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Cytokine profiles during cowpox virus (CPV) strain Brighton and vaccinia virus (VV) strain Western Reserve infections were characterized in intranasal (i.n.) and intraperitoneal (i .p.) models in BALB/c mice. The time-course of induction and effects of cidofovir treatment on interferon (IFN)-γ, IFN-γ inducible protein (IP)-10, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 were determined. The four models have distinct patterns of cytokine induction. CPV i.p. and VV i.n. infections showed increased induction throughout the time studied. CPV i.n. infection resulted in delayed induction of IFN-γ and IP-10. Cytokine levels were fairly constant during VV i.p. infections. Cidofovir treatment (100 mg/kg/day i.p. for 2 days) significantly reduced certain cytokine levels in the four models. Treatment did not affect IP-10 in the CPV i.n. model; IFN-γ and IP-10 in the CPV i.p. model; or IL-6, IP- 10, and MCP-1 in the VV i.p. model. Characterization of cytokine responses has implications for understanding the immune responses and pathogeneses of viral infections in these models.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 49 pages. Includes Vita. Includes bibliographical references.

Einleitung Die Kuhpockenvirusinfektion (CPXV-Infektion) ist eine sporadisch auftretende, meldepflichtige Erkrankung mit zoonotischem Potenzial. Sie wurde in den letzten sechs Jahrzehnten in vielen Säugetierspezies inkl. des Menschen beschrieben und erforscht. Sie äußert sich in zwei Verlaufsformen: mild, lokalisiert, selbstlimitierend oder generalisiert, dramatisch bis letal. Als Erregerreservoir dienen dem Virus wildlebende Nager (Wühlmäuse). Aktuell ist für diese Infektion keine kausale Therapie zugelassen, Prävention kann mittels prophylaktischer Impfung mit dem Modifizierten Vaccinia Virus Ankara (MVA)-Impfstoff erzielt werden. Für Neuweltkameliden wurde die CPXV-Infektion bisher nur dreimal beschrieben. Detaillierte Erkenntnisse zur Epidemiologie, klinischem Erscheinungsbild und Prävention dieser Infektion bei Neuweltkameliden fehlen. Vier unabhängige Cluster von CPXV-Infektion in ostdeutschen Alpakabeständen in nur fünf Jahren, bekannt geworden durch fünf Alpakas mit generalisierter CPXV-Infektion mit jeweils letalem Ausgang als Patienten der Klinik für Klauentiere Leipzig, gaben Anlass, die Infektion bei dieser Spezies genauer zu untersuchen. Ziele der Untersuchungen Ziel der Studie war, auf Basis einer Literaturrecherche einen Überblick über das Wesen der CPXV-Infektion und ihre Relevanz bei Neuweltkameliden zu gewinnen. Anhand von Bestandsuntersuchungen in vier Alpakabeständen sollten Informationen zur Epidemiologie der CPXV-Infektion mit Erregernachweis im Einzeltier, Erregerverbreitung in der Herde und Erregerreservoir gesammelt werden. Ebenso wurde in zwei der vier Alpakabestände eine Bestandsimpfung mit dem MVA-Impfstoff durchgeführt, um die Verträglichkeit und die Immunogenität dieses Impfstoffes beim Alpaka zu überprüfen. Tiere, Material und Methoden Vier Alpakabestände (107 Alpakas) wurden zweimal im Abstand von 19 – 54 Tagen besucht. Anhand der klinischen Untersuchung und verschiedener Proben (Blut, Tupferproben, Krusten von Hautläsionen, Kot) wurden CPXV-infizierte Tiere identifiziert. Wildlebende Nager als potenzielles Erregerreservoir wurden in der Umgebung der Bestände gefangen. Ein indirekter Immunfluoreszenztest (IFA) wurde zum Nachweis CPXV-spezifischer Antikörper aus dem Serum (Alpakas) bzw. Serum/Transsudat (Nager) eingesetzt, eine real-time PCR diente zum Nachweis von CPXV-spezifischer DNA aus Kot/Tupferproben/Krusten (Alpakas) bzw. Organproben (Nager). In zwei Alpakabeständen wurden 94 Tiere mit dem MVA-Impfstoff zweimal im Abstand von vier Wochen geimpft. Eine Ausnahmegenehmigung nach §17c Abs. 4 Nr. 2 Buchstabe a) des Tierseuchengesetzes vom 22. Juni 2004 (BGBl.I S. 1260) lag für beide Bestände vor. Vier Wochen nach jeder Impfung und 6 bzw. 12 Monate nach der 2. Impfung wurden von allen Tieren Serum (für IFA) und Tupferproben (für PCR) entnommen. Etwaige Impfreaktionen wurden vier Wochen nach jeder Impfung aufgezeichnet. Bei 14 Crias wurde 2 – 12 Wochen post partum eine Serumprobe auf spezifische maternale Antikörper untersucht. Ergebnisse Insgesamt wurden 28 von 107 Tieren mittels IFA und/oder PCR als CPXV-infiziert identifiziert. Die Seroprävalenz pro Bestand schwankte zwischen 16,1 % und 81,2 %. Auch beim Alpaka traten zwei klinische Verlaufsformen auf: mild, lokalisiert, selbstlimitierend vs. generalisiert, letal. In zwei Beständen wurden CPXV-spezifische Antikörper in den gefangenen Nagern gefunden. Im dritten Bestand konnte CPXV aus einer Feldmaus (Microtus arvalis) isoliert werden. Vollgenomsequenzierung dieses Isolates und der Vergleich mit dem Genom von CPXV aus einem Alpaka des gleichen Bestandes ergaben 99,997 % Übereinstimmung. In diesem Bestand wurde CPXV an einer Bürste zur Fellpflege nachgewiesen, die Erregerübertragung durch indirekten Tierkontakt und tierbezogene Utensilien erscheint bei Alpakas am wahrscheinlichsten. Die MVA-Impfung war auch bei Alpakas sicher und gut verträglich. Nach zweimaliger Grundimmunisierung konnte eine Antikörper-Seroprävalenz pro Bestand von 81,3 % bzw. 91,7 % nachgewiesen werden. Im Laufe eines Jahres sank diese auf 15,6 % bzw. 45,8 %, Neuerkrankungen wurden nicht detektiert. In 50,0 % der beprobten Crias wurden maternale Antikörper nachgewiesen. Schlussfolgerungen Das ubiquitäre Erregerreservoir Wühlmaus in Verbindung mit an Popularität zunehmenden Alpakas, als vollempfängliche Spezies für CPXV weisen auf ein gesteigertes Risiko für zukünftige zoonotische CPXV-Infektionen hin. Die MVA-Impfung schützt Alpakas erfolgreich vor einer generalisierten CPXV-Infektion. Die Dauer des Impfschutzes und geeignete Auffrischungs-Impfregime gilt es in Langzeitstudien zu erforschen.:Inhalt 1 Einleitung 2 Literaturübersicht 2.1 Das Alpaka 2.1.1 Zoologische Einordnung, Herkunft, Nutzung 2.1.2 Haltung 2.1.3 Viruserkrankungen bei Neuweltkameliden in Europa 2.2 Die Kuhpockenvirusinfektion 2.2.1 Ätiologie 2.2.2 Wirtsspektrum und Erregerreservoir 2.2.3 Diagnostik 2.2.4 Klinisches Erscheinungsbild 2.2.5 Differentialdiagnosen 2.2.6 Therapie/Prophylaxe 2.3 Der MVA-Impfstoff 2.4 Schlussfolgerung aus der Literaturrecherche und Zielstellung 3 Publikation 1 4 Publikation 2 5 Publikation 3 6 Diskussion 7 Zusammenfassung 8 Summary 9 Literaturverzeichnis 10 Danksagung / Introduction Cowpox virus (CPXV) infection is a reportable and potentially zoonotic disease that occurs sporadically in a variety of animals and in humans. It has been extensively researched and described in both domestic and zoo animals as well as in humans. Although infected individuals generally have a mild form of disease, cases of fatal generalized CPXV infection have also been described. Prevention by prophylactic vaccination using modified vaccinia virus Ankara (MVA) vaccine is the only method of protecting animals against disease. CPXV infection has been described in South American camelids, but data on its epidemiology, clinical features and prevention are lacking. Four CPXV outbreaks occurred in unrelated alpaca herds in Eastern Germany in a five-year period. The diagnosis in those herds was based on five cases with severe, generalized and lethal CPXV infection referred to the Department for Ruminants and Swine in Leipzig. The outbreaks provided us with an opportunity to better understand CPXV-infection in this species. Aims of the study The first aim of the study was to conduct a literature review of the clinical features of CPXV infection in South American Camelids and to compare their clinical signs with those of other animal species. The second goal was to evaluate the epidemiology of CPXV infection in four alpaca herds by evaluating the mode of virus transmission and the occurrence of CPXV in individual alpacas and in putative reservoir hosts. The third goal was to determine the safety and immunogenicity of MVA vaccine in alpacas by using in a prime-boost MVA vaccination regimen in two alpaca herds. Material and methods Four alpaca herds (107 animals) were evaluated on two separate occasions, and samples (serum, swab samples, crusts of suspicious pox lesions, feces) were collected to identify CPXV-infected animals. Wild small mammals were trapped on the alpaca farms to investigate the potential source of infection. Serum (alpacas, rodents) and/or transudate (rodents) samples were used to detect CPXV-specific antibodies using an indirect immunofluorescence assay (IFA). Swab samples, crusts and feces (alpacas) or organ tissue (rodents) were used for the detection of CPXV-specific DNA in a real-time PCR. A total of 94 animals from two alpaca herds were vaccinated twice with MVA vaccine. A special exemption was obtained from the relevant Ministries of the Federal States under German law. Blood samples (serum, IFA) and swab samples (PCR) were collected 4 weeks after prime and boost vaccination as well as 6 and 12 months after boost vaccination. In 14 crias, 1 blood sample was collected 2 – 12 weeks after birth to determine the presence of specific maternal antibodies (serum, IFA). Results A total of 28 of 107 animals were diagnosed with CPXV using IFA and/or PCR. Herd seroprevalence ranged from 16.1% to 81.2%. The clinical signs in infected animals were mostly mild, localized and self-limiting, but 5 animals had generalized signs and died. In two herds, CPXV-specific antibodies were found in the local rodent population. In the third herd, CPXV was isolated from a common vole (Microtus arvalis); full genome sequencing and comparison with the genome of CPXV from an alpaca on the same farm revealed a 99.997% match. Virus transmission through indirect contact seems likely because CPXV-specific DNA was detected on a brush used for grooming. MVA vaccine was well tolerated and safe in vaccinated alpacas. Seroprevalence after booster vaccination was 81.3% in one herd and 91.7% in the other. Detectable antibody titers declined to 15.6% and 45.8% over a 12-month period after booster vaccination. New CPXV infections were not detected in this period. Specific maternal antibodies were detected in 50.0% of newborn crias. Conclusions With the recent increase in their popularity, alpacas may pose an increased risk of zoonotic disease spread because of their susceptibility to CPXV infection and their relatively close proximity to reservoir hosts such as rodents. Prevention of generalized CPXV infection in alpacas using MVA vaccine appears feasible. The duration of immunity and appropriate booster vaccination regimens need to be verified in long-term studies.:Inhalt 1 Einleitung 2 Literaturübersicht 2.1 Das Alpaka 2.1.1 Zoologische Einordnung, Herkunft, Nutzung 2.1.2 Haltung 2.1.3 Viruserkrankungen bei Neuweltkameliden in Europa 2.2 Die Kuhpockenvirusinfektion 2.2.1 Ätiologie 2.2.2 Wirtsspektrum und Erregerreservoir 2.2.3 Diagnostik 2.2.4 Klinisches Erscheinungsbild 2.2.5 Differentialdiagnosen 2.2.6 Therapie/Prophylaxe 2.3 Der MVA-Impfstoff 2.4 Schlussfolgerung aus der Literaturrecherche und Zielstellung 3 Publikation 1 4 Publikation 2 5 Publikation 3 6 Diskussion 7 Zusammenfassung 8 Summary 9 Literaturverzeichnis 10 Danksagung

<p>Orthopoxviruses encode many genes that are not essential for viral replication, which often account for differences in pathogenesis among otherwise closely related orthopoxviruses. Although dendritic cells (DCs) are essential to the generation of an effective anti-viral immune response, the effects of different orthopoxviruses on DC function is poorly understood. The objective of these studies was to determine the effect of different orthopoxviruses on DCs. Cowpox virus (CPXV) is ideally suited to this purpose because it encodes the largest and most representative set of accessory genes among orthopoxviruses, it is endemic in mouse populations, and can infect humans. </p><p>We hypothesized that CPXV would have novel mechanisms of evading the immune response that other orthopoxviruses lack, which may exert maximal effect in the context of antigen presenting cells such as DCs, allowing for discovery of novel viral strategies of immune evasion. To test this, CPXV was used to infected mouse bone marrow-derived DCs (BMDCs), and the effect of the virus on DC survival, expression of T-cell costimulatory molecules and cytokine production was determined. The effects of vaccinia virus strain Western Reserve (VV), the prototype of the species, and modified vaccinia virus strain Ankara (MVA), a promising vaccine vector, on mouse BMDCs were also determined. Confirming the hypothesis that CPXV would have different effects on mouse BMDCs from other orthopoxviruses, BMDCs infected with CPXV survived longer in culture than those infected with MVA or VV. In addition, CPXV specifically downregulated MHC I, MHC II, CD40, and CD86, and induced production of significant levels of IL-6 and IL-10.</p><p>Because IL-10 has many suppressive effects on the immune system, inducing IL-10 may provide a selective advantage to CPXV in vivo. To examine the role of IL-10 in a CPXV infection, wild type and IL-10 deficient mice were infected intranasally with CPXV. The effect of CPXV infection on disease morbidity, viral loads, inflammation and the protective immune response was determined. As expected, IL-10 was important in controlling inflammation during CPXV infection, but there was no effect on viral replication or clearance. Surprisingly, IL-10 was important in generation of a protective memory response to CPXV, which may reflect a novel role for IL-10 in the immune response.</p> / Dissertation

De nombreuses espèces de rongeurs montrent des variations cycliques de leurs densités. Ces cycles ont un rôle important dans l'émergence de zoonoses en augmentant les contacts entre l'Homme et l'animal. Cette thèse s'inscrit dans le domaine de l'écologie de la santé qui étudie les interactions entre santé humaine, santé animale et dynamique des écosystèmes. Dans ce cadre, j'ai étudié la communauté de parasites d'une communauté de rongeurs à dynamique cyclique afin d'identifier les réservoirs d'agents de zoonoses et les parasites potentiellement impliqués dans les cycles de cette communauté. Je me suis intéressée à trois espèces de campagnols et à deux espèces de mulots en Franche-Comté. Les résultats et les suivis épizootiologiques réalisés permettent d'inférer les facteurs de risques biotiques et abiotiques associés à l'émergence de ces zoonoses.<br />Trois agents de zoonoses sont présents : deux hantavirus (virus Puumala et Tula) et le virus Cowpox. La dispersion et le comportement social des rongeurs sont importants pour la transmission de ces virus spécifiques (hantavirus) et non spécifique (virus Cowpox). Ces virus sont majoritairement trouvés en milieu forestier. Les communautés de parasites détectées en forêt et en prairie sont différentes. Les infestations par des helminthes sont plus nombreuses en prairie qu'en forêt. Une étude immunogénétique montre l'existence d'allèles de susceptibilité et de résistance aux agents de zoonoses étudiés. Des helminthes ou des acariens pourraient aussi intervenir dans l'infection par ces virus. Un de ces helminthes pourrait être impliqué dans la dynamique de ses hôtes. Mes travaux expérimentaux et de modélisation montrent l'impact du nématode non spécifique Trichuris arvicolae sur la reproduction du campagnol des champs et son rôle régulateur pour les populations d'arvicolinés. <br />Cette thèse contribue à montrer l'importance de la biodiversité et de l'écologie des communautés pour évaluer et gérer les risques pour l'Homme vis-à-vis de zoonoses.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Two vaccinia viruses (VACV) genetically and phenotypically divergent were isolated, in a mixed infection, from a horse lesion during an outbreak of vesicular and exanthematous disease in horses in Southern Brazil and termed Pelotas 1 (P1V) and Pelotas 2 (P2V). This thesis describes studies performed to investigate the pathogenesis of P1V and P2V infection in rabbits and guinea pigs, and to analyze the sequence of genes potentially involved in their phenotype. Chapter 1 investigated the dose-dependent susceptibility of rabbits to P1V and P2V after intranasal (IN) inoculation. Groups of weaning rabbits were inoculated with three doses of each VACV isolate (102.5 TCID50, 104.5 TCID50 e 106.5 TCID50/rabbit). The inoculation resulted in severe respiratory distress and death of most inoculated rabbits regardless the viral strain. Clinical signs started three to six days post-inoculation (pi) and culminated in death or euthanasia at days 5 to 10 pi. Viremia was detected in animals of all groups. All rabbits surviving the infection beyond day 9 pi developed neutralizing antibodies. Interstitial pneumonia, necrossupurative bronchopneumonia and diarrhea were observed in animals which died or were euthanized in extremis. These results demonstrate that P1V and P2V are virulent for rabbits and show no apparent differences in phenotype in this species. Chapter 2 describes the investigation of the susceptibility of rabbits to intradermal (ID) inoculation to VACV, in single or mixed infection. All inoculated animals developed skin lesions characterized by hyperemia, papules, vesicles pustules and ulcers. Infectious virus was detected in cutaneous lesions, lungs and intestine of animals that died during acute infection. These results demonstrate that rabbits develop cutaneous disease and systemic infection after P1V and P2V ID inoculation. Apparently, co-infected animals developed lesions more severe than those submitted to single virus infection. In chapter 3, the susceptibility and the potential of transmission of P1V and P2V by guinea pigs were investigated. For that, guinea pigs were inoculated IN with both P1V and P2V (106 TCID50/ml). The guinea pigs did not showed clinical signs but developed viremia, shed virus in secretions and seroconverted to VACV. Nevertheless, the virus was not transmitted to guinea pig sentinels maintained in close contact or when exposed to food and feces contaminated with VACV. In Chapter 4, four genes involved in virus phenotype/virulence (C7L, K2L, N1L e B1R) were submitted to nucleotide sequencing and analysis. A 15 nucleotide (nt) deletion in K2L gene was identified in P2V. The same pattern of nucleotide deletion was also detected in other genogroup 1 Brazilian VACV isolates. Point mutations were identified in K2L, C7L and N1R genes from P2V isolates when compared to P1V and to a standard VACV strain. The molecular analysis of these genes would not allow the establishment of association between the sequences/genotype and phenotype. However, this analysis indicate that the 15 nt deletion in K2L gene may be used as a molecular marker for genogroup 1 Brazilian VACV isolates. In summary, the results obtained in these studies demonstrate: i. P1V and P2V produce systemic and cutaneous disease in rabbits but they do not exhibit evident differences in virulence for rabbits; ii. Guinea pigs are susceptible to mixed P1V an P2V infection but apparently do not effectively transmit the virus; iii. P1V and P2V present some sequence differences in virulence genes and that a 15 nt deletion in K2L gene may be used as a molecular marker to distinguish between VACV genogroups. / Duas amostras de vírus vaccínia (VACV) geneticamente e fenotipicamente distintas foram isoladas de um mesmo animal em um surto de doença vesicular e exantemática em equinos no Rio Grande do Sul, e denominados Pelotas 1 (P1V) e Pelotas 2 (P2V). Esta tese descreve estudos realizados para investigar a patogenia dos isolados P1V e P2V em coelhos e cobaias, e analisar a sequência de genes potencialmente envolvidos no fenótipo desses isolados. O Capítulo 1 relata a investigação da susceptibilidade dose-dependente de coelhos ao P1V e P2V. Os animais foram inoculados pela via intranasal (IN) com três doses (102,5 DICC50, 104,5 DICC50 e 106,5DICC50/coelho) de cada um dos isolados. A inoculação resultou em enfermidade respiratória grave e morte na maioria dos coelhos, independente do isolado utilizado. Os sinais clínicos iniciaram nos dias 3 e 6 pós-inoculação (pi) e culminaram com a morte ou eutanásia dos animais, 5 a 10 dias pi. Viremia foi detectada em coelhos de todos os grupos. Anticorpos neutralizantes foram detectados em todos os animais que sobreviveram além do dia 9 pi. Pneumonia intersticial com broncopneumonia necrossupurativa e conteúdo líquido intestinal foram lesões observadas em animais inoculados com o P1V ou P2V que evoluíram para a morte ou foram motivo para a eutanásia in extremis. Esses resultados demonstram que P1V e P2V são virulentos para coelhos e não apresentam diferenças evidentes de patogenia nessa espécie. No Capítulo 2 foi investigada a susceptibilidade de coelhos após inoculação de VACV pela via intradérmica (ID). Para isso, os coelhos foram inoculados com um dos isolados ou com ambos. Todos os coelhos inoculados apresentaram lesões de pele caracterizadas por hiperemia, pápulas, vesículas, pústulas e úlceras. Excreção viral foi detectada nas lesões cutâneas e também em amostras de pulmão e intestino de animais que morreram durante a fase aguda da infecção. Os resultados desta inoculação demonstraram que coelhos desenvolvem doença cutânea e sistêmica após a inoculação ID de P1V e P2V. Algumas evidências indicam que os coelhos co-infectados desenvolveram lesões mais severas do que na infecção simples. No Capítulo 3, investigou-se a susceptibilidade e o potencial de transmissibilidade dos isolados P1V e P2V por cobaias. Para isso, cobaias foram inoculadas pela via intranasal (IN) com uma mistura dos isolados P1V e P2V (106 DICC50/ml). As cobaias não apresentaram sinais clínicos, porém excretaram o vírus nas secreções nasais, desenvolveram viremia e soroconverteram para VACV. Apesar disso, o vírus não foi transmitido a sentinelas por contato direto, indireto (aerossóis) ou por água e alimentos contaminados com fezes deliberadamente infectadas com o vírus. No Capítulo 4, quatro genes (C7L, K2L, N1L e B1R) envolvidos no fenótipo do VACV foram amplificados por PCR, sequenciados e submetidos à análise molecular. Uma deleção de 15 nucleotídeos (nt) no gene K2L foi identificada no P2V. Essa mesma deleção também foi identificada em isolados brasileiros do VACV pertencentes ao genogrupo 1. Mutações pontuais foram identificadas nos genes K2L, C7L e N1L no P2V comparando-se com o P1V e cepas de referência do VACV. A análise molecular desses genes não permite associar essas deleções/mutações presentes no P2V com o fenótipo, mas sugere que a deleção de 15 nt no gene K2L possa ser utilizado como marcador molecular de isolados de VACV do genogrupo 1. Em resumo, os resultados obtidos nesses experimentos demonstram que: i. P1V e P2V produzem doença sistêmica e cutânea em coelhos, mas não diferem fenotipicamente nessas espécies; ii. cobaias são susceptíveis à infecção mista pelo P1V e P2V, mas aparentemente não transmitem o vírus com eficiência; iii. P1V e P2V apresentam algumas diferenças em genes de virulência, sendo que a deleção de 15 nt no gene K2L pode ser utilizada como marcador de genogrupos de VACV.