The End of a 60-year Riddle: Identification and Genomic Characterization of an Iridovirus, the Causative Agent of White Fat Cell Disease in Zooplankton.

The planktonic freshwater crustacean of the genus Daphnia are a model system for biomedical research and, in particular, invertebrate-parasite interactions. Up until now, no virus has been characterized for this system. Here we report the discovery of an iridovirus as the causative agent of White Fat Cell Disease (WFCD) in Daphnia WFCD is a highly virulent disease of Daphnia that can easily be cultured under laboratory conditions. Although it has been studied from sites across Eurasia for more than 60 years, its causative agent had not been described, nor had an iridovirus been connected to WFCD before now. Here we find that an iridovirus-the Daphnia iridescent virus 1 (DIV-1)-is the causative agent of WFCD. DIV-1 has a genome sequence of about 288 kbp, with 39% G+C content and encodes 367 predicted open reading frames. DIV-1 clusters together with other invertebrate iridoviruses but has by far the largest genome among all sequenced iridoviruses. Comparative genomics reveal that DIV-1 has apparently recently lost a substantial number of unique genes but has also gained genes by horizontal gene transfer from its crustacean host. DIV-1 represents the first invertebrate iridovirus that encodes proteins to purportedly cap RNA, and it contains unique genes for a DnaJ-like protein, a membrane glycoprotein and protein of the immunoglobulin superfamily, which may mediate host-pathogen interactions and pathogenicity. Our findings end a 60-year search for the causative agent of WFCD and add to our knowledge of iridovirus genomics and invertebrate-virus interactions.

Occurrence, pathology, and ultrastructure of iridovirus and cytoplasmic polyhedrosis viruses in daphnids from the Czech Republic.

Iridescent (IVs, family Iridoviridae, genus Iridovirus) and cytoplasmic polyhedrosis viruses (CPVs; family Reoviridae, genus Cypovirus) are well known in insects, with thirteen IV species recognized from various orders, and sixteen CPV species known from lepidopterans. In 1975, an IV and CPV were reported in the daphnid, Simocehpalus expinosus, in Florida, but other reported daphnid virus infections seem to be rare. Here we report infected daphnids from woodland and carp ponds in the Czech Republic, Daphnia curvirostris with an IV, and D. pulex and D. ambigua, with CPVs. This suggests these viruses are more common in daphnids, the rarity of reports due to few surveys.

Statistical Mechanics of Zooplankton.

Statistical mechanics provides the link between microscopic properties of many-particle systems and macroscopic properties such as pressure and temperature. Observations of similar "microscopic" quantities exist for the motion of zooplankton, as well as many species of other social animals. Herein, we propose to take average squared velocities as the definition of the "ecological temperature" of a population under different conditions on nutrients, light, oxygen and others. We test the usefulness of this definition on observations of the crustacean zooplankton Daphnia pulicaria. In one set of experiments, D. pulicaria is infested with the pathogen Vibrio cholerae, the causative agent of cholera. We find that infested D. pulicaria under light exposure have a significantly greater ecological temperature, which puts them at a greater risk of detection by visual predators. In a second set of experiments, we observe D. pulicaria in cold and warm water, and in darkness and under light exposure. Overall, our ecological temperature is a good discriminator of the crustacean's swimming behavior.

Persistence of an amphibian ranavirus in aquatic communities.

Host-parasite dynamics can be strongly influenced by interactions with other members of the biotic community, particularly when the parasite spends some fraction of its life in the environment unprotected by its host. Ranaviruses-often lethal viruses of cold-blooded vertebrate hosts transmitted by direct contact, and via water and fomites-offer an interesting system for understanding these community influences. Previous laboratory studies have shown that ranaviruses can persist for anywhere from days to years, depending on the conditions, with much longer times under sterile conditions. To address the role of the biotic community and particulate matter on ranavirus persistence, we experimentally inoculated filter-sterilized, UV-treated, and unmanipulated pond water with a Frog virus 3 (FV3)-like Ranavirus and took samples over 78 d, quantifying viral titers with real-time quantitative PCR and plaque assays. Viral counts dropped quickly in all treatments, by an order of magnitude in under a day in unmanipulated pond water and in 8 d in filter-sterilized pond water. In a second experiment, we measured viral titers over 24 h in virus-spiked spring water with Daphnia pulex. Presence of D. pulex reduced the concentration of infectious ranavirus, but not viral DNA, by an order of magnitude in 24 h. D. pulex themselves did not accumulate the virus. We conclude that both microbial and zooplanktonic communities can play an important role in ranavirus epidemiology, rapidly inactivating ranavirus in the water and thereby minimizing environmental transmission. We suspect that interactions with the biotic community will be important for most pathogens with environmental resting or transmission stages.

Selectively maintained paleoviruses in Holarctic water fleas reveal an ancient origin for phleboviruses.

The ecological model, Daphnia pulex (Cladocera: Daphniidae), is broadly distributed in Holarctic freshwater habitats and has been the subject of multidisciplinary study for over half a century, but never has a natural RNA virus infection been reported in daphnids. Here we report on a group of paleoviruses related to RNA dependent RNA polymerase in the genome of D. pulex. Phylogenetic analysis suggests that these paleoviruses are derived from a viral lineage within the genus Phlebovirus. Comparison of the genomic sequences flanking individual paleoviruses reveal that some are orthologous viral insertions having been present in the common ancestor of the D. pulex species complex, which is millions of years old. Still, we detected some sites that have the signature of purifying selection. In contrast, other paleoviruses in this group seem to be unique to specific host lineages and even contain undisrupted open reading frames, suggesting either more recent acquisition, or selective maintenance.

Accumulation and inactivation of avian influenza virus by the filter-feeding invertebrate Daphnia magna.

The principal mode of avian influenza A virus (AIV) transmission among wild birds is thought to occur via an indirect fecal-oral route, whereby individuals are exposed to virus from the environment through contact with virus-contaminated water. AIV can remain viable for an extended time in water; however, little is known regarding the influence of the biotic community (i.e., aquatic invertebrates) on virus persistence and infectivity in aquatic environments. We conducted laboratory experiments to investigate the ability of an aquatic filter-feeding invertebrate, Daphnia magna, to accumulate virus from AIV-dosed water under the hypothesis that they represent a potential vector of AIV to waterfowl hosts. We placed live daphnids in test tubes dosed with low-pathogenicity AIV (H3N8 subtype isolated from a wild duck) and sampled Daphnia tissue and the surrounding water using reverse transcription-quantitative PCR (RT-qPCR) at 3- to 120-min intervals for up to 960 min following dosing. Concentrations of viral RNA averaged 3 times higher in Daphnia tissue than the surrounding water shortly after viral exposure, but concentrations decreased exponentially through time for both. Extracts from Daphnia tissue were negative for AIV by cell culture, whereas AIV remained viable in water without Daphnia present. Our results suggest daphnids can accumulate AIV RNA and effectively remove virus particles from water. Although concentrations of viral RNA were consistently higher in Daphnia tissue than the water, additional research is needed on the time scale of AIV inactivation after Daphnia ingestion to fully elucidate Daphnia's role as a potential vector of AIV infection to aquatic birds.

An experimental study of the population and evolutionary dynamics of Vibrio cholerae O1 and the bacteriophage JSF4.

Studies of Vibrio cholerae in the environment and infected patients suggest that the waning of cholera outbreaks is associated with rise in the density of lytic bacteriophage. In accordance with mathematical models, there are seemingly realistic conditions where phage predation could be responsible for declines in the incidence of cholera. Here, we present the results of experiments with the El Tor strain of V. cholerae (N16961) and a naturally occurring lytic phage (JSF4), exploring the validity of the main premise of this model: that phage predation limits the density of V. cholerae populations. At one level, the results of our experiments are inconsistent with this hypothesis. JSF4-resistant V. cholerae evolve within a short time following their confrontation with these viruses and their populations become limited by resources rather than phage predation. At a larger scale, however, the results of our experiments are not inconsistent with the hypothesis that bacteriophage modulate outbreaks of cholera. We postulate that the resistant bacteria that evolved play an insignificant role in the ecology or pathogenicity of V. cholerae. Relative to the phage-sensitive cells from whence they are derived, the evolved JSF4-resistant V. cholerae have fitness costs and other characters that are likely to impair their ability to compete with the sensitive cells in their natural habitat and may be avirulent in human hosts. The results of this in vitro study make predictions that can be tested in natural populations of V. cholerae and cholera-infected patients.

Flavobacterium psychrophilum, invasion into and shedding by rainbow trout Oncorhynchus mykiss.

The infection route of Flavobacterium psychrophilum into rainbow trout Oncorhynchus mykiss was studied using bath and cohabitation challenges as well as oral challenge with live feed as a vector. Additionally, the number of bacterial cells shed by infected fish into the surrounding water was determined in the cohabitation experiment and in challenge experiments at 3 different water temperatures. The experiments showed that skin and skin mucus abrasion dramatically enhanced the invasion of F. psychrophilum into the affected fish in bath and cohabitation challenges. Disruption of the skin is discussed as an important invasion route for F. psychrophilum into the fish. The shedding rate of F. psychrophilum by infected fish was associated with water temperature and the mortality of the infected fish. High numbers of F. psychrophilum cells were released into the water by dead rainbow trout during a long time period compared to the numbers of cells shed by live fish. The results emphasise the importance of removing dead and moribund fish from rearing tanks in order to diminish the infection pressure against uninfected fish in commercial fish farms. In immunohistochemical examinations of organs and tissues of orally infected fish, F. psychrophilum cells were detected in only 1 fish out of 31 studied. Mortality of the orally challenged fish was not observed in the experiment.