Author Correction: Ecology, evolution and spillover of coronaviruses from bats.

In the past two decades, three coronaviruses with ancestral origins in bats have emerged and caused widespread outbreaks in humans, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the first SARS epidemic in 2002–2003, the appreciation of bats as key hosts of zoonotic coronaviruses has advanced rapidly. More than 4,000 coronavirus sequences from 14 bat families have been identified, yet the true diversity of bat coronaviruses is probably much greater. Given that bats are the likely evolutionary source for several human coronaviruses, including strains that cause mild upper respiratory tract disease, their role in historic and future pandemics requires ongoing investigation. We review and integrate information on bat–coronavirus interactions at the molecular, tissue, host and population levels. We identify critical gaps in knowledge of bat coronaviruses, which relate to spillover and pandemic risk, including the pathways to zoonotic spillover, the infection dynamics within bat reservoir hosts, the role of prior adaptation in intermediate hosts for zoonotic transmission and the viral genotypes or traits that predict zoonotic capacity and pandemic potential. Filling these knowledge gaps may help prevent the next pandemic

Immunology going wild : genetic variation and immunocompetence in the mallard (Anas platyrhynchos)

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Evaluating Effects of AIV Infection Status on Ducks Using a Flow Cytometry-Based Differential Blood Count

ABSTRACT Ducks have recently received a lot of attention from the research community due to their importance as natural reservoirs of avian influenza virus (AIV). Still, there is a lack of tools to efficiently determine the immune status of ducks. The purpose of this work was to develop an automated differential blood count for the mallard duck (Anas platyrhynchos), to assess reference values of white blood cell (WBC) counts in this species, and to apply the protocol in an AIV field study. We established a flow cytometry-based duck WBC differential based on a no-lyse no-wash single-step one-tube technique, applying a combination of newly generated monoclonal antibodies with available duck-specific as well as cross-reacting chicken markers. The blood cell count enables quantification of mallard thrombocytes, granulocytes, monocytes, B cells, CD4+ T cells (T helper) and CD8+ cytotoxic T cells. The technique is reproducible, accurate, and much faster than traditional evaluations of blood smears. Stabilization of blood samples enables analysis up to 1 week after sampling, thus allowing for evaluation of blood samples collected in the field. We used the new technique to investigate a possible influence of sex, age, and AIV infection status on WBC counts in wild mallards. We show that age has an effect on the WBC counts in mallards, as does sex in juvenile mallards. Interestingly, males naturally infected with low pathogenic AIV showed a reduction of lymphocytes (lymphocytopenia) and thrombocytes (thrombocytopenia), which are both common in influenza A infection in humans. IMPORTANCE Outbreaks of avian influenza in poultry and humans are a global public health concern. Aquatic birds are the primary natural reservoir of avian influenza viruses (AIVs), and strikingly, AIVs mainly cause asymptomatic or mild infection in these species. Hence, immunological studies in aquatic birds are important for investigating variation in disease outcome of different hosts to AIV and may aid in early recognition and a better understanding of zoonotic events. Unfortunately, immunological studies in these species were so far hampered by the lack of diagnostic tools. Here, we present a technique that enables high-throughput white blood cell (WBC) analysis in the mallard and report changes in WBC counts in wild mallards naturally infected with AIV. Our protocol permits large-scale immune status monitoring in a widespread wild and domesticated duck species and provides a tool to further investigate the immune response in an important reservoir host of zoonotic viruses

Health monitoring in birds using bio-loggers and whole blood transcriptomics

Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards (Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases.publishe

Comparative Genomics of the Waterfowl Innate Immune System.

Funder: International Max Planck Research School of Organismal BiologyFunder: Universities of the State of Baden-WürttembergAnimal species differ considerably in their ability to fight off infections. Finding the genetic basis of these differences is not easy, as the immune response is comprised of a complex network of proteins that interact with one another to defend the body against infection. Here, we used population- and comparative genomics to study the evolutionary forces acting on the innate immune system in natural hosts of the avian influenza virus (AIV). For this purpose, we used a combination of hybrid capture, next- generation sequencing and published genomes to examine genetic diversity, divergence, and signatures of selection in 127 innate immune genes at a micro- and macroevolutionary time scale in 26 species of waterfowl. We show across multiple immune pathways (AIV-, toll-like-, and RIG-I -like receptors signalling pathways) that genes involved genes in pathogen detection (i.e., toll-like receptors) and direct pathogen inhibition (i.e., antimicrobial peptides and interferon-stimulated genes), as well as host proteins targeted by viral antagonist proteins (i.e., mitochondrial antiviral-signaling protein, [MAVS]) are more likely to be polymorphic, genetically divergent, and under positive selection than other innate immune genes. Our results demonstrate that selective forces vary across innate immune signaling signalling pathways in waterfowl, and we present candidate genes that may contribute to differences in susceptibility and resistance to infectious diseases in wild birds, and that may be manipulated by viruses. Our findings improve our understanding of the interplay between host genetics and pathogens, and offer the opportunity for new insights into pathogenesis and potential drug targets

Health monitoring in birds using bio-loggers and whole blood transcriptomics

Monitoring and early detection of emerging infectious diseases in wild animals is of crucial global importance, yet reliable ways to measure immune status and responses are lacking for animals in the wild. Here we assess the usefulness of bio-loggers for detecting disease outbreaks in free-living birds and confirm detailed responses using leukocyte composition and large-scale transcriptomics. We simulated natural infections by viral and bacterial pathogens in captive mallards (Anas platyrhynchos), an important natural vector for avian influenza virus. We show that body temperature, heart rate and leukocyte composition change reliably during an acute phase immune response. Using genome-wide gene expression profiling of whole blood across time points we confirm that immunostimulants activate pathogen-specific gene regulatory networks. By reporting immune response related changes in physiological and behavioural traits that can be studied in free-ranging populations, we provide baseline information with importance to the global monitoring of zoonotic diseases

A Comparison of Host Responses to Infection with Wild-Type Avian Influenza Viruses in Chickens and Tufted Ducks

ABSTRACT Cross-species transmission of influenza A virus (IAV) from wild waterfowl to poultry is the first step in a chain of events that can ultimately lead to exposure and infection of humans. Herein, we study the outcome of infection with eight different mallard-origin IAV subtypes in two different avian hosts: tufted ducks and chickens. We found that infection and shedding patterns as well as innate immune responses were highly dependent on viral subtypes, host species, and inoculation routes. For example, intraoesophageal inoculation, commonly used in mallard infection experiments, resulted in no infections in contrast to oculonasal inoculation, suggesting a difference in transmission routes. Despite H9N2 being endemic in chickens, inoculation of mallard-origin H9N2 failed to cause viable infection beyond 1 day postinfection in our study design. The innate immune responses were markedly different in chickens and tufted ducks, and despite the presence of retinoic acid-inducible gene-I (RIG-I) in tufted duck transcriptomes, it was neither up nor downregulated in response to infection. Overall, we have revealed the heterogeneity of infection patterns and responses in two markedly different avian hosts following a challenge with mallard-origin IAV. These virus-host interactions provide new insights into important aspects of interspecies transmission of IAV. IMPORTANCE Our current findings highlight important aspects of IAV infection in birds that have implications for our understanding of its zoonotic ecology. In contrast to mallards where the intestinal tract is the main site of IAV replication, chickens and tufted ducks show limited or no signs of intestinal infection suggesting that the fecal-oral transmission route might not apply to all bird IAV host species. Our results indicate that mallard-origin IAVs undergo genetic changes upon introduction into new hosts, suggesting rapid adaptation to a new environment. However, similar to the mallard, chickens and tufted ducks show a limited immune response to infection with low pathogenic avian influenza viruses. These findings and future studies in different IAV hosts are important for our understanding of barriers to IAV transmission between species and ultimately from the wild reservoir to humans

Ecology, evolution and spillover of coronaviruses from bats.

In the past two decades, three coronaviruses with ancestral origins in bats have emerged and caused widespread outbreaks in humans, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the first SARS epidemic in 2002-2003, the appreciation of bats as key hosts of zoonotic coronaviruses has advanced rapidly. More than 4,000 coronavirus sequences from 14 bat families have been identified, yet the true diversity of bat coronaviruses is probably much greater. Given that bats are the likely evolutionary source for several human coronaviruses, including strains that cause mild upper respiratory tract disease, their role in historic and future pandemics requires ongoing investigation. We review and integrate information on bat-coronavirus interactions at the molecular, tissue, host and population levels. We identify critical gaps in knowledge of bat coronaviruses, which relate to spillover and pandemic risk, including the pathways to zoonotic spillover, the infection dynamics within bat reservoir hosts, the role of prior adaptation in intermediate hosts for zoonotic transmission and the viral genotypes or traits that predict zoonotic capacity and pandemic potential. Filling these knowledge gaps may help prevent the next pandemic