Observing and modeling global warming impacts in Virginia

VIMS climate change white paper: Observing and modeling global warming impacts in Virgini

Immune responses of the Egyptian Rousette Bat - Deciphering the unique immunity of an important reservoir host for zoonotic viruses

Die Ordnung der Fledertiere (Chiroptera) macht etwa 20% aller Säugetierarten aus und zieht wegen ihres einzigartigen Viroms weltweit großes Interesse auf sich. Fledertiere können, unter Ausprägung geringer oder keiner Krankheitsanzeichen, eine Vielzahl von hochpathogenen zoonotischen Viren wie Filo , Lyssa und Coronaviren beherbergen. Angesichts der Virom-Vielfalt, ihrer Fähigkeit zum autonomen Flug und ihrer globalen Verbreitung ist das Verständnis der Immunität von Fledertieren von entscheidender Bedeutung, um die Einschätzung künftiger Spillover-Ereignisse und Risiken zu optimieren. Der Mangel an Fledertier-spezifischem oder kreuzreaktivem Material und standardisierten Modellsystemen erschwert dies jedoch bis heute. Darüber hinaus hat die schiere Anzahl der Arten zu isolierten Datensätzen geführt, was Interpretation und Identifizierung allgemeiner und artspezifischer Immunmechanismen erschwert. Um die Immunität der Fledertiere gezielt zu adressieren, benötigt es koordinierte Forschungsansätze zur möglichst umfassenden Untersuchung vieler Arten. In dieser Arbeit wird eine eingehende Untersuchung der angeborenen und adaptiven Immunmechanismen des Nilflughundes (Rousettus aegyptiacus, Egyptian Rousette bat [ERB]) vorgestellt. Detaillierte Stabilitätsanalysen identifizierten EEF1A1 als überlegenes Referenzgen gegenüber ACTB und GAPDH, deren Expression bei Temperaturerhöhung oder Anwesenheit von Typ-I-Interferon (IFN) instabil ist. Die Expression eines Referenzgens muss auch unter diesen für Fledertiere physiologischen Bedingungen stabil sein. Zur Untersuchung der molekularen angeborenen Immunität, wurden Zelllinien aus dem Nasenepithel, dem Riech-system und dem Großhirn generiert. Um die Immunreaktionen von primären Epithelzellen einzubeziehen, die für Immunität an Orten der initialen Virusinfektion entscheidend sind, wurden Zellen aus Nasenepithel, Trachea, Lunge und Dünndarm generiert. Die Zellidentitäten wurden mit umfassenden Analysen der von jeder Zelllinie exprimierten Transkripte und Proteine bestimmt. Die Fähigkeit jeder Zelllinie, Typ I- und III-IFN zu produzieren, wurde bei 37°C und 40°C nach Stimulation mit viralen Mimetika untersucht. Dabei zeigten sich zelltypabhängige Unterschiede in der Fähigkeit, IFN zu exprimieren. Darüber hinaus war die konstitutive Expression von Typ-I- und III-IFN bei 40°C erhöht. Um die angeborene Immunität von ERB bei Infektionen mit zoonotischen Viren zu charakterisieren, wurden Zellen aus dem Nasenepithel, dem Riechsystem und dem Gehirn mit verschiedenen Lyssaviren infiziert. Darüber hinaus zeigten sich auffällige Unterschiede in der Suszeptibilität, da Zellen aus dem Nasenepithel weniger, Zellen aus dem Riechepithel hingegen vermehrt suszeptibel waren. Ferner konnte gezeigt werden, dass das Lagos bat Lyssavirus (LBV) aufgrund einer fehlenden IFN-Expression in infizierten Zellen vermutlich fortgeschrittene Immunoevasions-Eigenschaften besitzt. Da die aktuelle SARS-CoV-2-Pandemie Fledertiere weiter in den Fokus der Zoonoseforschung gerückt hat, wurden primäre Epithelzellen und Tiere mit diesem Virus infiziert, um ERB-spezifische Immuntranskripte in Zellen und Geweben zu untersuchen. Diese Studien ergaben eine auffallend frühe IFNG-Expression im Respirationstrakt infizierter Individuen. Darüber hinaus wurde das Leukozytenprofil in Peripherie und verschiedenen Geweben bei adulten und juvenilen ERB mittels Durchflusszytometrie und Sequenzierung auf Einzelzellebene (scRNA-seq) analysiert. Die Durchflusszytometrie ergab eine signifikant höhere Anzahl von Granulozyten bei adulten, sowie eine höhere Anzahl von B-Zellen bei juvenilen Tieren. Das scRNA-seq ermöglichte eine detaillierte Identifizierung verschiedener Leukozyten-Untergruppen, unter anderem NKT-ähnlichen Zellen und einer PLAC8-exprimierenden B-Zell-Population. Eine funktionelle Charakterisierung von phagozytierenden Zellen und Lymphozyten aus adulten und juvenilen ERB ergab keine signifikanten Unterschiede in zellulärer Funktionalität. Zusammenfassend lässt sich sagen, dass die vorliegende Arbeit die Eignung aller etablierten ERB-Zelllinien für Untersuchungen der Fledertier-Immunität in vitro gezeigt hat. Zu den Kernergebnissen zählt die Analyse der IFN-Expression im Ruhezustand, bei Stimulation oder Virusinfektion. Darüber hinaus konnten mit Hilfe etablierter qRT-PCR-Protokolle die konstitutive und temperaturabhängige Erhöhung der IFN-Expression sowie die Expression immunbezogener Transkripte in mit SARS-CoV-2 infizierten ERB bestimmt werden. Mithilfe von optimierten scRNA-seq Technologien und Durchflusszytometrie konnten Frequenzen und absolute Zellzahlen in ERB unterschiedlichen Alters bestimmt werden, was z. B. altersabhängige Schwankungen in der Zusammensetzung des Leukozytenprofils aufzeigte.The order of bats (Chiroptera) account for ~20% of all mammalian species and attracted immense global attention due to their identification as important viral reservoir. Bats can harbour a plethora of high-impact zoonotic viruses, such as filoviruses, lyssaviruses, and coronaviruses without displaying clinical signs of disease themselves. Given this striking diversity of the bat virome, their ability of self-powered flight, and global distribution, understanding chiropteran immunity is essential to facilitate assessment of future spillover events and risks. However, scarcity of bat-specific or cross-reactive tools and standardized model systems impede progress until today. Furthermore, the richness of species led to generation of isolated datasets, hampering data interpretation and identification of general immune mechanisms, applicable for various chiropteran suborders/families. The key to unlocking bat immunity are coordinated research approaches that comprehensively define immunity in several species. In this work, an in-depth study of innate and adaptive immune mechanisms in the fructivorous Egyptian Rousette bat (Rousettus aegyptiacus, ERB) is presented. Detailed stability analyses identified EEF1A1 as superior reference gene to ACTB, and GAPDH, which rendered unstable upon temperature increase or presence of type-I-IFN. Since the body core temperatures of pteropid bats reach from 35°C to 41°C and it has been postulated that bats display constitutive expression of IFNs, a suitable reference gene has to be stable under these physiologically relevant conditions. To study cellular innate immunity in detail, cell lines from the nasal epithelium, the olfactory compartment and the cerebrum were generated. To include immune responses of epithelia cells, essential for immunity at sites of primary viral infection, primary epithelia cells from the nasal epithelium, trachea, lung and small intestine were generated. Cellular identities were determined by comprehensive analyses of transcripts and proteins expressed by each cell line. The capacity of each cell line to produce type-I- and III-IFNs was assessed at 37°C and 40°C upon stimulation with viral mimetics. This revealed cell type-dependent differences is the capability to express IFNs upon stimulation. Furthermore, the constitutive expression of type-I- and III-IFNs was significantly elevated in higher temperatures and quantified at mRNA copy levels. To characterize ERB innate immunity upon infection with high-impact zoonotic viruses, cells from the nasal epithelium, the olfactory system, and the brain were infected with several lyssaviruses. This revealed striking differences in susceptibility: cells from the nasal epithelium rendered least whereas cells from the olfactory epithelium rendered most susceptible to viral infection and replication. Additionally, due to a lack of IFN expression in infected cells, it could be shown that LBV possibly possesses advanced strategies to ensure successful replication in ERB cells. Since the current SARS-CoV-2 pandemic put bats even further in the focus of zoonotic research, primary epithelial cells and animals were infected with this virus to monitor ERB-specific immune transcripts in cells and tissues. These studies revealed a notably early IFNG expression in the respiratory tract of infected individuals. To understand immunomaturation in bats, the immune cell landscape in periphery and various tissue in adult and juvenile ERB was analyzed by flow cytometry and scRNA-seq, revealing intriguing, age-dependent variations in the abundance of granulocytes and lymphocytes. Flow cytometry revealed a significantly higher number of granulocytes in adults, as well as higher numbers of B cells in juveniles. scRNA-seq allowed detailed identification of different leukocyte subsets, uncovering the presence of highly-abundant NKT-like cells and a unique PLAC8 expressing B cell population. A functional characterization of phagocytic cells and lymphocytes derived from adult and juvenile ERB revealed no significant differences in cellular functionality. In conclusion, the presented work demonstrated suitability of all established ERB cell lines to study bat immunity in vitro, which led to striking findings regarding IFN expression at steady state, or upon stimulation or viral infection. In addition, established qRT-PCR protocols allowed definition of constitutive and temperature-dependent elevation of IFN expression magnitudes, as well as insights into expression of immune-related transcripts in SARS-CoV-2 infected ERB. Finally, based on optimized scRNA-seq technologies and flow cytometry, frequencies and absolute cell counts could be determined in ERB of different ages, revealing e.g. age-dependent variations in leukocyte profile compositions

Baseline of Physiological Body Temperature and Hematological Parameters in Captive Rousettus aegyptiacus and Eidolon helvum Fruit Bats

The discovery of bats as reservoir hosts for a number of highly pathogenic zoonotic agents has led to an increasing interest of infectious disease research in experimental studies with bats. Therefore, we established breeding colonies of Rousettus aegyptiacus and Eidolon helvum fruit bats, which both have been identified as reservoir hosts for relevant zoonotic disease agents, such as Marburg virus and Lagos bat virus. Since 2013, individuals of both species have been recruited to the Friedrich-Loeffler-Institut (FLI) from zoological gardens in Europe, to where these species had been introduced from the wild several decades ago. The aviaries have been designed according to national recommendations published by the Federal Ministry of Agriculture. Under these conditions, both species have been reproducing for years. To better understand the physiology of these animals, and to generate baseline knowledge for infection experiments, we monitored the body core temperatures of R. aegyptiacus bats in the aviaries, and found a circadian variation between 34 degrees C and 41.5 degrees C. We also determined the hematological parameters of both species, and detected specific differences between both bat species. For values of clinical chemistry, no correlation to age or sex was observed. However, species-specific differences were detected since ALT, BUN and CREA were found to be significantly higher in R. aegyptiacus and GLU and TP were significantly higher in E. helvum bats. A higher hematocrit, hemoglobin and red blood cell level was observed in subadult R. aegyptiacus, with hemoglobin and red blood cells also being significantly increased compared to E. helvum. Lymphocytes were found to be the dominant white blood cells in both species and are higher in female E. helvum. Neutrophil granulocytes were significantly higher in E. helvum bats. This underlines the necessity to define baseline profiles for each bat species prior to their use in experimental challenge

Establishment of a Suitable Diagnostic Workflow to Ensure Sensitive Detection of African Swine Fever Virus Genome in Porcine Semen

The rapid spread of African swine fever virus (ASFV), causing severe and often lethal disease in domestic pigs and Eurasian wild boar, continues to be a threat to pig populations and dependent industries. Despite scientific achievements that have deepened our understanding of ASFV pathogenesis, alternative transmission routes for ASFV remain to be elucidated. We previously demonstrated the efficient transmission of ASFV from infected boars to naïve recipient gilts via artificial insemination, thereby highlighting the importance of surveillance of boar semen prior to its shipment. Since the accurate and reliable detection of even low amounts of ASFV in boar semen is key to disease prevention and control, we established a suitable diagnostic workflow to efficiently detect the ASFV genome in boar semen. Here, we assessed the sensitivity of various routine nucleic acid extraction kits as well as qPCR protocols in detecting the ASFV genome in the blood and semen of infected boars. The feasibility of the respective kits and methods for future use in boar studs was also considered. Variability in sensitivity mostly concerned samples with low to very low amounts of the ASFV genome. Ultimately, we defined a well-suited workflow for precisely detecting the ASFV genome in boar semen as early as 2 days post ASFV infection

Humoral Immune Response of Thai Dogs after Oral Vaccination against Rabies with the SPBN GASGAS Vaccine Strain

Applied research is crucial in pushing the boundaries and finding a solution to the age-old problem of dog-mediated rabies. Although oral vaccination of dogs is considered to have great potential in mass dog vaccination campaigns and could have far-reaching benefits, it is perhaps the most ignored of all available tools in efforts to eliminate dog-mediated rabies, not least because of limited data on immunogenicity, efficacy, and safety of potential oral rabies vaccine candidates. In this study, the long-term immunogenicity in local Thai dogs after oral administration of the highly attenuated 3rd generation rabies virus vaccine strain SPBN GASGAS was assessed. The oral rabies vaccine was administered to dogs by either direct oral administration (n = 10) or by offering a vaccine loaded intestine bait (n = 15). The humoral immune response was then compared to three groups of dogs; a group that received a parenteral delivered inactivated rabies vaccine (n = 10), a group offered a placebo intestine bait (n = 7), and a control group (n = 4) for an observation period of 365 days. There was no significant difference in the immune response of dogs that received oral and parenteral vaccine in terms of magnitude, kinetics, and persistence of both rabies virus (RABV) neutralizing (RFFIT) and binding (ELISA) antibodies. Although the single parenteral injection of an inactivated rabies vaccine mounted a slightly higher humoral immune response than the orally delivered live vaccine, RABV specific antibodies of both types were still detectable after one year in most animals for all treatment groups and resulted in no difference in seropositivity. Characterization of rabies specific antibodies revealed two main classes of antibodies involved in the immune response of dogs vaccinated. While IgM antibodies were the first to appear, the succeeding IgG response was mainly IgG2 dominated independent of the vaccine type used. The results support the view that SPBN GASGAS induces a sustained detectable immune response in local dogs both after direct oral administration and via bait application

Composting of Wild Boar Carcasses in Lithuania Leads to Inactivation of African Swine Fever Virus in Wintertime

African swine fever (ASF) continues to spread and persist in the Eurasian wild boar population. The infection pressure resulting from infected carcasses in the environment can be a major contributor to disease persistence and spread. For this reason, it is crucial to find a safe and efficient method of carcass disposal under different circumstances. In the presented study, we investigated open-air composting of carcasses under winter conditions in northeastern Europe, i.e., Lithuania. We can demonstrate that the ASF virus (ASFV) is inactivated in both entire wild boar carcasses and pieces thereof in a time- and temperature-dependent manner. Composting piles reached up to 59.0 °C, and ASFV was shown to be inactivated. However, the ASFV genome was still present until the end of the 112-day sampling period. While further studies are needed to explore potential risk factors (and their mitigation), such as destruction of composting piles by scavengers or harsh weather conditions, composting seems to present a valid method to inactivate the ASFV in wild boar carcasses where rendering or other disposal methods are not feasible. In summary, composting provides a new tool in our toolbox of ASF control in wild boar and can be considered for carcass disposal

Artificial Insemination as an Alternative Transmission Route for African Swine Fever Virus

The rapid spread of the African swine fever virus (ASFV), causing severe disease with often high fatality rates in Eurasian suids, prevails as a threat for pig populations and dependent industries worldwide. Although advancing scientific progress continually enhances our understanding of ASFV pathogenesis, alternative transmission routes for ASFV have yet to be assessed. Here, we demonstrate that ASFV can efficiently be transferred from infected boars to naïve recipient gilts through artificial insemination (AI). In modern pig production, semen from boar studs often supplies many sow herds. Thus, the infection of a boar stud presents the risk of rapidly and widely distributing ASFV within or between countries. Daily blood and semen collection from four boars after intramuscular inoculation with ASFV strain ‘Estonia 2014’ resulted in the detection of ASFV genomes in the semen as early as 2 dpi, in blood at 1 dpi while semen quality remained largely unaffected. Ultimately, after insemination with extended semen, 7 of 14 gilts were ASFV positive by 7 days post insemination, and all gilts were ASFV positive by 35 days post insemination. Twelve out of 13 pregnant gilts aborted or resorbed at the onset of fever. A proportion of fetuses originating from the remaining gilt showed both abnormalities and replication of ASFV in fetal tissues. Thus, we present evidence for the efficient transmission of ASFV to gilts via AI and also to implanted embryos. These results underline the critical role that boar semen could play in ASFV transmission