PhD Thesis Demeter Zoltan

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MHC major histocompatibility complex min minute MLV modified live virus mM micromolar mRNA messenger RNA N nucleocapsid (gene/protein) nm nanometer ORF open reading frame P phospholipid (gene/protein) p.i. post infection PCR polymerase chain reaction RFLP restriction fragment length polymorphism RNA ribonucleic acid RPV raccoon parvovirus RT-PCR reverse transcription polymerase chain reaction sec second SN serum neutralization SNP single nucleotide polymorphism UK United Kingdom USA United States of America UV ultraviolet VN virus neuralization VP viral protein µl microlitre µm micrometer °C degrees Celsius 6
Summary Samples taken from various species from 2004 to 2008 were analyzed for the presence of canine distemper virus (CDV), type 2 canine parvovirus (CPV2), or feline panleukopenia virus (FPV). The samples were collected from animals showing signs of clinical illness (clinical samples), or were collected from animals that succumbed following clinical signs suggestive of a viral infection, or when pathological changes indicative of such a disease were found during necropsy (necropsy samples). In case of clinical samples, the diagnosis was based on various molecular techniques applying polymerase chain reaction (PCR) based techniques, while in case of succumbed animals a wide spectrum of diagnostic methods was employed, such as macroscopic and routine light microscopic investigations, electron microscopy, and PCR-based techniques concluding with nucleic acid sequencing and subsequent phylogenetic analysis. The genetic analysis of Hungarian CDVs revealed the presence of strains belonging to several lineages: European, Arctic and European wildlife. The virus strains clustered in the Arctic group of CDVs were also demonstrated to be responsible for the endemic infection at the Dog Shelter of the City Council of Budapest. On the other hand, CDV infection was demonstrated in several other species from Hungary: fox (Vulpes vulpes), raccoon (Procyon lotor) and ferret (Mustela putorius furo). A restriction fragment length polymorphism (RFLP) assay was also developed for the fast differentiation of vaccine and wild-type CDVs. The practical trials of the PsiI-based RFLP revealed that the virus strain present in one of the currently used vaccines reacted as a wild-type strain. Following the diagnostic PCR reactions, out of the 214 analyzed samples 58 (27.1 %) proved to be positive for CDV. Based on the subsequent nucleic acid sequencing and phylogenetic analysis, the incriminated strain was not clustered in the group of vaccine strains (America-1), as expected based on the product description provided by the manufacturer, but it was more closely related to pathogenic strains of different geographical origins. Vaccine batches of the same manufacturer were purchased in different countries (Israel, Malta, and USA), and batches dating back to 1992 and 1994 were also included in the analysis. The genetic analyses revealed that all these batches contained the exact same virus strains as the Hungarian vaccines purchased in 2006. Parvovirus (FPV/CPV2) genetic material was successfully demonstrated by PCR in 72 (31.3 %) out of the 230 analyzed samples (in 17 cats, 1 lion, 1 Asian palm civet and 53 dogs). The initial genotyping attempts of 20 type 2 canine parvovirus (CPV2) strains from Hungary, using a previously described MboII-based RFLP test, revealed that 7
Page 1 and 2: Szent István University Postgradua
Page 3 and 4: Contents Abbreviations ............
Page 5: aa amino acid Asn asparagine Asp as
Page 9 and 10: Összefoglaló (Summary in Hungaria
Page 11 and 12: 1. Introduction Based on modern sci
Page 13 and 14: 3.1.1 General informations 3. Liter
Page 15 and 16: stomach, and mononuclear cells of t
Page 17 and 18: (noninflammatory demyelination), ho
Page 19 and 20: according to the area of CNS involv
Page 21 and 22: Edward Jenner was probably the firs
Page 23 and 24: comparison, a study conducted by Bo
Page 25 and 26: 3.2.2.1 Neonatal animals Infection
Page 27 and 28: 3.2.4 Pathology Gross pathologic ch
Page 29 and 30: 3.3.1 General informations 3.3 Type
Page 31 and 32: ecovered from the intestinal-associ
Page 33 and 34: 3.3.5 Diagnosis, treatment and prev
Page 35 and 36: een overtaken by CPV2b and/or CPV2c
Page 37 and 38: Table 1: Description of the analyze
Page 39 and 40: purposes by a circus lion tamer fro
Page 41 and 42: Table 4: Primers used for the diagn
Page 43 and 44: 4.5.3.2 Classical PCR assays The 50
Page 45 and 46: 5.1.1 CDV infections 5. Results 5.1
Page 47 and 48: 5.1.2 FPV infections During necrops
Page 49 and 50: 5.2.1 CDV infection Most relevant c
Page 51 and 52: 5.3 Electron microscopy In the urin
Page 53 and 54: amplicons obtained from field virus
Page 55 and 56: 5.4.3 Sequence analysis and phyloge
Page 57 and 58:
Fig. 19: Phylogenetic tree construc
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P gene Vanguard A75/17 Ferret Onder
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(98.90 %) isolated from a naturally
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Fig. 22: Phylogenetic tree construc
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5.4.3.2 FPV strains PCR positive sa
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The alignment of the Hungarian CPV2
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America. The similarity of the Hung
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Only one strain from each subgroup
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Vanguard (Pfizer Animal Health, USA
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performed in order to reveal whethe
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a relatively quick evolutionary pat
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7. New Scientific Results Most impo
Page 81 and 82:
APPEL, M.J.G.: Canine distemper vir
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DE YBANEZ R.R., VELA C., CORTES E.,
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HAAS L., MARTENS W., GREISER-WILKE
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KIMURA, M.: A simple method for est
Page 89 and 90:
MEURS K.M., FOX P.R., MAGNON A.M.,
Page 91 and 92:
PARRISH C.R.: Pathogenesis of felin
Page 93 and 94:
VANDEVELDE M., ZURBRIGGEN A., HIGGI
Page 95 and 96:
10. Other Publications in Peer Revi
Page 97 and 98:
Academy of Sciences and the Doctora
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PhD Thesis Demeter Zoltan

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