Understanding virus evolution is a fundamental goal of virology, evolutionary biology, and disease epidemiology. We provide a detailed analysis of evolution and origin of Cryphonectria hypovirus 1 ...(CHV1) populations in Europe, based on the complete genome sequence of all European subtypes. Phylogenetic analyses divided European strains into two closely related clades. Strains of the subtype I belong to the first, while strains of the subtypes F1, D and E belong to the second clade suggesting that the subtypes F1, D and E are more closely related than previously thought. Strains of the subtype F2 appeared to be recombinant; subtypes F1/D/E contributed a larger fraction of sequence while subtype I contributed a smaller fraction. The p29 was the most variable domain, while the replication-associated large ORF B protein was the most conserved domain within the CHV1. Low sequence similarity, predominant negative selection and frequent recombination characterise the evolution of CHV1.
•Subtypes F1, D and E are more closely related than previously thought.•Strains of the subtype F2 are recombinant originating from subtypes F1/D/E and I.•CHV1 protein domains vary considerably in genetic diversity and selection pressure.•Negative selection and frequent recombination characterise CHV1 evolution.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Forest ecosystems are highly threatened by the simultaneous effects of climate change and invasive pathogens. Chestnut blight, caused by the invasive phytopathogenic fungus
, has caused severe damage ...to European chestnut groves and catastrophic dieback of American chestnut in North America. Within Europe, the impacts of the fungus are widely mitigated through biological control that utilizes the RNA mycovirus: Cryphonectria hypovirus 1 (CHV1). Viral infections, similarly to abiotic factors, can cause oxidative stress in their hosts leading to physiological attrition through stimulating ROS (reactive oxygen species) and NOx production.
To fully understand the interactions leading to the biocontrol of chestnut blight, it is vital to determine oxidative stress damage arising during CHV1 infection, especially considering that other abiotic factors, like long-term cultivation of model fungal strains, can also impact oxidative stress. Our study compared CHV1-infected
isolates from two Croatian wild populations with CHV1-infected model strains (EP713, Euro7 and CR23) that have experienced long-term laboratory cultivation.
We determined the level of oxidative stress in the samples by measuring stress enzymes' activity and oxidative stress biomarkers. Furthermore, for the wild populations, we studied the activity of fungal laccases, expression of the laccase gene
, and a possible effect of CHV1 intra-host diversity on the observed biochemical responses. Relative to the wild isolates, the long-term model strains had lower enzymatic activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), and higher content of malondialdehyde (MDA) and total non-protein thiols. This indicated generally higher oxidative stress, likely arising from their decades-long history of subculturing and freeze-thaw cycles. When comparing the two wild populations, differences between them in stress resilience and levels of oxidative stress were also observed, as evident from the different MDA content. The intra-host genetic diversity of the CHV1 had no discernible effect on the stress levels of the virus-infected fungal cultures. Our research indicated that an important determinant modulating both
expression and laccase enzyme activity is intrinsic to the fungus itself, possibly related to the vc type of the fungus, i.e., vegetative incompatibility genotype.
Laccase activity reduction in the chestnut blight fungus
usually accompanies the hypovirulence caused by the infection of fungus with
(CHV1). However, the different methods utilized for assessing ...this phenomenon has produced varied and often conflicting results. Furthermore, the majority of experimental setups included only one prototypic system, further confounding the results. Considering the diversity of fungal isolates, viral strains, and variability of their effects on the phytopathogenic process observed in nature, our goal was to ascertain if laccase activity variability is affected by (1) different
isolates infected with several CHV1 strains, and (2) growth conditions. We have demonstrated that some CHV1 strains, contrary to previous assumptions, increase the activity of
laccases. The specific fungal isolates used in the experiments and culture conditions also affected the results. Furthermore, we showed that two commonly used laccase substrates, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,4-dimethoxyphenol, cannot be used interchangeably in
laccase activity measurements. Our results illustrate the importance of conducting this type of study in experimental systems and culture conditions that resemble natural conditions as much as possible to be able to infer the most relevant conclusions applicable to natural populations.
Abstract
Intra-host dynamics are a core component of virus evolution but most intra-host data come from a narrow range of hosts or experimental infections. Gaining broader information on the ...intra-host diversity and dynamics of naturally occurring virus infections is essential to our understanding of evolution across the virosphere. Here we used PacBio long-read HiFi sequencing to characterize the intra-host populations of natural infections of the RNA mycovirus Cryphonectria hypovirus 1 (CHV1). CHV1 is a biocontrol agent for the chestnut blight fungus (Cryphonectria parasitica), which co-invaded Europe alongside the fungus. We characterized the mutational and haplotypic intra-host virus diversity of thirty-eight natural CHV1 infections spread across four locations in Croatia and Switzerland. Intra-host CHV1 diversity values were shaped by purifying selection and accumulation of mutations over time as well as epistatic interactions within the host genome at defense loci. Geographical landscape features impacted CHV1 inter-host relationships through restricting dispersal and causing founder effects. Interestingly, a small number of intra-host viral haplotypes showed high sequence similarity across large geographical distances unlikely to be linked by dispersal.
Cryphonectria parasitica is an invasive fungal pathogen that causes blight disease on chestnut trees. Its destructive effect can be controlled with naturally occurring mycovirus Cryphonectria ...hypovirus 1 (CHV1). To date, the spread of C. parasitica and CHV1 in Europe is fairly well documented, but there are still several unexplored regions. Thus, we sampled blight cankers from four sweet chestnut populations in Bay of Kotor and Lake Skadar regions in Montenegro. We determined vegetative compatibility (vc) type and mating-type diversity using molecular vic and MAT1 genotyping, as well as confirming the presence of CHV1 by RT-PCR. We identified 11 vc types, with EU-12 being the dominant one represented by 58.2% of all fungal isolates. The Shannon diversity index ranged from 0.93 to 1.47. Both mating types of C. parasitica were found in all four populations. The prevalence of CHV1 ranged from 15% to 40%. All CHV1 isolates belonged to Italian subtype I of CHV1 and were closely related, with relatively recent common ancestors. Our results indicate a longer presence of C. parasitica and CHV1 in Montenegro than previously thought. Natural biocontrol with CHV1 seems to be well established. However, it has the potential for deterioration; thus, close monitoring is required.
Background and purpose: The next challenge after development of COVID-19 vaccines is reaching adequate vaccine coverage, which could be hampered by COVID-19 vaccine hesitancy. The aim of this study ...was to assess general attitude towards vaccination among biology students, as well as prevalence of COVID-19 vaccine acceptance. Since participants of this study could be employed in educational institutions in the future, their knowledge and attitudes towards vaccination could have a role in forming the attitudes of the next generations.Materials and methods: Biology students from two Universities in Croatia were questioned using an anonymous online survey about their attitude towards vaccination in general and COVID-19 vaccination at the moment and from the perspective of a future teacher. Data analysis included descriptive statistics, Chi-square test, Fisher's exact test and binary logistic regression.Results: Mostly positive opinion about vaccination was revealed among Croatian biology students, while 67.3% expressed willingness to vaccinate against COVID-19. COVID-19 vaccine compliance was not associated with gender, level of education or University they attended. However, opinion about safety and effectiveness of COVID-19 vaccines, perceiving vaccination as important for stopping current pandemic and feeling exposed to COVID-19 infection significantly affected the odds for accepting COVID-19 vaccine. Many students were aware of the opportunity to influence vaccination decisions of the next generations by providing knowledge regarding infectious diseases, immunity and vaccines as future biology teachers.Conclusions: Increasing willingness and competence of biology students and biology teachers to promote vaccine-positive attitudes could be promising long-term strategy for decreasing vaccine hesitancy.
Biotic stress caused by virus infections induces epigenetic changes in infected plants and animals, but this is the first report on methylation pattern changes in a fungus after mycovirus infection. ...As a model pathosystem for mycovirushost interactions, we used Cryphonectria hypovirus 1 (CHV1) and its host fungus Cryphonectria parasitica, in which deregulation of methylation cycle enzymes upon virus infection was observed previously. Six CHV1 strains of different subtypes were transferred into three different C. parasitica isolates in order to assess the effect of different CHV1 strains and/or subtypes on global cytosine methylation level in infected fungus, using methylation-sensitive amplification polymorphism (MSAP). Infection with CHV1 affected the methylation pattern of the C. parasitica genome; it increased the number and diversity of methylated, hemi-methylated, and total MSAP markers found in infected fungal isolates compared to virus-free controls. The increase in methylation levels correlated well with the CHV1-induced reduction of fungal growth in vitro, indicating that C. parasitica genome methylation upon CHV1 infection, rather than being the defensive mechanism of the fungus, is more likely to be the virulence determinant of the virus. Furthermore, the severity of CHV1 effect on methylation levels of infected C. parasitica isolates depended mostly on individual CHV1 strains and on the combination of host and virus genomes, rather than on the virus subtype. These novel findings broaden our knowledge about CHV1 strains which could potentially be used in human-aided biocontrol of chestnut blight, a disease caused by C. parasitica in chestnut forest ecosystems and orchards.
Full text
Available for:
BFBNIB, EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NMLJ, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Chestnut blight has spread throughout Europe since the introduction of its causal agent Cryphonectria parasitica over 70 years ago. In our study, we have analysed diversity of vegetative ...compatibility (vc) and microsatellite genotypes of C. parasitica, as well as sequence diversity of Cryphonectria hypovirus 1 (CHV1) in six populations from Switzerland, Croatia and North Macedonia. Resampling of local populations that were already investigated more than a decade ago allowed us to analyse the spatial and temporal population structure across an invasive range of the pathogen in Europe. Regardless which genetic marker was used, the over 60 year-old Swiss and Croatian populations had a high population diversity, while more recent North Macedonian populations were mostly clonal. These diversity differences between the investigated populations remained stable over time. A high diversity of CHV1 was observed in all three countries, with North Macedonian strains forming a separate cluster from strains obtained in other countries. No correlation between vc diversity and CHV1 prevalence was observed, suggesting a well-established and maintained natural hypovirulence in all countries, further corroborated by an observed increase in genetic diversity of Croatian C. parasitica populations over time, without collapse of CHV1 prevalence.
Cryphonectria parasitica is a phytopathogenic fungus introduced from Eastern Asia to North America and to Europe, where it causes chestnut blight, a devastating disease of chestnut trees. The disease ...can be biologically controlled utilising the mycovirus Cryphonectria hypovirus 1 (CHV1), which changes the physiology of the host, reducing its virulence towards chestnut. We measured fungal growth in vitro and activities of glutathione S-transferase, catalase and super-oxide dismutase, enzymes involved in oxidative stress response, to elucidate the effects of CHV1 infection on the host. Six CHV1 strains of different subtypes and three fungal isolates were used in different combinations to better represent natural conditions, where higher genetic diversity of both fungus and virus is expected. The infection with different CHV1 strains decreased in vitro growth rate of infected fungal isolates and increased activity of their stress enzymes in most of the studied fungus/virus combinations, indicating increased oxidative stress following CHV1 infection. All our field CHV1 strains belong to the Italian subtype, but while strain M56-1 had equal or even stronger effect on its fungal host than prototypic strain EP713 of French subtype F1, strain B11 had no effect. Thus, the severity of the observed effects depended on a particular virus strain, fungal isolate, and the combination of the two, rather than solely on the virus subtype. Since previous research showed discordance between accumulation of mRNA and stress-related proteins in CHV1 infected C. parasitica, our results emphasise the importance of enzymes’ activity measurements as an invaluable extension of transcriptomic and proteomic analyses.
Full text
Available for:
BFBNIB, EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NMLJ, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
PDF
