Hungarian Isolates Research Articles

Phylogenetic analysis of Hungarian goose parvovirus isolates and vaccine strains

Polymerase chain reaction and sequencing were used to analyse goose parvovirus field isolates and vaccine strains. Two fragments of the genome were amplified. Fragment “A” represents a region of VP3 gene, while fragment “B” represents a region upstream of the VP3 gene, encompassing part of the VP1 gene. In the region of fragment “A” the deduced amino acid sequence of the strains was identical, therefore differentiation among strains could be done only at the nucleotide level, which resulted in the formation of three groups: Hungarian, West-European and Asian strains. In the region of fragment “B”, separation of groups could be done by both nucleotide and deduced amino acid sequence level. The nucleotide sequences resulted in the same groups as for fragment “A” but with a different clustering pattern among the Hungarian strains. Within the “Hungarian” group most of the recent field isolates fell into one cluster, very closely related or identical to each other, indicating a very slow evolutionary change. The attenuated strains and field isolates from 1979/80 formed a separate cluster. When vaccine strains and field isolates were compared, two specific amino acid differences were found that can be considered as possible markers for vaccinal strains. Sequence analysis of fragment “B” seems to be a suitable method for differentiation of attenuated vaccine strains from virulent strains.

An Unusual Feature at the N‐terminal end of the Coat Protein of Maize dwarf mosaic virus isolated in Hungary

AbstractMaize dwarf mosaic is the most widespread virus disease affecting corn production in Hungary. In attempts to identify the causal virus by test plant reactions, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), only Maize dwarf mosaic virus (MDMV) was detected. To further characterize Hungarian isolates of MDMV, one isolate from each of the sweet corn varieties Dallas, Royalty and GH23‐85 was selected for sequence analysis of its coat protein (CP) gene. The three Hungarian isolates shared CP amino acid sequence similarities of 95–98% not only with one another but also with MDMV isolates from other countries. However, the N‐terminus of the CP of the ‘Dallas’ isolate was unusual in containing a stretch of 13 additional amino acids. This is the first report of variation in the size of the N‐terminus of the MDMV CP.

Canine onchocercosis in Greece: report of further 20 cases and molecular characterization of the parasite and its Wolbachia endosymbiont

Recently, sporadic cases of subconjunctival Onchocerca infection have been reported in dogs in Greece and Hungary. Herein we report further cases from Greece and the results of the molecular analysis of Onchocerca sp. removed from Greek dogs and its Wolbachia endosymbionts. Twenty dogs of various breeds, 1–11 years of age with subconjunctival onchocercosis (4 cases each in right or left eye, 12 cases in both eyes) were presented having similar manifestations. Periorbital swelling, exophthalmos, lacrimation, discharge, photophobia, conjunctival congestion, corneal edema, protrusion of the nictitating membrane, and subconjunctival granuloma or cyst formation were the most important clinical signs. After surgical excision of the periocular masses containing the worms, all animals recovered fully from onchocercosis. Based on the similarities of the clinical picture of the Greek and Hungarian cases, the similar morphology of the Greek and Hungarian isolates, and the identical sequences of the cytochrome oxidase gene of the filarial parasites and that of the 16S ribosomal RNA gene from their Wolbachia endosymbionts, the Onchocerca sp. isolated from dogs in Greece and Hungary appears to belong to the same species.

Antigenic and genetic diversity of early European isolates of Infectious bursal disease virus prior to the emergence of the very virulent viruses: early European epidemiology of Infectious bursal disease virus revisited?

Eleven Polish and Hungarian isolates of Infectious bursal disease virus (IBDVs) obtained in the 70/80s (early IBDV) and in the 90s (recent IBDV) were characterized in an Antigen-Capture-ELISA with a panel of neutralizing monoclonal antibodies (Mabs), and by nucleotide sequencing of the VP2 variable domain (vVP2). The viruses were compared with reference IBDV strains, among others with Faragher 52/70 (F52/70, classical, isolated 1970), 89163 (typical very virulent-vvIBDV, isolated 1989) and 91168 (antigenically modified vvIBDV, isolated 1991). Only one of the early isolates (Hungarian strain P1) proved antigenically and genetically similar to F52/70. Other early isolates exhibited no reactivity versus Mabs 3, 4, 5 and/or 8 and had a common previously unrecognized combination of amino acid changes in vVP2. The recent isolates all proved antigenically and genetically related to typical vvIBDV strain 89163, except the Polish isolate 93/35 which proved related to the 91168 strain although no epidemiological relationship had been documented between these viruses in the field. Phylogenetic analysis confirmed that the non-P1 early IBDVs represent a previously unrecognized group among serotype 1 IBDVs. It is discussed whether these early isolates are derivatives of the F52/70-like viruses that might still be present in the field, or whether they represent early IBDV strains that might have been present prior to and progressively replaced by the F52/70-like viruses, as the latter have been replaced by vvIBDVs in the late eighties.

Characterisation of Hungarian porcine circovirus 2 genomes associated with PMWS and PDNS cases.

The authors report the data of the first survey on the incidence of post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS) in Hungary. A PCR method specific for the detection of porcine circovirus 2 (PCV-2) was developed, which proved to be suitable for diagnostic purposes. PCR screening of organ samples from pigs suspected to be affected with PMWS or PDNS revealed the presence of PCV-2 in 80% of the cases. Six PCV-2 genomes from Hungarian isolates were completely sequenced. Phylogenetic comparison with all the available PCV-2 sequences showed that porcine circoviruses circulating in Hungary are more variable than in several other European countries. Two Hungarian strains clustered together with the Spanish strains forming a distinct group; two others fell in a common group with the French, UK, and Dutch strains, whereas another two strains showed the closest relationship to two of the three known German PCV-2 sequences.

Sequence Variability of Helper Component Protein of Potato Virus Y Identified by Thermodynamic Methods

An extent of helper component protein (HC-Pro) sequence variability within virus population of single Czech isolate of potato virus YNTN (PVYNTN) Nicola was identified by temperature-gradient gel electrophoresis (TGGE) and heteroduplex analysis. HC-Pro region was approximated with 6 pairs of primers derived from Hungarian PVYNTN isolate (sequence AC M95491). Immunocapture reverse transcription - polymerase chain reaction (RT-PCR) was used to obtain six mixtures of individual overlapping polymerase chain reaction (PCR) products. cDNA libraries were prepared by cloning of purified PCR products in pCR-Script vector and screened by heteroduplex analyses. 15 different subfragments within the HC-Pro region were isolated and sequenced. In comparison to AC M95491, 19 nucleotide changes were identified, 13 led to amino acid (aa) changes. In comparison to available post-transcriptional gene silencing (PTGS) specific suppressor HC-Pro sequences of potyviruses we found out 4 aa changes in conserved regions.

Characterization of Hungarian isolates of zucchini yellow mosaic virus (ZYMV, potyvirus) transmitted by seeds of Cucurbita pepo var Styriaca.

Zucchini yellow mosaic virus (ZYMV) has emerged as an important pathogen of cucurbits within the last few years in Hungary. The Hungarian isolates show a high biological variability, have specific nucleotide and amino acid sequences in the N-terminal region of coat protein and form a distinct branch in the phylogenetic tree. The virus is spread very efficiently in the field by several aphid species in a non-persistent manner. It can be transmitted by seed in holl-less seeded oil pumpkin (Cucurbita pepo (L) var Styriaca), although at a very low rate. Three isolates from seed transmission assay experiments were chosen and their nucleotide sequences of coat proteins have been compared with the available CP sequences of ZYMV. According to the sequence analysis, the Hungarian isolates belong to the Central European branch in the phylogenetic tree and, together with the ZYMV isolates from Austria and Slovenia, share specific amino acids at positions 16, 17, 27 and 37 which are characteristic only to these isolates. The phylogenetic tree suggests the common origin of distantly distributed isolates which can be attributed to widespread seed transmission.

Standard and Swedish variant types of the hybrid alder Phytophthora attacking alder in Hungary.

A new Phytophthora disease of common alder (Alnus glutinosa) similar to that previously reported in several countries in Europe has been observed in Hungary. Based on these earlier studies, the alder Phytophthora was considered likely to be a hybrid between P cambivora and a P fragariae-like species: across Europe a range of new alder Phytophthora is spreading that comprise a range of heteroploid hybrids including a 'standard' hybrid type and several other hybrid types termed 'variants'. Phenotypic and molecular features of the pathogen in Hungary were characterised and compared with isolates from elsewhere. The morphologies of five isolates from one region (Hévíz) resembled the common, 'standard' type, whereas the three isolates from another region (Hanság) exhibited traits similar to those of one of the 'variant' types, ie the Swedish 'variant'. Molecular markers of these two groups of Hungarian isolates also represented a good fit to those of the standard type and the Swedish variant, respectively. Isozyme patterns and profiles of restriction fragments of the entire internal transcribed spacer (ITS) region or mitochondrial DNAs and of RAPD-PCR products did not differ within a group, but distinct polymorphisms were exhibited between the two groups of isolates. Southern analysis of random amplified polymorphic DNA (RAPD) revealed the homologous nature of co-migrating bands of P cambivora and the isolates of alder Phytophthora. Furthermore, restriction fragment profiles of the ITS region of ribosomal DNAs and the mtDNAs were consistent with reported biparental origin of alder Phytophthora. The hybrid status of these continuously evolving pathogens raises many issues and challenges concerning efficient control measures.

Site-specific mutagenesis analysis of PBP 1A from a penicillin-cephalosporin-resistant pneumococcal isolate.

Our mutagenesis study has investigated all amino acid mutations in the penicillin-binding domain of PBP 1A from Hungarian pneumococcal isolate 3191 to determine the importance of every mutation in the development of penicillin and cefotaxime resistance. Our data reveal that mutations at amino acid positions 574 to 577 and position 539 cause penicillin and cefotaxime resistance.

In vitro sensitivity of Hungarian Actinobaculum suis strains to selected antimicrobials.

In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC50 values in the range of 0.05-3.125 micrograms/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC50 value of oxytetracycline and spectinomycin was 6.25 and 12.5 micrograms/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC50 values were in the range of 25-100 micrograms/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman's rho 0.789, p < 0001). MIC values of the type strain and MIC50 values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.

Characterization of Plum Pox Virus PPV-BT-H Isolated from Naturally Infected Blackthorn (Prunus spinosa L.) in Hungary

Plum pox virus (PPV) was found naturally infecting blackthorn (Prunus spinosa L.) plants in different regions in Hungary. The virus was identified on the basis of reactions with indicator plants, by DAS-ELISA tests and by RT-PCR. PPV isolated from blackthorn (PPV-BT-H) caused yellow lesions with a necrotic centre on Chenopodium foetidum L. indicating that it belongs to the intermediate pathotype. The coat protein gene of the blackthorn isolate was cloned, sequenced and compared with other PPV sequences. The BT-H isolate and the Hungarian plum isolate SK68 belong to different subclusters of the M group in contrast to the Hungarian almond isolate (PA) which belongs to the D group. Detecting PPV on blackthorn demonstrated that this plant may be an important source and reservoir for PPV in Hungary.

Alterations in MurM, a Cell Wall Muropeptide Branching Enzyme, Increase High-Level Penicillin and Cephalosporin Resistance in Streptococcus pneumoniae

We report that alteration in MurM, an enzyme involved in the biosynthesis of branched-stem cell wall muropeptides, is required for maximal expression of penicillin and cefotaxime resistance in the pneumococcus. Hungarian isolate 3191 (penicillin MIC, 16 microg/ml; cefotaxime MIC, 4 microg/ml) was a source of donor DNA in transformation experiments. Penicillin-binding protein DNA was insufficient to transform recipient strain R6 to full resistance. Further transformation with altered murM DNA was required for full expression of donor penicillin and cefotaxime resistance.

Serological, Pathological and Molecular Characterisation of Hungarian Pepper Mild Mottle Tobamovirus (PMMoV) Isolates

Last year pepper growers observed symptoms referring to virus-infection in pepper plantations in plastic tunnels. Infected plants showed mosaic symptoms or mottling of the leaves, while on the fruits necrotic spots developed. These symptoms referred to a tobamovirus infection. Collected samples were examined by serological and pathological methods, followed by the biological characterisation of the isolates. For serological studies the DAS-ELISA method was used, in which the pathogen was identified as pepper mild mottle tobamovirus. During the pathological examination different host-plants have been used including some pepper varieties containing different L genes (L + –L 4). It was found, that the Hungarian isolates belonged to the P 1,2 pathotype and were closely related to the Spanish isolate (PMMV-S). PCR- studies proved the presence of the PMMoV P 1,2 pathotype in Hungary as well.

CHARACTERIZATION OF HUNGARIAN ISOLATES OF APPLE CHLOROTIC LEAF SPOT VIRUS

Apple chlorotic leaf spot trichovirus is considered one of the most economically important viruses affecting both pome and stone fruit species. High biological and molecular variability is known to occur among ACLSV isolates of different countries. Occurrence of ACLSV on apple in Hungary is known since the age of ‘60s. Severe symptoms, not characteristic for any known virus disease of stone fruits, were observed on peach in 1974 and on wild cherry in 1982. Later it was suspected that those symptoms were caused by atypical isolates of ACLSV. Therefore the above virus isolates were studied and compared to ACLVS isolates from apple. IC-RT-PCR, Western blot analyses as well as on cloning and sequencing of PCR products were performed. On the basis of electrophoretic mobility of the coat protein, which ranged between 19.7 and 22.7 kDa, Hungarian isolates could be classified, with different percentages, into three distinct groups. Six of 12 isolates belonged to Cis, 5 belonged to Bit and one mixed infection (Cen+Bit) was found. Virus cDNA was amplified by IC-RT-PCR. Amplified fragments of two apple, one peach and two cherry isolates were cloned, sequenced and compared. Homology of the compared isolates ranged between 81-94%.

Comparative Amino Acid Analysis of Coat Proteins of Plum Pox Virus Isolates

ABSTRACTSeven PPV isolates (PPV-A, PPV-P, PPV-C, PPV-NL, PPV-P48, PPV-P45 and PPV-II/7) from different natural hosts (apricot, plum, peach and sour cherry) were compared on the base of amino acid sequences of their coat protein genes. The results obtained allowed the division of that isolates into two subgroups—A and B. Two of the isolates, PPV-C and PPV-NL showed high level of identity with D and AT isolates and were placed into subgroup A. PPV-P48, PPV-P45 and PPV-II/7 shared significant sequence homology also with D and AT strains, but they all had deletions in their coat proteins and formed a detached subcloster into subgroup A. PPV-A and PPV-P isolates were placed into subgroup B, on the base of high level of identities with the Hungarian isolate SK-68.

Genetic typing of classical swine fever virus isolates from the territory of the Czech Republic.

Epizootiological studies based on genetic typing were performed using 14 isolates from outbreaks of classical swine fever (CSF) in domestic pigs and wild boar in the Czech Republic which occurred between 1991 and 1998. They were compared with Austrian, Slovakian, Hungarian, Polish and German isolates. The aim of this study was to characterise the CSF virus isolates and find out the possible relationships between the outbreaks in domestic pigs and wild boar, and to map the spread of the virus in the Czech Republic. For this, fragments of the 5' nontranslated region (5' NTR) and of the E2 glycoprotein gene were sequenced and used for genetic typing. The analysis of both fragments of the genome showed that the Czech isolates belong to two CSF subgroups within group 2, namely to subgroups 2.2 and 2.3. A close relation was found with Austrian isolates from 1992 and 1994, belonging to subgroup 2.2. The isolates in subgroup 2.3 formed a very homogeneous group, although they originated from different regions of the country. They seem identical to two Slovakian isolates from 1998, and differed from Hungarian isolates from 1992. Epizootiological links became evident when the epidemiological data were compared.

First Report of Phytophthora Root and Collar Rot of Alder in Hungary.

In June 1999, a disease associated with mortality of Alnus glutinosa, was observed in a 12- to 18-year-old peatland plantation in northwest Hungary. The root and collar rot symptoms were similar to those caused by Phytophthora cambivora in tree species other than alders. Nine isolations were made from diseased roots and soil samples using the Rhododendron leaf baiting method. Three isolates recovered from two sites, approximately 2 km apart, exhibited similar growth and morphology in vitro and were pathogenic to 2-year-old trees of A. glutinosa following inoculation of root collars. All three isolates had amphigynous long, two-celled antheridia. The mean diameter of oogonia ranged from 39.5 to 64.6 μm. They also produced nonpapillate, ellipsoid, non-caducous sporangia 26.9 to 50.5 μm long and 19.3 to 38.5 μm wide with broad exit pores in soil filtrate. These characteristics were similar to those reported for Phytophthora on alder from elsewhere in Europe and for P. cambivora that is not a pathogen of alder (1,2). However, Hungarian isolates from alder, in contrast to P. cambivora, were homothallic like previously recorded isolates from alder, formed nonornamented oogonia and developed colonies at lower optimum (approximately 25°C) and maximum (approximately 30°C) temperatures on carrot agar. A comparison with Phytophthora from alder from other countries (courtesy of C. M. Brasier) showed that the Hungarian isolates have smooth-walled oogonia typical of Swedish isolates rather than the ornamented oogonia of U.K. isolates, but have the appressed, slightly woolly colony morphology like U.K. isolates rather than the fluffy growth found in Swedish isolates. Moreover, cellulose acetate electrophoresis of glucose-6-phosphate isomerase revealed one homodimer band in Hungarian isolates that was identical with that of the Swedish isolate from alder P876 and isolates P1010 and P1011 of P. cambivora (courtesy of C. M. Brasier). This band comigrated with the middle one of the five-banded U.K. standard isolate P772. Molecular evidence (2) indicates that the Phytophthora from alder with its unusual characteristics is not a species in the strict sense but comprises natural hybrids that may have originated in an interspecific hybridization event between a P. cambivora-like species and an unknown species similar to P. fragariae. On this basis, the Hungarian Phytophthora from alder might have evolved similarly. It remains to be determined whether the pathogen was introduced or has developed independently.

Molecular genotyping of Candida species with special respect to Candida (Torulopsis) glabrata strains by arbitrarily primed PCR.

A set of 46 epidemiologically related or unrelated Candida (Torulopsis) glabrata isolates from four different medical centres in Germany and Hungary, and the type strain of this species, were genetically typed by arbitrarily primed PCR (AP-PCR). The resulting band patterns of C. glabrata strains were compared with those of other species of the genus Candida including C. albicans, C. guilliermondii, C. kefyr, C. parapsilosis, C. tropicalis and C. krusei. After preliminary trials of various reaction parameters and control experiments to test the reproducibility of this method, it was found that consistently reproducible amplification patterns were obtained only when rigorously optimised and standardised reaction conditions were employed. Discriminatory abilities were studied with 29 generated 10-mer oligonucleotides of different G+C content. Typing of clinical isolates with the optimised AP-PCR protocol was then performed with the primer 50-1, with a G+C content of 50%. Sufficiently discriminatory polymorphisms were observed among the band patterns of the Candida species included. The gel electrophoresis patterns of each species showed an adequate similarity. Variations in minor bands were characteristic for comparison at the isolate level. Only three AP-PCR genotypes were identified among the clinical isolates of C. glabrata tested. Two of these genotypes were closely related and appeared to be widespread within German and Hungarian isolates. The third genotype of C. glabrata showed a distinct band pattern. With optimised, validated and standardised assay conditions, the feasibility, sensitivity and rapidity of AP-PCR may offer a discriminatory method for genotyping of yeasts in epidemiological studies, as well as in the control of nosocomial infections.

Morphologic, host specificity, and molecular characterization of a Hungarian Cryptosporidium meleagridis isolate.

This study was undertaken in order to characterize Cryptosporidium meleagridis isolated from a turkey in Hungary and to compare the morphologies, host specificities, organ locations, and small-subunit RNA (SSU rRNA) gene sequences of this organism and other Cryptosporidium species. The phenotypic differences between C. meleagridis and Cryptosporidium parvum Hungarian calf isolate (zoonotic genotype) oocysts were small, although they were statistically significant. Oocysts of C. meleagridis were successfully passaged in turkeys and were transmitted from turkeys to immunosuppressed mice and from mice to chickens. The location of C. meleagridis was the small intestine, like the location of C. parvum. A comparison of sequence data for the variable region of the SSU rRNA gene of C. meleagridis isolated from turkeys with other Cryptosporidium sequence data in the GenBank database revealed that the Hungarian C. meleagridis sequence is identical to a C. meleagridis sequence recently described for a North Carolina isolate. Thus, C. meleagridis is a distinct species that occurs worldwide and has a broad host range, like the C. parvum zoonotic strain (also called the calf or bovine strain) and Cryptosporidium felis. Because birds are susceptible to C. meleagridis and to some zoonotic strains of C. parvum, these animals may play an active role in contamination of surface waters not only with Cryptosporidium baileyi but also with C. parvum-like parasites.

Optimized ribotyping protocol applied to Hungarian Bordetella bronchiseptica isolates: identification of two novel ribotypes

We reported previously that ribotype patterns generated with PvuII and a probe derived from the Escherichia coli rrnB gene could be used to differentiate isolates of Bordetella bronchiseptica. In the present study we report modifications made to the original ribotyping procedure that permit detection in the formerly characterized isolates of an additional 8 fragments with homology to rrnB. Ribotypes were redefined to include these fragments. Although this modification did not permit the detection of novel ribotypes from the previously characterized isolates, it did result in a more accurate reclassification of five of these isolates to other existing ribotypes. It was hypothesized that the additional fragments could form the basis for novel ribotypes in future analyses, and this was supported by the subsequent evaluation of 101 previously uncharacterized pig, rabbit, and dog B. bronchiseptica isolates from Hungary. A total of six different patterns were detected from this group, including two previously not identified that were designated ribotypes 17 and 18. The profile of ribotype 17 includes a novel fragment not associated with any other ribotype. A subset of the fragments constituting ribotype 18, essential for its differentiation from other ribotypes, is only detectable under the modified conditions reported here. Hungarian swine isolates are highly clonal, since 98.2% were identified as ribotype 3. Similarly, 83.7% of rabbit isolates from Hungary are also ribotype 3. Cluster analysis revealed that despite the existence of numerous ribotypes, B. bronchiseptica isolates display limited heterogeneity. The ability to detect additional ribotypes under the modified conditions described in this study strengthens the usefulness of ribotyping as an epidemiologic tool.