Sacbrood viruses cross-infection between Apis cerana and Apis mellifera: Rapid detection, viral dynamics, evolution and spillover risk assessment.

Recent outbreaks of sacbrood virus (SBV) have caused serious epizootic disease in Apis cerana populations across Asia including Taiwan. Earlier phylogenetic analyses showed that cross-infection of AcSBV and AmSBV in both A. cerana and A. mellifera seems common, raising a concern of cross-infection intensifying the risk of disease resurgence in A. cerana. In this study, we analyzed the dynamics of cross-infection in three different types of apiaries (A. mellifera-only, A. cerana-only and two species co-cultured apiaries) over one year in Taiwan. Using novel, genotype-specific primer sets, we showed that SBV infection status varies across apiaries: AmSBV-AM and AcSBV-AC were the major genotype in the A. mellifera-only and the A. cerana-only apiaries, respectively, while AmSBV-AC and AcSBV-AC were the dominant genotypes in the co-cultured apiaries. Interestingly, co-cultured apiaries were among the only apiary type that harbored all variants and dual infections (i.e., AC and AM genotype co-infection in a single sample), indicating the interactions between hosts may form a conduit for cross-infection. The cross-infection between the two honey bee species appears to occur in a regular cycle with temporal fluctuation of AmSBV-AC and AcSBV-AC prevalence synchronized to each other in the co-cultured apiaries. Artificial infection of AcSBV in A. mellifera workers showed the suppression of viral replication, suggesting the potential of A. mellifera serving as a AcSBV reservoir that may contribute to virus spillover. Furthermore, the survival rate of A. cerana larvae was significantly reduced after artificial infections of both SBVs, indicating fitness costs of cross-infection on A. cerana and thus a high risk of disease resurgence in co-cultured apiaries. Our field and laboratory data provide baseline information that facilitates understanding of the risk of SBV cross-infection, and highlights the urgent need of SBV monitoring in co-cultured apiaries.

X-ray inactivation of RNA viruses without loss of biological characteristics.

In the event of an unpredictable viral outbreak requiring high/maximum biosafety containment facilities (i.e. BSL3 and BSL4), X-ray irradiation has the potential to relieve pressures on conventional diagnostic bottlenecks and expediate work at lower containment. Guided by Monte Carlo modelling and in vitro 1-log10 decimal-reduction value (D-value) predictions, the X-ray photon energies required for the effective inactivation of zoonotic viruses belonging to the medically important families of Flaviviridae, Nairoviridae, Phenuiviridae and Togaviridae are demonstrated. Specifically, it is shown that an optimized irradiation approach is attractive for use in a multitude of downstream detection and functional assays, as it preserves key biochemical and immunological properties. This study provides evidence that X-ray irradiation can support emergency preparedness, outbreak response and front-line diagnostics in a safe, reproducible and scalable manner pertinent to operations that are otherwise restricted to higher containment BSL3 or BSL4 laboratories.

Heterobasidion Partitivirus 13 Mediates Severe Growth Debilitation and Major Alterations in the Gene Expression of a Fungal Forest Pathogen.

The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the boreal forest region. In this study, we showed that the alphapartitivirus Heterobasidion partitivirus 13 from Heterobasidion annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, of which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and Heterobasidion parviporum The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing medium, while only two of six H. annosum isolates tested showed significant debilitation. Based on reverse transcription-quantitative PCR (RT-qPCR) analysis, the response toward HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates.IMPORTANCE A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry, as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of €800 million in Europe. We used virus curing and reintroduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a low growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.

Applications of omics approaches to the development of microbiological risk assessment using RNA virus dose-response models as a case study.

The last decade has seen a huge increase in the amount of 'omics' data available and in our ability to interpret those data. The aim of this paper was to consider how omics techniques can be used to improve and refine microbiological risk assessment, using dose-response models for RNA viruses, with particular reference to norovirus through the oral route as the case study. The dose-response model for initial infection in the gastrointestinal tract is broken down into the component steps at the molecular level and the feasibility of assigning probabilities to each step assessed. The molecular mechanisms are not sufficiently well understood at present to enable quantitative estimation of probabilities on the basis of omics data. At present, the great strength of gene sequence data appears to be in giving information on the distribution and proportion of susceptible genotypes (for example due to the presence of the appropriate pathogen-binding receptor) in the host population rather than in predicting specificities from the amino acid sequences concurrently obtained. The nature of the mutant spectrum in RNA viruses greatly complicates the application of omics approaches to the development of mechanistic dose-response models and prevents prediction of risks of disease progression (given infection has occurred) at the level of the individual host. However, molecular markers in the host and virus may enable more broad predictions to be made about the consequences of exposure in a population. In an alternative approach, comparing the results of deep sequencing of RNA viruses in the faeces/vomitus from donor humans with those from their infected recipients may enable direct estimates of the average probability of infection per virion to be made.

Genetic 'budget' of viruses and the cost to the infected host: a theory on the relationship between the genetic capacity of viruses, immune evasion, persistence and disease.

The nature of the pathogen-host relationship is recognized as being a dynamic coevolutionary process where the immune system has required ongoing adaptation and improvement to combat infection. Under survival pressure from sophisticated immune responses, adaptive processes for microbes, including viruses, have manifested as immune evasion strategies. This paper proposes a theory that virus immune evasion can be broadly classified into 'acquisition' or 'erroneous replication' strategies. Acquisition strategies are characteristic of large genome dsDNA viruses, which (i) replicate in the cell nucleus; (ii) have acquired host genes that can be used to directly manipulate responses to infection; (iii) are often latent for the lifetime of the host; and (iv) have little or no serious impact on health. Alternatively, erroneous replication strategies are characteristic of small genome RNA viruses, which are recognized as being the cause of many serious diseases in humans. It is proposed that this propensity for disease is due to the cytoplasmic site of replication and truncated temporal relationship with the host, which has limited or removed the evolutionary opportunity for RNA viruses to have acquired host genes. This has resulted in RNA viruses relying on error-prone replication strategies which, while allowing survival and persistence, are more likely to lead to disease due to the lack of direct viral control over potentially host-deleterious inflammatory and immune responses to infection.

Cost of host radiation in an RNA virus.

Although host radiation allows a parasite to expand its ecological niche, traits governing the infection of multiple host types can decrease fitness in the original or alternate host environments. Reasons for this reduction in fitness include slower replication due to added genetic material or modifications, fitness trade-offs across host environments, and weaker selection resulting from simultaneous adaptation to multiple habitats. We examined the consequences of host radiation using vesicular stomatitis virus (VSV) and mammalian host cells in tissue culture. Replicate populations of VSV were allowed to evolve for 100 generations on the original host (BHK cells), on either of two novel hosts (HeLa and MDCK cells), or in environments where the availability of novel hosts fluctuated in a predictable or random way. As expected, each experimental population showed a substantial fitness gain in its own environment, but those evolved on new hosts (constant or fluctuating) suffered reduced competitiveness on the original host. However, whereas evolution on one novel host negatively correlated with performance on the unselected novel host, adaptation in fluctuating environments led to fitness improvements in both novel habitats.

[Variability and development of viral populations: assessment and implications]. / Variabilité et evolution des populations virales: bilan et implications.

RNA virus populations consist of complex distributions of closely related but not identical genomes known as viral quasi-species. The quasi-species concept describes the dynamics of these genomes subjected to a continuous process of variation, competition, and selection. Quasi-species dynamics has broad implications not only in the understanding of the molecular mechanisms underlying adaptation of RNA viruses but also in the design of strategies for control and prevention of viral disease. Viral load and genetic heterogeneity have a determinant influence on the adaptation of RNA virus to their environment. Vaccines designed to control diseases caused by highly variable viruses must contain several B and T epitopes to provide an ample and diversified immune response. Similarly, antiviral drugs should be used in combination therapy to minimize selection of resistant viruses. The theoretical model of quasi-species has opened the way for new antiviral therapies based on augmentation of the mutation rate during replication of viral RNA. Finally the quasi-species concept provides the basis for defining the selective factors that could influence the evolution of RNA virus and promote the emergence or reemergence of viral diseases.