receptor-deficient mice could not be protected by poly(I:C). The poly(I:C)-induced IFN response resulted in the expression of antiviral enzymes, which suppressed FV replication. Also, the virus-specific T cell response was augmented. Interestingly, it did not enhance the number of virus-specific CD4(+) and CD8(+) T cells, but rather the functional properties of these cells, such as cytokine production and cytotoxic activity. The results demonstrate a direct antiviral and immunomodulatory effect of poly(I:C) and, therefore, suggests its potential for clinical treatment of retroviral infections.     

Two novel parvoviruses in frugivorous New and Old World bats

Bats, a globally distributed group of mammals with high ecological importance, are increasingly recognized as natural reservoir hosts for viral agents of significance to human and animal health. In the present study, we evaluated pools of blood samples obtained from two phylogenetically distant bat families, in particular from flying foxes (Pteropodidae), Eidolon helvum in West Africa, and from two species of New World leaf-nosed fruit bats (Phyllostomidae), Artibeus jamaicensis and Artibeus lituratus in Central America. A sequence-independent virus discovery technique (VIDISCA) was used in combination with high throughput sequencing to detect two novel parvoviruses: a PARV4-like virus named Eh-BtPV-1 in Eidolon helvum from Ghana and the first member of a putative new genus in Artibeus jamaicensis from Panama (Aj-BtPV-1). Those viruses were circulating in the corresponding bat colony at rates of 7-8%. Aj-BtPV-1 was also found in Artibeus lituratus (5.5%). Both viruses were detected in the blood of infected animals at high concentrations: up to 10E8 and to 10E10 copies/ml for Aj-BtPV-1 and Eh-BtPV-1 respectively. Eh-BtPV-1 was additionally detected in all organs collected from bats (brain, lungs, liver, spleen, kidneys and intestine) and spleen and kidneys were identified as the most likely sites where viral replication takes place. Our study shows that bat parvoviruses share common ancestors with known parvoviruses of humans and livestock. We also provide evidence that a variety of Parvovirinae are able to cause active infection in bats and that they are widely distributed in these animals with different geographic origin, ecologies and climatic ranges.     

The influence of bat ecology on viral diversity and reservoir status

Repeated emergence of zoonotic viruses from bat reservoirs into human populations demands predictive approaches to preemptively identify virus‐carrying bat species. Here, we use machine learning to examine drivers of viral diversity in bats, determine whether those drivers depend on viral genome type, and predict undetected viral carriers. Our results indicate that bat species with longer life spans, broad geographic distributions in the eastern hemisphere, and large group sizes carry more viruses overall. Life span was a stronger predictor of deoxyribonucleic acid viral diversity, while group size and family were more important for predicting ribonucleic acid viruses, potentially reflecting broad differences in infection duration. Importantly, our models predict 54 bat species as likely carriers of zoonotic viruses, despite not currently being considered reservoirs. Mapping these predictions as a proportion of local bat diversity, we identify global regions where efforts to reduce disease spillover into humans by identifying viral carriers may be most productive.     

A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special?

Bats are the natural reservoirs of a number of high-impact viral zoonoses. We present a quantitative analysis to address the hypothesis that bats are unique in their propensity to host zoonotic viruses based on a comparison with rodents, another important host order. We found that bats indeed host more zoonotic viruses per species than rodents, and we identified life-history and ecological factors that promote zoonotic viral richness. More zoonotic viruses are hosted by species whose distributions overlap with a greater number of other species in the same taxonomic order (sympatry). Specifically in bats, there was evidence for increased zoonotic viral richness in species with smaller litters (one young), greater longevity and more litters per year. Furthermore, our results point to a new hypothesis to explain in part why bats host more zoonotic viruses per species: the stronger effect of sympatry in bats and more viruses shared between bat species suggests that interspecific transmission is more prevalent among bats than among rodents. Although bats host more zoonotic viruses per species, the total number of zoonotic viruses identified in bats (61) was lower than in rodents (68), a result of there being approximately twice the number of rodent species as bat species. Therefore, rodents should still be a serious concern as reservoirs of emerging viruses. These findings shed light on disease emergence and perpetuation mechanisms and may help lead to a predictive framework for identifying future emerging infectious virus reservoirs.     

Deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases

Studies have demonstrated that ~60%–80% of emerging infectious diseases (EIDs) in humans originated from wild life. Bats are natural reservoirs of a large variety of viruses, including many important zoonotic viruses that cause severe diseases in humans and domestic animals. However, the understanding of the viral population and the ecological diversity residing in bat populations is unclear, which complicates the determination of the origins of certain EIDs. Here, using bats as a typical wildlife reservoir model, virome analysis was conducted based on pharyngeal and anal swab samples of 4440 bat individuals of 40 major bat species throughout China. The purpose of this study was to survey the ecological and biological diversities of viruses residing in these bat species, to investigate the presence of potential bat-borne zoonotic viruses and to evaluate the impacts of these viruses on public health. The data obtained in this study revealed an overview of the viral community present in these bat samples. Many novel bat viruses were reported for the first time and some bat viruses closely related to known human or animal pathogens were identified. This genetic evidence provides new clues in the search for the origin or evolution pattern of certain viruses, such as coronaviruses and noroviruses. These data offer meaningful ecological information for predicting and tracing wildlife-originated EIDs.     

Paramyxovirus Activation and Inhibition of Innate Immune Responses

Paramyxoviruses represent a remarkably diverse family of enveloped nonsegmented negative-strand RNA viruses, some of which are the most ubiquitous disease-causing viruses of humans and animals. This review focuses on paramyxovirus activation of innate immune pathways, the mechanisms by which these RNA viruses counteract these pathways, and the innate response to paramyxovirus infection of dendritic cells (DC). Paramyxoviruses are potent activators of extracellular complement pathways, a first line of defense that viruses must face during natural infections. We discuss mechanisms by which these viruses activate and combat complement to delay neutralization. Once cells are infected, virus replication drives type I interferon (IFN) synthesis that has the potential to induce a large number of antiviral genes. Here we describe four approaches by which paramyxoviruses limit IFN induction: by limiting synthesis of IFN-inducing aberrant viral RNAs, through targeted inhibition of RNA sensors, by providing viral decoy substrates for cellular kinase complexes, and through direct blocking of the IFN promoter. In addition, paramyxoviruses have evolved diverse mechanisms to disrupt IFN signaling pathways. We describe three general mechanisms, including targeted proteolysis of signaling factors, sequestering cellular factors, and upregulation of cellular inhibitors. DC are exceptional cells with the capacity to generate adaptive immunity through the coupling of innate immune signals and T cell activation. We discuss the importance of innate responses in DC following paramyxovirus infection and their consequences for the ability to mount and maintain antiviral T cells.     

Type I interferon production during herpes simplex virus infection is controlled by cell-type-specific viral recognition through Toll-like receptor 9, the mitochondrial antiviral signaling protein pathway, and novel recognition systems

Recognition of viruses by germ line-encoded pattern recognition receptors of the innate immune system is essential for rapid production of type I interferon (IFN) and early antiviral defense. We investigated the mechanisms of viral recognition governing production of type I IFN during herpes simplex virus (HSV) infection. We show that early production of IFN in vivo is mediated through Toll-like receptor 9 (TLR9) and plasmacytoid dendritic cells, whereas the subsequent alpha/beta IFN (IFN-α/β) response is derived from several cell types and induced independently of TLR9. In conventional DCs, the IFN response occurred independently of viral replication but was dependent on viral entry. Moreover, using a HSV-1 UL15 mutant, which fails to package viral DNA into the virion, we found that entry-dependent IFN induction also required the presence of viral genomic DNA. In macrophages and fibroblasts, where the virus was able to replicate, HSV-induced IFN-α/β production was dependent on both viral entry and replication, and ablated in cells unable to signal through the mitochondrial antiviral signaling protein pathway. Thus, during an HSV infection in vivo, multiple mechanisms of pathogen recognition are active, which operate in cell-type- and time-dependent manners to trigger expression of type I IFN and coordinate the antiviral response.     

Reciprocal immunomodulation in a schistosome and hepatotropic virus coinfection model

Human coinfection with the helminth parasite Schistosoma mansoni and hepatitis B and hepatitis C viruses is associated with increased hepatic viral burdens and severe liver pathology. In this study we developed a murine S. mansoni/lymphocytic choriomeningitis virus (LCMV) coinfection model that reproduces the enhanced viral replication and liver pathology observed in human coinfections, and used this model to explore the mechanisms involved. Viral coinfection during the Th2-dominated granulomatous phase of the schistosome infection resulted in induction of a strong LCMV-specific T cell response, with infiltration of high numbers of LCMV-specific IFN-gamma-producing CD8+ cells into the liver. This was associated with suppression of production of the Th2 cytokines dominant during S. mansoni infection and a rapid increase in morbidity, linked to hepatotoxicity. Interestingly, the liver of coinfected mice was extremely susceptible to viral replication. This correlated with a reduced intrahepatic type I IFN response following virus infection. Schistosome egg Ags were found to suppress the type I IFN response induced in murine bone marrow-derived dendritic cells by polyinosinic-polycytidylic acid. These results suggest that suppression of the antiviral type I IFN response by schistosome egg Ags in vivo predisposes the liver to enhanced viral replication with ensuing immunopathological consequences, findings that may be paralleled in human schistosome/hepatotropic virus coinfections.