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Mud volcanoes are geological structures in the oceans that have key roles in the functioning of the global ecosystem. Information on the dynamics of benthic viruses and their interactions with prokaryotes in mud volcano ecosystems is still completely lacking. We investigated the impact of viral infection on the mortality and assemblage structure of benthic prokaryotes of five mud volcanoes in the Mediterranean Sea. Mud volcano sediments promote high rates of viral production (1.65-7.89 × 109 viruses g-1 d-1), viral-induced prokaryotic mortality (33% cells killed per day) and heterotrophic prokaryotic production (3.0-8.3 μgC g-1 d-1 ) when compared with sediments outside 10 the mud volcano area. The viral shunt (i.e., the microbial biomass converted into dissolved organic matter as a result of viral infection, and thus diverted away from higher trophic levels) provides 49 mgC m-2 d-1, thus fuelling the metabolism of uninfected prokaryotes and contributing to the total C budget. Bacteria are the dominant components of prokaryotic assemblages in surface sediments of mud volcanoes, whereas archaea dominate the subsurface sediment layers. Multivariate multiple regression analyses show that prokaryotic assemblage composition is not only dependent on the geochemical features and processes of mud volcano ecosystems, but also on synergistic interactions between bottom-up (i.e., trophic resources) and top-down (i.e., viral-induced prokaryotic mortality) controlling factors. Overall, these findings highlight the significant role of the viral shunt in sustaining the metabolism of prokaryotes and shaping their assemblage structure in mud volcano sediments, and they provide new clues for our understanding of the functioning of cold-seep ecosystems.

Background Over the last 30 years, extensive studies have revealed the crucial roles played by microbes in aquatic ecosystems. It has been shown that bacteria, viruses and protozoan grazers are dominant in terms of abundance and biomass. The frequent interactions between these microbiological compartments are responsible for strong trophic links from dissolved organic matter to higher trophic levels, via heterotrophic bacteria, which form the basis for the important biogeochemical roles of microbial food webs in aquatic ecosystems. To gain a better understanding of the interactions between bacteria, viruses and flagellates in lacustrine ecosystems, we investigated the effect of protistan bacterivory on bacterial abundance, production and structure [determined by 16S rRNA PCR-DGGE], and viral abundance and activity of two lakes of contrasting trophic status. Four experiments were conducted in the oligotrophic Lake Annecy and the mesotrophic Lake Bourget over two seasons (early spring vs. summer) using a fractionation approach. In situ dark vs. light incubations were performed to consider the effects of the different treatments in the presence and absence of phototrophic activity. Results The presence of grazers (i.e. < 5-μm small eukaryotes) affected viral production positively in all experiments, and the stimulation of viral production (compared to the treatment with no eukaryotic predators) was more variable between lakes than between seasons, with the highest value having been recorded in the mesotrophic lake (+30%). Viral lysis and grazing activities acted additively to sustain high bacterial production in all experiments. Nevertheless, the stimulation of bacterial production was more variable between seasons than between lakes, with the highest values obtained in summer (+33.5% and +37.5% in Lakes Bourget and Annecy, respectively). The presence of both predators (nanoflagellates and viruses) did not seem to have a clear influence upon bacterial community structure according to the four experiments. Conclusions Our results highlight the importance of a synergistic effect, i.e. the positive influence of grazers on viral activities in sustaining (directly and indirectly) bacterial production and affecting composition, in both oligotrophic and mesotrophic lakes.

Viral and bacterial communities were studied at two different areas (coastal and open sea) in the Middle Adriatic during the summer period 2008. Viral number was obtained by epifluorescent microscopy and viral production rates were determined from first-order regressions of viral abundance versus time for triplicate incubations. Viral turnover rates were estimated by dividing viral production rates by viral abundance. Abundance of bacteria was obtained by flow cytometer. Relationship between viruses and bacteria was calculated from virus to bacteria ratio (VBR). Viral abundance (2, 77 - 6, 31 x 106 VLP ml-1) and production (2, 28 - 6, 26 x 106 VLP ml-1 h-1) in the coastal sea area were lower than in the open sea area where viral abundance ranged from 2, 75 to 8, 39 x 106 VLP ml-1 and viral production from 4, 14 to 7, 27 x 106 VLP ml-1 h-1. Viral turnover rates at coastal sea ranged from 1, 41 to 2, 00 h and from 0, 39 to 0, 70 h at open sea. VBR in the coastal sea area ranged from 5, 49 to 7, 54 and in the open sea area from 7, 44 to 36, 46. Domination of the viruses over bacteria was evident in both observed areas, but it was greater in oligotrophic open sea suggesting that the viruses were more important factor in controlling bacterial abundance in the open than in the coastal sea area.

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ± 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.