Evidence linking APOBEC3B genesis and evolution of innate immune antagonism by gamma-herpesvirus ribonucleotide reductases.

Viruses have evolved diverse mechanisms to antagonize host immunity such as direct inhibition and relocalization of cellular APOBEC3B (A3B) by the ribonucleotide reductase (RNR) of Epstein-Barr virus. Here, we investigate the mechanistic conservation and evolutionary origin of this innate immune counteraction strategy. First, we find that human gamma-herpesvirus RNRs engage A3B via largely distinct surfaces. Second, we show that RNR-mediated enzymatic inhibition and relocalization of A3B depend upon binding to different regions of the catalytic domain. Third, we show that the capability of viral RNRs to antagonize A3B is conserved among gamma-herpesviruses that infect humans and Old World monkeys that encode this enzyme but absent in homologous viruses that infect New World monkeys that naturally lack the A3B gene. Finally, we reconstruct the ancestral primate A3B protein and demonstrate that it is active and similarly engaged by the RNRs from viruses that infect humans and Old World monkeys but not by the RNRs from viruses that infect New World monkeys. These results combine to indicate that the birth of A3B at a critical branchpoint in primate evolution may have been a driving force in selecting for an ancestral gamma-herpesvirus with an expanded RNR functionality through counteraction of this antiviral enzyme.

Pulmonary Granuloma Is Not Always the Tuberculosis Hallmark: Pathology of Tuberculosis Stages in New World and Old World Monkeys Naturally Infected with the Mycobacterium tuberculosis Complex.

We present the pathology of monkeys naturally infected with Mycobacterium tuberculosis complex from five different colonies in Rio de Janeiro, Brazil. On the basis of gross and histopathological findings, the lesions were classified into chronic-active, extrapulmonary, early-activation or latent-reactivation stages. Typical granulomatous pneumonia was seen in 46.6% of cases (six rhesus monkeys [Macaca mulatta] and one Uta Hick's bearded saki [Chiropotes utahickae]). The absence of pulmonary granulomas did not preclude a diagnosis of tuberculosis (TB): classical granulomatous pneumonia was observed in the chronic-active and latent-reactivation stages but not in the extrapulmonary and early-activation stages. The early-activation stage was characterized by interstitial pneumonia with a predominance of foamy macrophages and molecular and immunohistochemical evidence of M. tuberculosis complex infection. TB should be considered as a cause of interstitial pneumonia in New World Monkeys. We recommend the use of immunohistochemistry and molecular analysis for diagnosis of TB, even when typical macroscopic or histological changes are not observed.

The draft genome of mandrill (Mandrillus sphinx): An Old World monkey.

Mandrill (Mandrillus sphinx) is a primate species, which belongs to the Old World monkey (Cercopithecidae) family. It is closely related to human, serving as a model for human health related research. However, the genetic studies on and genomic resources of mandrill are limited, especially in comparison to other primate species. Here we produced 284 Gb data, providing 96-fold coverage (considering the estimated genome size of 2.9 Gb), to construct a reference genome for the mandrill. The assembled draft genome was 2.79 Gb with contig N50 of 20.48 Kb and scaffold N50 of 3.56 Mb. We annotated the mandrill genome to find 43.83% repeat elements, as well as 21,906 protein-coding genes. The draft genome was of good quality with 98% gene annotation coverage by Benchmarking Universal Single-Copy Orthologs (BUSCO). Based on comparative genomic analyses of  the Major Histocompatibility Complex (MHC) of the immune system in mandrill and human, we found that 17 genes in the mandrill that have been associated with disease phenotypes in human such as Lung cancer, cranial volume and asthma, barbored amino acids changing mutations. Gene family analyses revealed expansion of several genes, and several genes associated with stress environmental adaptation and innate immunity responses exhibited signatures of positive selection. In summary, we established the first draft genome of  the mandrill of value for studies on evolution and human health.

Cytokine-Mediated Tissue Injury in Non-human Primate Models of Viral Infections.

Viral infections trigger robust secretion of interferons and other antiviral cytokines by infected and bystander cells, which in turn can tune the immune response and may lead to viral clearance or immune suppression. However, aberrant or unrestricted cytokine responses can damage host tissues, leading to organ dysfunction, and even death. To understand the cytokine milieu and immune responses in infected host tissues, non-human primate (NHP) models have emerged as important tools. NHP have been used for decades to study human infections and have played significant roles in the development of vaccines, drug therapies and other immune treatment modalities, aided by an ability to control disease parameters, and unrestricted tissue access. In addition to the genetic and physiological similarities with humans, NHP have conserved immunologic properties with over 90% amino acid similarity for most cytokines. For example, human-like symptomology and acute respiratory syndrome is found in cynomolgus macaques infected with highly pathogenic avian influenza virus, antibody enhanced dengue disease is common in neotropical primates, and in NHP models of viral hepatitis cytokine-induced inflammation induces severe liver damage, fibrosis, and hepatocellular carcinoma recapitulates human disease. To regulate inflammation, anti-cytokine therapy studies in NHP are underway and will provide important insights for future human interventions. This review will provide a comprehensive outline of the cytokine-mediated exacerbation of disease and tissue damage in NHP models of viral infections and therapeutic strategies that can aid in prevention/treatment of the disease syndromes.

Functional characterization of enhancer evolution in the primate lineage.

BACKGROUND: Enhancers play an important role in morphological evolution and speciation by controlling the spatiotemporal expression of genes. Previous efforts to understand the evolution of enhancers in primates have typically studied many enhancers at low resolution, or single enhancers at high resolution. Although comparative genomic studies reveal large-scale turnover of enhancers, a specific understanding of the molecular steps by which mammalian or primate enhancers evolve remains elusive. RESULTS: We identified candidate hominoid-specific liver enhancers from H3K27ac ChIP-seq data. After locating orthologs in 11 primates spanning around 40 million years, we synthesized all orthologs as well as computational reconstructions of 9 ancestral sequences for 348 active tiles of 233 putative enhancers. We concurrently tested all sequences for regulatory activity with STARR-seq in HepG2 cells. We observe groups of enhancer tiles with coherent trajectories, most of which can be potentially explained by a single gain or loss-of-activity event per tile. We quantify the correlation between the number of mutations along a branch and the magnitude of change in functional activity. Finally, we identify 84 mutations that correlate with functional changes; these are enriched for cytosine deamination events within CpGs. CONCLUSIONS: We characterized the evolutionary-functional trajectories of hundreds of liver enhancers throughout the primate phylogeny. We observe subsets of regulatory sequences that appear to have gained or lost activity. We use these data to quantify the relationship between sequence and functional divergence, and to identify CpG deamination as a potentially important force in driving changes in enhancer activity during primate evolution.

Non-invasive assessment of adrenocortical function as a measure of stress in the endangered golden langur.

The golden langur (Trachypithecus geei) is an endangered primate endemic to northern India and Bhutan. The main stressors to the species are habitat degradation and fragmentation. Non-invasive fecal glucocorticoid metabolite (fGCM) analysis is a powerful tool for assessing stress associated with environmental disturbances in wildlife. However, interspecific differences in glucocorticoid metabolism require careful selection of the antibody used in their quantification. The goals of this study were to: 1) validate an enzyme immunoassay (EIA) to determine fecal GC metabolite (fGCM) concentrations in the golden langur and 2) compare fGCM concentrations between golden langurs living under different environmental conditions. We compared five enzyme immunoassays for determining stress related physiological responses in golden langurs by performing an adrenocorticotropic stimulation test in both sexes. Our validations identified an 11-oxoaetiocholanolone EIA detecting 11, 17 dioxoandrostanes as the most suitable assay for monitoring adrenocortical activity in the species. FGCM concentrations from semi-captive temple langurs were significantly higher than respective stress steroid concentrations in free-ranging or pet/zoo langurs. This study presents a validated practical method for non-invasive monitoring of adrenocortical function in this rare and declining species.

ORONASAL SQUAMOUS CELL CARCINOMAS IN FRANÇOIS' LANGURS (TRACHYPITHECUS FRANCOISI).

Squamous cell carcinomas (SCCs) are common oronasal tumors in nonhuman primates. In this study, 11 cases of oronasal SCC in François' langurs ( Trachypithecus francoisi ) are described. Five initial cases were discovered on review of the North American François' langur studbook, with a potential familial pattern observed. The studbook was used to identify related individuals, and records were requested for review. Six additional cases were documented, and samples from all cases were submitted for microscopic review, as well as polymerase chain reaction (PCR), immunohistochemistry (IHC), and in situ hybridization (ISH), for generic papillomaviruses and PCR for herpesviruses because either virus may cause SCC in humans and other nonhuman primates. Affected langurs commonly presented with facial swelling or ocular discharge but frequently did not have clinical signs, and carcinomas were diagnosed during routine examinations. Carcinomas were located in the oral or nasal cavities affecting the oral mucosa, tongue, hard palate, or oropharynx. Histologically, SCCs comprised anastomosing cords and nests of neoplastic epithelial cells that differentiated synchronously and asynchronously from peripheral basal type cells to central squamous-type cells and were occasionally oriented around accumulations of necrotic cell debris. Nuclear pleomorphism, anisokaryosis, prominent nucleoli, occasional mitoses, and a scirrhous response were common features. All animals tested negative for both viruses, except two langurs that were positive for generic papillomavirus by PCR, but no papillomavirus was detected by either IHC or ISH. In most cases, affected animals died within 5 mo of diagnosis.

Anterograde or Retrograde Transsynaptic Circuit Tracing in Vertebrates with Vesicular Stomatitis Virus Vectors.

Viruses have been used as transsynaptic tracers, allowing one to map the inputs and outputs of neuronal populations, due to their ability to replicate in neurons and transmit in vivo only across synaptically connected cells. To date, their use has been largely restricted to mammals. In order to explore the use of such viruses in an expanded host range, we tested the transsynaptic tracing ability of recombinant vesicular stomatitis virus (rVSV) vectors in a variety of organisms. Successful infection and gene expression were achieved in a wide range of organisms, including vertebrate and invertebrate model organisms. Moreover, rVSV enabled transsynaptic tracing of neural circuitry in predictable directions dictated by the viral envelope glycoprotein (G), derived from either VSV or rabies virus (RABV). Anterograde and retrograde labeling, from initial infection and/or viral replication and transmission, was observed in Old and New World monkeys, seahorses, jellyfish, zebrafish, chickens, and mice. These vectors are widely applicable for gene delivery, afferent tract tracing, and/or directional connectivity mapping. Here, we detail the use of these vectors and provide protocols for propagating virus, changing the surface glycoprotein, and infecting multiple organisms using several injection strategies.

Taxonomy proposal for Old World monkey adenoviruses: characterisation of several non-human, non-ape primate adenovirus lineages.

A species classification regarding Old World monkey adenoviruses is proposed. We determined the nucleotide sequences of PCR-amplified fragments from the genes of the IVa2, DNA-dependent DNA polymerase, penton base, and hexon proteins from every simian adenovirus (SAdV) serotype that originated from Old World monkeys for which the full genome sequence had not yet been published. We confirmed that the majority of Old Word monkey SAdVs belong to two previously established species. Interestingly, one is the most recently established human AdV species, Human mastadenovirus G, which includes a single human virus, HAdV-52, as well as SAdV-1, -2, -7, -11, -12, and -15. The other approved species, Simian mastadenovirus A includes SAdV-3, -4, -6, -9, -10, -14, and -48. Several SAdVs (SAdV-5, -8, -49, -50) together with baboon AdV-1 and rhesus monkey AdV strains A1139, A1163, A1173, A1258, A1285, A1296, A1312, A1327 and A1335 have been proposed to be classified into an additional species, Simian mastadenovirus B. Another proposed species, Simian mastadenovirus C has been described for SAdV-19, baboon AdV-2/4 and -3. Our study revealed the existence of four additional AdV lineages. The corresponding new candidate species are Simian mastadenovirus D (for SAdV-13), Simian mastadenovirus E (for SAdV-16), Simian mastadenovirus F (for SAdV-17 and -18), and Simian mastadenovirus G (for SAdV-20). Several biological and genomic properties, such as the host origin, haemagglutination profile, number of fibre genes, and G+C content of the genome, strongly support this classification. Three SAdV strains originating from the American Type Culture Collection turned out to be mixtures of at least two virus types, either of the same species (SAdV-12 and -15 types from Human mastadenovirus G) or of two different species (SAdV-5 types from Simian mastadenovirus B and Human mastadenovirus G).

Host Range Restriction of Epstein-Barr Virus and Related Lymphocryptoviruses.

Epstein-Barr-related herpesviruses, or lymphocryptoviruses (LCV), naturally infect humans and nonhuman primates (NHP), but their host range is not well characterized. Using LCV and B cells from multiple species of Hominidae and Cercopithecidae, we show that LCV can immortalize B cells from some nonnative species but that growth transformation is restricted to B cells from their own family of hominoids or Old World NHP, suggesting a high degree of LCV adaptation to their natural primate host.

Diversity and prevalence of gastrointestinal parasites in seven non-human primates of the Taï National Park, Côte d'Ivoire.

Parasites and infectious diseases are well-known threats to primate populations. The main objective of this study was to provide baseline data on fecal parasites in the cercopithecid monkeys inhabiting Côte d'Ivoire's Taï National Park. Seven of eight cercopithecid species present in the park were sampled: Cercopithecus diana, Cercopithecus campbelli, Cercopithecus petaurista, Procolobus badius, Procolobus verus, Colobus polykomos, and Cercocebus atys. We collected 3142 monkey stool samples between November 2009 and December 2010. Stool samples were processed by direct wet mount examination, formalin-ethyl acetate concentration, and MIF (merthiolate, iodine, formalin) concentration methods. Slides were examined under microscope and parasite identification was based on the morphology of cysts, eggs, and adult worms. A total of 23 species of parasites was recovered including 9 protozoa (Entamoeba coli, Entamoeba histolytica/dispar, Entamoeba hartmanni, Endolimax nana, Iodamoeba butschlii, Chilomastix mesnili, Giardia sp., Balantidium coli, and Blastocystis sp.), 13 nematodes (Oesophagostomum sp., Ancylostoma sp., Anatrichosoma sp., Capillariidae Gen. sp. 1, Capillariidae Gen. sp. 2, Chitwoodspirura sp., Subulura sp., spirurids [cf Protospirura muricola], Ternidens sp., Strongyloides sp., Trichostrongylus sp., and Trichuris sp.), and 1 trematode (Dicrocoelium sp.). Diversity indices and parasite richness were high for all monkey taxa, but C. diana, C. petaurista, C. atys, and C. campbelli exhibited a greater diversity of parasite species and a more equitable distribution. The parasitological data reported are the first available for these cercopithecid species within Taï National Park.

Evolution of primate α and θ defensins revealed by analysis of genomes.

Defensins are endogenous peptides with cysteine-rich antimicrobial ability that contribute to host defence against bacterial, fungal and viral infections. There are three subfamilies of defensins in primates: α, ß and θ-defensins. α-defensins are most present in neutrophils and Paneth cells; ß-defensins are involved in protecting the skin and the mucous membranes of the respiratory, genitourinary and gastrointestinal tracts; and θ-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin, which are first isolated from rhesus macaques. All three kinds of defensins have six conserved cysteines, three intramolecular disulfide bonds, a net positive charge, and ß-sheet regions. α and θ-defensins are closely related, comparative amino acid sequences showed that the difference between them is that θ-defensins have an additional stop codon limits the initial defensin domain peptides to 12 residues. Humans, chimpanzees and gorillas do not produce θ-defensin peptides due to a premature stop codon present in the signal sequence of all θ-defensin pseudogenes. By using comprehensive computational searches, here we report the discovery of complete repertoires of the α and θ-defensin gene family in ten primate species. Consistent with previous studies, our phylogenetic analyses showed all primate θ-defensins evident formed one distinct clusters evolved from α-defensins. ß-defensins are ancestors of both α and θ-defensins. Human has two copies of DEFA1 and DEFT1P, and two extra DEFA3 and DEFA10P genes compared with gorilla. As different primates inhabit in quite different ecological niches, the production of species-specific α and θ-defensins and these highly evolved θ-defensins in old world monkeys would presumably allow them to better respond to the specific microbial challenges that they face.

The essential detail: the genetics and genomics of the primate immune response.

Next-generation sequencing technologies have led to rapid progress in the fields of human and nonhuman primate (NHP) genomics. The less expensive and more efficient technologies have enabled the sequencing of human genomes from multiple populations and the sequencing of many NHP species. NHP genomes have been sequenced for two main reasons: (1) their importance as animal models in biomedical research and (2) their phylogenetic relationship to humans and use in derivative evolutionary studies. NHPs are valuable animal models for a variety of diseases, most notably for human immunodeficiency virus/acquired immunodeficiency syndrome research, and for vaccine development. Knowledge about the variation in primate immune response loci can provide essential insights into relevant immune function. However, perhaps ironically considering their central role in infectious disease, the accumulation of sequence detail from genomic regions harboring immune response loci, such as the major histocompatibility complex and killer immunoglobulin-like receptors, has been slow. This deficiency is, at least in part, due to the highly repetitive and polymorphic nature of these regions and is being addressed by the application of special approaches to targeted sequencing of the immune response genomic regions. We discuss one such targeting approach that has successfully yielded complete phased genomic sequences from complex genomic regions and is now being used to resequence macaque and other primate major histocompatibility complex regions. The essential detail contained within the genomics of the NHP immune response is now being assembled, and the realization of precise comparisons between NHP and human immune genomics is close at hand, further enhancing the NHP animal model in the search for effective treatments for human disease.

Differential evolutionary fate of an ancestral primate endogenous retrovirus envelope gene, the EnvV syncytin, captured for a function in placentation.

Syncytins are envelope genes of retroviral origin that have been co-opted for a role in placentation. They promote cell-cell fusion and are involved in the formation of a syncytium layer--the syncytiotrophoblast--at the materno-fetal interface. They were captured independently in eutherian mammals, and knockout mice demonstrated that they are absolutely required for placenta formation and embryo survival. Here we provide evidence that these "necessary" genes acquired "by chance" have a definite lifetime with diverse fates depending on the animal lineage, being both gained and lost in the course of evolution. Analysis of a retroviral envelope gene, the envV gene, present in primate genomes and belonging to the endogenous retrovirus type V (ERV-V) provirus, shows that this captured gene, which entered the primate lineage >45 million years ago, behaves as a syncytin in Old World monkeys, but lost its canonical fusogenic activity in other primate lineages, including humans. In the Old World monkeys, we show--by in situ analyses and ex vivo assays--that envV is both specifically expressed at the level of the placental syncytiotrophoblast and fusogenic, and that it further displays signs of purifying selection based on analysis of non-synonymous to synonymous substitution rates. We further show that purifying selection still operates in the primate lineages where the gene is no longer fusogenic, indicating that degeneracy of this ancestral syncytin is a slow, lineage-dependent, and multi-step process, in which the fusogenic activity would be the first canonical property of this retroviral envelope gene to be lost.

Relatively recent evolution of pelage coloration in Colobinae: phylogeny and phylogeography of three closely related langur species.

To understand the evolutionary processes leading to the diversity of Asian colobines, we report here on a phylogenetic, phylogeographical and population genetic analysis of three closely related langurs, Trachypithecus francoisi, T. poliocephalus and T. leucocephalus, which are all characterized by different pelage coloration predominantly on the head and shoulders. Therefore, we sequenced a 395 bp long fragment of the mitochondrial control region from 178 T. francoisi, 54 T. leucocephalus and 19 T. poliocephalus individuals, representing all extant populations of these three species. We found 29 haplotypes in T. francoisi, 12 haplotypes in T. leucocephalus and three haplotypes in T. poliocephalus. T. leucocephalus and T. poliocephalus form monophyletic clades, which are both nested within T. francoisi, and diverged from T. francoisi recently, 0.46-0.27 (T. leucocephalus) and 0.50-0.25 million years ago (T. poliocephalus). Thus, T. francoisi appears as a polyphyletic group, while T. leucocephalus and T. poliocephalus are most likely independent descendents of T. francoisi that are both physically separated from T. francoisi populations by rivers, open sea or larger habitat gaps. Since T. francoisi populations show no variability in pelage coloration, pelage coloration in T. leucocephalus and T. poliocephalus is most likely the result of new genetic mutations after the split from T. francoisi and not of the fixation of different characters derived from an ancestral polymorphism. This case study highlights that morphological changes for example in pelage coloration can occur in isolated populations in relatively short time periods and it provides a solid basis for studies in related species. Nevertheless, to fully understand the evolutionary history of these three langur species, nuclear loci should be investigated as well.

Animal models for the study of hepatitis C virus infection and related liver disease.

Hepatitis C virus (HCV) causes liver-related death in more than 300,000 people annually. Treatments for patients with chronic HCV are suboptimal, despite the introduction of directly acting antiviral agents. There is no vaccine that prevents HCV infection. Relevant animal models are important for HCV research and development of drugs and vaccines. Chimpanzees are the best model for studies of HCV infection and related innate and adaptive host immune responses. They can be used in immunogenicity and efficacy studies of HCV vaccines. The only small animal models of robust HCV infection are T- and B- cell deficient mice with human chimeric livers. Although these mice cannot be used in studies of adaptive immunity, they have provided new insights into HCV neutralization, interactions between virus and receptors, innate host responses, and therapeutic approaches. Recent progress in developing genetically humanized mice is exciting, but these models only permit studies of specific steps in the HCV life cycle and have limited or no viral replication.

A comparison of the neuroendocrine mechanisms underlying the initiation of the preovulatory LH surge in the human, Old World monkey and rodent.

As recognized for decades, the role of the rodent hypothalamus in timing the LH surge is deterministic and mediated by a GnRH discharge that is generated by an obligatory interaction in the preoptic area (POA) between a threshold level of estradiol and a circadian neural signal: a view consistent with contemporary kisspeptinocentric models of the estrous cycle. In higher primates, generation of the LH surge is emancipated from control by the POA. Woman represents the exemplar of the system in higher primates, as the LH surge appears to unfold in the absence of a midcycle GnRH discharge being generated instead by facilitatory interaction between a pulsatile GnRH input to the pituitary and an action of ovarian estradiol. The neurobiology of GnRH pulse generation is only beginning to emerge but from a translational perspective this aspect of hypothalamic function is critical for understanding the human menstrual cycle and how it may be perturbed.

High seroprevalence of enterovirus infections in apes and old world monkeys.

To estimate population exposure of apes and Old World monkeys in Africa to enteroviruses (EVs), we conducted a seroepidemiologic study of serotype-specific neutralizing antibodies against 3 EV types. Detection of species A, B, and D EVs infecting wild chimpanzees demonstrates their potential widespread circulation in primates.

African monkeys are infected by Plasmodium falciparum nonhuman primate-specific strains.

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.

Airborne Mycobacterium avium infection in a group of red-shanked douc langurs (Pygathrix nemaeus nemaeus).

BACKGROUND: This report describes an airborne Mycobacterium avium (MA)-infection in two red-shanked douc langurs (Pygathrix nemaeus nemaeus) from Cologne zoo. METHODS: The two individuals and their tissues were investigated clinically (including x-rays), in pathology, in pathohistology, in classical bacteriology and by polymerase chain reaction (PCR). RESULTS: Clinically, one individual displayed emaciation and a positive reaction in an intrapalpebral testing for M. bovis/MA. The other individual was without any symptoms and did not show any reaction in the intrapalpebral test. In x-ray photos of the lungs, calcified nodules were detected. In pathology, calcified and necrotic nodules were observed within the lungs and the bronchial lymph nodes. In pathohistology, both classical tuberculous granulomas, and few acid fast rods were seen in Ziehl-Neelsen-stain. However, classical bacteriology could not demonstrate mycobacteria. In PCR, MA-infection could be confirmed in one individual using the bronchial lymph nodes. CONCLUSIONS: It was an airborne infection; however, the definite source of infection in these cases remained unclear. Animals in contact to the langurs (house sparrows and mice) as well as water used in the building are the most promising candidates. The risk for a zoonotic transmission in these cases has been calculated to be low.