The core genome multi-locus sequence typing of Mycoplasma anserisalpingitidis.

BACKGROUND: Mycoplasma anserisalpingitidis is a waterfowl pathogen that mainly infects geese, can cause significant economic losses and is present worldwide. With the advance of whole genome sequencing technologies, new methods are available for the researchers; one emerging methodology is the core genome Multi-Locus Sequence Typing (cgMLST). The core genome contains a high percentage of the coding DNA sequence (CDS) set of the studied strains. The cgMLST schemas are powerful genotyping tools allowing for the investigation of potential epidemics, and precise and reliable classification of the strains. Although whole genome sequences of M. anserisalpingitidis strains are available, to date, no cgMLST schema has been published for this species. RESULTS: In this study, Illumina short reads of 81 M. anserisalpingitidis strains were used, including samples from Hungary, Poland, Sweden, and China. Draft genomes were assembled with the SPAdes software and analysed with the online available chewBBACA program. User made modifications in the program enabled analysis of mycoplasmas and provided similar results as the conventional SeqSphere+ software. The threshold of the presence of CDS in the strains was set to 93% due to the quality of the draft genomes, resulting in the most accurate and robust schema. Three hundred thirty-one CDSs constituted our cgMLST schema (representing 42,77% of the whole CDS set of M. anserisalpingitidis ATCC BAA-2147), and a Neighbor joining tree was created using the allelic profiles. The correlation was observed between the strains' cgMLST profile and geographical origin; however, strains from the same integration but different locations also showed close relationship. Strains isolated from different tissue samples of the same animal revealed highly similar cgMLST profiles. CONCLUSIONS: The Neighbor joining tree from the cgMLST schema closely resembled the real-life spatial and temporal relationships of the strains. The incongruences between background data and the cgMLST profile in the strains from the same integration can be because of the higher probability of contacts between the flocks. This schema can help with the epidemiological investigation and can be used as a basis for further studies.

Novel Orthobunyavirus Causing Severe Kidney Disease in Broiler Chickens, Malaysia, 2014-2017.

During 2014-2017, we isolated a novel orthobunyavirus from broiler chickens with severe kidney lesions in the state of Kedah, Malaysia; we named the virus Kedah fatal kidney syndrome virus (KFKSV). Affected chickens became listless and diarrheic before dying suddenly. Necropsies detected pale and swollen kidneys with signs of gout, enlarged and fragile livers, and pale hearts. Experimental infection of broiler chickens with KFKSV reproduced the disease and pathologic conditions observed in the field, fulfilling the Koch's postulates. Gene sequencing indicated high nucleotide identities between KFKSV isolates (99%) and moderate nucleotide identities with the orthobunyavirus Umbre virus in the large (78%), medium (77%), and small (86%) genomic segments. KFKSV may be pathogenic for other host species, including humans.

Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates.

Mycoplasma gallisepticum is among the most economically significant mycoplasmas causing production losses in poultry. Seven melt-curve and agarose gel-based mismatch amplification mutation assays (MAMAs) and one PCR are provided in the present study to distinguish the M. gallisepticum vaccine strains and field isolates based on mutations in the crmA, gapA, lpd, plpA, potC, glpK, and hlp2 genes. A total of 239 samples (M. gallisepticum vaccine and type strains, pure cultures, and clinical samples) originating from 16 countries and from at least eight avian species were submitted to the presented assays for validation or in blind tests. A comparison of the data from 126 samples (including sequences available at GenBank) examined by the developed assays and a recently developed multilocus sequence typing assay showed congruent typing results. The sensitivity of the melt-MAMA assays varied between 101 and 104M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 103 to 105 template copies/reaction, and no cross-reactions occurred with other Mycoplasma species colonizing birds. The presented assays are also suitable for discriminating multiple strains in a single sample. The developed assays enable the differentiation of live vaccine strains by targeting two or three markers/vaccine strain; however, considering the high variability of the species, the combined use of all assays is recommended. The suggested combination provides a reliable tool for routine diagnostics due to the sensitivity and specificity of the assays, and they can be performed directly on clinical samples and in laboratories with basic PCR equipment.

Characterization of the genomic sequence of a novel CRESS DNA virus identified in Eurasian jay (Garrulus glandarius).

Circular replication associated protein (Rep)-encoding ssDNA (CRESS DNA) viruses have diverse genomic architecture and are widely distributed in different ecosystems. In this study we characterized the complete genomic sequence of a novel circovirus-like virus, Garrulus glandarius associated circular virus-1 (GgaCV-1). The genome size (1971 nt) and other features (the nonanucleotide, rolling circle replication motif and SF3 helicase motif) are also reminiscent of circoviruses. Similar genomes with uni-directionally localized and overlapping rep and cap genes are typical of type V CRESS DNA viruses that were identified in invertebrates and environmental samples of aquatic ecosystems. GgaCV-1 showed the highest aa identity with partial rep sequences detected in bat feces (77%) and with the rep (54%) and cap (42%) of Lake Sarah-associated circular virus-23 of New Zealand freshwater mussel origin. A dietary origin for GgaCV-1 could not be excluded as the virus was detected in the cloacal swab specimen of an Eurasian jay. Further studies may help to reveal the linkage among variable organisms regarding virus transmission.

Genome sequence of a mallard duck origin cyclovirus, DuACyV-1.

The genome sequence of a novel avian cyclovirus is described in this study. The genome size and orientation of predicted genes was similar to those described in other vertebrate and insect origin cycloviruses. The greatest genome sequence identity was shared with a dragonfly cyclovirus (nt, 60.6%). Phylogenetic analysis showed marginal relatedness with another avian cyclovirus, the chicken associated cyclovirus 1. In contrast, along a short fragment of the replication-associated protein coding gene (rep) (spanning nt 1240-1710) the duck origin cyclovirus was very similar to human origin and honey bee origin rep sequences (human - TN4, 98%; honey bee - hb10, 100%). Related cyclovirus strains existing amongst various animal species living in diverse ecosystems and separated by large geographic distances show the need for additional studies to better understand the ecology and epidemiology of cycloviruses.

Correlation between host specificity and genetic diversity for the muscle-dwelling fish parasite Myxobolus pseudodispar: examples of myxozoan host-shift?

Myxobolus pseudodispar Gorbunova, 1936 (Myxozoa) is capable of infecting and developing mature myxospores in several cyprinid species. However, M. pseudodispar isolates from different fish show up to 5% differences in the SSU rDNA sequences. This is an unusually large intraspecific difference for myxozoans and only some of the muscle-dwelling myxozoan species possess such a high genetic variability. We intended to study the correlation between the host specificity and the phylogenetic relationship of the parasite isolates, and to find experimental proof for the putatively wide host range of M. pseudodispar with cross-infection experiments and phylogenetic analyses based on SSU rDNA. The experimental findings distinguished 'primary' and less-susceptible 'secondary' hosts. With some exceptions, M. pseudodispar isolates showed a tendency to cluster according to the fish host on the phylogenetic tree. Experimental and phylogenetic findings suggest the cryptic nature of the species. It is likely that host-shift occurred for M. pseudodispar and the parasite speciation in progress might explain the high genetic diversity among isolates which are morphologically indistinguishable.

Molecular genetic investigations on Balantidium ctenopharyngodoni Chen, 1955, a parasite of the grass carp (Ctenopharyngodon idella).

Balantidium ctenopharyngodoni is a common ciliate in Hungary, infecting the hindgut of grass carp (Ctenopharyngodon idella), a cyprinid fish of Chinese origin. Although data have already been presented on its occasional pathogenic effect on the endothelium of the host, generally it is a harmless inhabitant of the gut. Phylogenetic analysis of the 18S rDNA and ITS fragments of this protozoan proved that it is in the closest phylogenetic relationship with endocommensalist and symbiont ciliates of mammals feeding on large volumes of green forage, in a similar way as Balantidium spp. known from algae-eating marine fishes.

Decidual γδT cells of early human pregnancy produce angiogenic and immunomodulatory proteins while also possessing cytotoxic potential.

During pregnancy, the maternal immune system must allow and support the growth of the developing placenta while maintaining the integrity of the mother's body. The trophoblast's unique HLA signature is a key factor in this physiological process. This study focuses on decidual γδT cell populations and examines their expression of receptors that bind to non-classical HLA molecules, HLA-E and HLA-G. We demonstrate that decidual γδT cell subsets, including Vδ1, Vδ2, and double-negative (DN) Vδ1-/Vδ2- cells express HLA-specific regulatory receptors, such as NKG2C, NKG2A, ILT2, and KIR2DL4, each with varying dominance. Furthermore, decidual γδT cells produce cytokines (G-CSF, FGF2) and cytotoxic mediators (Granulysin, IFN-γ), suggesting functions in placental growth and pathogen defense. However, these processes seem to be controlled by factors other than trophoblast-derived non-classical HLA molecules. These findings indicate that decidual γδT cells have the potential to actively contribute to the maintenance of healthy human pregnancy.

Phylogeny of Hungarian EBLV-1 strains using whole-genome sequence data.

European bat lyssavirus 1 (EBLV-1) is a widespread lyssavirus across Europe, whose epizootic cycle is linked to a few bat species. Occasionally, EBLV-1 infection may occur in domestic animals and humans. EBLV-1 can be classified into two subtypes, where subtype EBLV-1a shows a wide geographic distribution between France and Russia whereas subtype EBLV-1b is distributed between Spain and Poland. In this study, we determined the genome sequence of two recent EBLV-1a strains detected in Hungary and analysed their adaptive evolution and phylodynamics. The data set that included 100 EBLV-1 genome sequences identified positive selection at selected sites in genes coding for viral proteins (N, codon 18; P, 141 and 155; G, 244 and 488; L, 168, 980, 1597 and 1754). A major genetic clade containing EBLV-1a isolates from Hungary, Slovakia, Denmark and Poland was estimated to have diverged during the 19th century whereas the divergence of the most recent ancestor of Hungarian and Slovakian isolates dates back to 1950 (time span, 1930 to 1970). Phylogeographic analysis of the EBLV-1a genomic sequences demonstrated strong evidence of viral dispersal from Poland to Hungary. This new information indicates that additional migratory flyways may help the virus spread, a finding that supplements the general theory on a west-to-east dispersal of EBLV-1a strains. Long-distance migrant bats may mediate the dispersal of EBLV-1 strains across Europe; however, structured surveillance and extended genome sequencing would be needed to better understand the epizootiology of EBLV-1 infections in Europe.

Whole genome sequencing of coagulase positive staphylococci from a dog-and-owner screening survey.

BACKGROUND: Staphylococcus aureus and S. pseudintermedius are the two most common coagulase positive staphylococci (CPS). S. aureus is more prevalent among humans, whereas S. pseudintermedius is more commonly isolated from dogs, however, both can cause various community and hospital acquired diseases in humans. METHODS: In the current study we screened 102 dogs and 84 owners in Hungary. We tested the antibiotic susceptibility of the strains and in order to get a better picture of the clonal relationship of the strains, we used pulsed-field gel electrophoresis. In addition, three pairs of isolates with identical PFGE patterns were whole genome sequenced, MLST and spa types were established. RESULTS: Carriage rate of S. aureus was 23.8% in humans and 4.9% in dogs and two cases of co-carriage were found among dogs and owners. S. pseudintermedius carriage rate was 2.4% and 34.3%, respectively, with only one co-carriage. The isolates were generally rather susceptible to the tested antibiotics, but high tetracycline resistance of S. pseudintermedius strains was noted. The co-carried isolates shared almost the same resistance genes (including tet(K), bla(Z), norA, mepR, lmrS, fosB) and virulence gene pattern. Apart from the common staphylococcal enzymes and cytotoxins, we found enterotoxins and exfoliative toxins as well. The two S. aureus pairs belonged to ST45-t630, ST45-t671 and ST15-t084, ST15-t084, respectively. The co-carried S. pseudintermedius isolates shared the same housekeeping gene alleles determining a novel sequence type ST1685. CONCLUSIONS: Based on the genomic data, dog-owner co-carried strains displayed only insignificant differences therefore provided evidence for potential human-to-dog and dog-to-human transmission.

Vaccine-associated rabies in red fox, Hungary.

Rabies vaccine strain was isolated from a red fox (Vulpes vulpes) with signs of neurological disorder during an oral vaccination campaign in 2015, Hungary. The whole genome sequence of the isolated strain shared >99.9% nucleotide sequence identity to the whole genomes of vaccines strains recently used in Hungarian oral vaccination campaigns. The neuroinvasive potential of rabies vaccines that leads to development of clinical manifestations is rarely seen among wild animals; however, the observed residual pathogenicity needs awareness of field experts and requires close monitoring of rabies cases in areas where elimination programs are implemented.

Methylation Status of the Adeno-Associated Virus Type 2 (AAV2).

To analyze the methylation status of wild-type adeno-associated virus type 2 (AAV2), bisulfite PCR sequencing (BPS) of the packaged viral genome and its integrated form was performed and 262 of the total 266 CG dinucleotides (CpG) were mapped. In virion-packaged DNA, the ratio of the methylated cytosines ranged between 0⁻1.7%. In contrast, the chromosomally integrated AAV2 genome was hypermethylated with an average of 76% methylation per CpG site. The methylation level showed local minimums around the four known AAV2 promoters. To study the effect of methylation on viral rescue and replication, the replication initiation capability of CpG methylated and non-CpG methylated AAV DNA was compared. The in vitro hypermethylation of the viral genome does not inhibit its rescue and replication from a plasmid transfected into cells. This insensitivity of the viral replicative machinery to methylation may permit the rescue of the integrated heavily methylated AAV genome from the host's chromosomes.

Complete Genome Sequences of Three Mycoplasma anserisalpingitis (Mycoplasma sp. 1220) Strains.

Mycoplasma anserisalpingitis is a goose pathogen. The main symptoms in affected flocks are inflammation of the cloaca and the reproductive organs, decreased egg production, and increased embryo mortality. Here, we report the complete genome sequences of the type strain (ATCC BAA-2147) and two clinical isolates.

Combination therapy of rabies-infected mice with inhibitors of pro-inflammatory host response, antiviral compounds and human rabies immunoglobulin.

Recent studies demonstrated that inhibitors of pro-inflammatory molecular cascades triggered by rabies infection in the central nervous system (CNS) can enhance survival in mouse model and that certain antiviral compounds interfere with rabies virus replication in vitro. In this study different combinations of therapeutics were tested to evaluate their effect on survival in rabies-infected mice, as well as on viral load in the CNS. C57Bl/6 mice were infected with Silver-haired bat rabies virus (SHBRV)-18 at virus dose approaching LD50 and LD100. In one experimental group daily treatments were initiated 4 h before-, in other groups 48 or 96 h after challenge. In the first experiment therapeutic combination contained inhibitors of tumour necrosis factor-α (infliximab), caspase-1 (Ac-YVAD-cmk), and a multikinase inhibitor (sorafenib). In the treated groups there was a notable but not significant increase of survival compared to the virus infected, non-treated mice. The addition of human rabies immunoglobulins (HRIG) to the combination in the second experiment almost completely prevented mortality in the pre-exposure treatment group along with a significant reduction of viral titres in the CNS. Post-exposure treatments also greatly improved survival rates. As part of the combination with immunomodulatory compounds, HRIG had a higher impact on survival than alone. In the third experiment the combination was further supplemented with type-I interferons, ribavirin and favipiravir (T-705). As a blood-brain barrier opener, mannitol was also administered. This treatment was unable to prevent lethal consequences of SHBRV-18 infection; furthermore, it caused toxicity in treated mice, presumably due to interaction among the components. In all experiments, viral loads in the CNS were similar in mice that succumbed to rabies regardless of treatment. According to the findings, inhibitors of detrimental host response to rabies combined with antibodies can be considered among the possible therapeutic and post-exposure options in human rabies cases.

Complete Genome Sequences of Mycoplasma anatis, M. anseris, and M. cloacale Type Strains.

Mycoplasma anatis, M. anseris, and M. cloacale are pathogens of waterfowl. Airsacculitis, nervous disease, and reproductive disorders are the main symptoms in the affected flocks. Here, we report the complete genome sequences of the M. anatis (NCTC 10156), M. anseris (ATCC 49234), and M. cloacale (NCTC 10199) type strains.

A single point mutation on the cucumber mosaic virus surface induces an unexpected and strong interaction with the F1 complex of the ATP synthase in Nicotiana clevelandii plants.

A previous study showed that a single amino acid difference in the cucumber mosaic virus (CMV) capsid protein (CP) elicits unusual symptoms. The wild-type strain (CMV-R) induces green mosaic symptoms and malformation while the mutant strain (CMV-R3E79R) causes chlorotic lesions on inoculated leaves and strong stunting with necrosis on systemic leaves. Virion preparations of CMV-R and CMV-R3E79R were partially purified from Nicotiana clevelandii A. Gray and analysed by two-dimensional gel electrophoresis. Their separated protein patterns showed remarkable differences at the 50-75 kDa range, both in numbers and intensity of spots, with more protein spots for the mutant CMV. Mass spectrometry analysis demonstrated that the virion preparations contained host proteins identified as ATP synthase alpha and beta subunits as well as small and large Rubisco subunits, respectively. Virus overlay protein binding assay (VOPBA), immunogold electron microscopy and modified ELISA experiments were used to prove the direct interaction between the virus particle and the N. clevelandii ATP synthase F1 motor complex. Protein-protein docking study revealed that the electrostatic change in the mutant CMV can introduce stronger interactions with ATP synthase F1 complex. Based on our findings we suggest that the mutation present in the CP can have a direct effect on the long-distance movement and systemic symptoms. In molecular view the mutant CMV virion can lethally block the rotation of the ATP synthase F1 motor complex which may lead to cell apoptosis, and finally to plant death.

Whole genome sequencing of a rare rotavirus from archived stool sample demonstrates independent zoonotic origin of human G8P[14] strains in Hungary.

Genotype P[14] rotaviruses in humans are thought to be zoonotic strains originating from bovine or ovine host species. Over the past 30 years only few genotype P[14] strains were identified in Hungary totaling<0.1% of all human rotaviruses whose genotype had been determined. In this study we report the genome sequence and phylogenetic analysis of a human genotype G8P[14] strain, RVA/Human-wt/HUN/182-02/2001/G8P[14]. The whole genome constellation (G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3) of this strain was shared with another Hungarian zoonotic G8P[14] strain, RVA/Human-wt/HUN/BP1062/2004/G8P[14], although phylogenetic analyses revealed the two rotaviruses likely had different progenitors. Overall, our findings indicate that human G8P[14] rotavirus detected in Hungary in the past originated from independent zoonotic events. Further studies are needed to assess the public health risk associated with infections by various animal rotavirus strains.

Whole genome sequencing and phylogenetic characterization of brown bullhead (Ameiurus nebulosus) origin ranavirus strains from independent disease outbreaks.

Ranaviruses are emerging pathogens associated with high mortality diseases in fish, amphibians and reptiles. Here we describe the whole genome sequence of two ranavirus isolates from brown bullhead (Ameiurus nebulosus) specimens collected in 2012 at two different locations in Hungary during independent mass mortality events. The two Hungarian isolates were highly similar to each other at the genome sequence level (99.9% nucleotide identity) and to a European sheatfish (Silurus glanis) origin ranavirus (ESV, 99.7%-99.9% nucleotide identity). The coding potential of the genomes of both Hungarian isolates, with 136 putative proteins, were shared with that of the ESV. The core genes commonly used in phylogenetic analysis of ranaviruses were not useful to differentiate the two brown bullhead ESV strains. However genome-wide distribution of point mutations and structural variations observed mainly in the non-coding regions of the genome suggested that the ranavirus disease outbreaks in Hungary were caused by different virus strains. At this moment, due to limited whole genome sequence data of ESV it is unclear whether these genomic changes are useful in molecular epidemiological monitoring of ranavirus disease outbreaks. Therefore, complete genome sequencing of further isolates will be needed to identify adequate genetic markers, if any, and demonstrate their utility in disease control and prevention.

Parental genetic diversity of brown trout (Salmo trutta m. fario) brood stock affects offspring susceptibility to whirling disease.

BACKGROUND: Whirling disease, caused by the myxozoan parasite Myxobolus cerebralis, has high economical and ecological importance worldwide. Susceptibility to the disease varies considerably among salmonid species. In brown trout (Salmo trutta) the infection is usually subclinical with low mortality, which increases the risk of parasite dissemination, especially when farm fish are used for stocking natural habitats. The influence of intraspecific genetic differences (especially the level of homozygosity) on susceptibility is unknown. Therefore, we examined the possible correlations between parental genetic diversity and offspring susceptibility of brown trout stocks to whirling disease. METHODS: Two brown trout brood stocks from a German and a Hungarian fish farm were genetically characterized using microsatellite and lineage-specific genetic markers. The individual inbreeding coefficient f and pairwise relatedness factor r were estimated based on eight microsatellite markers. Brood stock populations were divided into groups according to low and high f and r value estimates and subjected to selective fertilization. The offspring from these separate groups were exposed to M. cerebralis actinospores, and the infection prevalence and intensity was measured and statistically analysed. RESULTS: The analysis of phylogeographic lineage heritage revealed high heterogeneity in the Hungarian brood stock since > 50% of individuals were Atlantic-Danubian hybrids, while only pure Atlantic-descending specimens were detected in the German population. Based on f msat and r msat estimations, classified non-inbred (NIB), inbred (IB) and a group of closely related fish (REL) were created. The susceptibility of their offspring varied considerably. Although there was no significant difference in the prevalence of M. cerebralis infection, the mean intensity of infection differed significantly between NIB and IB groups. In REL and IB groups, a high variability was observed in infection intensity. No external clinical signs were observed in the exposed brown trout groups. CONCLUSIONS: Our findings indicate that the allelic diversity of brown trout brood stock may constitute a significant factor in disease susceptibility, i.e. the intensity of parasite infection in the subsequent generation.