Barbara Forró

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

BackgroundWhirling 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.MethodsTwo 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.ResultsThe 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 fmsat and rmsat 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.ConclusionsOur 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.

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.

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.

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.

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.

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.

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.

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.

Inosine-Arginine salt as a promising agent for in vitro activation of waterborne fish-pathogenic myxozoan actinospores

Mucus-derived nucleosides serve as key host cues for myxozoan actinospore fish host recognition, but to date their use for experimental actinospore activation in the laboratory or application in disease prevention has not progressed very far. One obstacle has been the low solubility of pure inosine and guanosine. To overcome this, we used inosine-arginine salt (ino-arg), which incorporates both high activation properties and high solubility. We tested its efficacy both in microassays directly observing reactions of actinospores of 2 distantly related myxozoan species, Myxobolus cerebralis and M. pseudodispar in comparison to inosine, as well as its actinospore-inactivation properties by premature polar capsule discharge in an infection experiment. Ino-arg was considerably more effective in eliciting polar capsule discharge and sporoplasm emission at much lower concentrations than pure inosine and, in contrast to the latter, remained dissolved in aqueous solution. Ino-arg exposure of M. pseudodispar actinospores resulted in polar capsule discharge and sporoplasm emission before host contact and subsequently in a lower infection rate in roach Rutilus rutilus.