Identification of a novel statovirus in a faecal sample from a calf with enteric disease.

A novel clade of RNA viruses was identified in the mammalian gastrointestinal tract by next-generation sequencing. Phylogenetically, these viruses are related to the genera Tombusviridae (plant viruses) and Flaviviridae, which includes mammalian, avian and insect hosts. Named in line with their characterization as stool-associated Tombus-like viruses, it is unclear if statoviruses infect mammals or are dietary in origin. Here, metagenomic sequencing of faecal material collected from a 10-week-old calf with enteric disease found that 20 % of the reads mapped to a de novo-assembled 4 kb contig with homology to statoviruses. Phylogenetic analysis of the statovirus genome found a clear evolutionary relationship with statovirus A, but, with only 47 % similarity, we propose that the statovirus sequence presents a novel species, statovirus F. A TaqMan PCR targeting statovirus F performed on faecal material found a cycle threshold of 11, suggesting a high titre of virus shed from the calf with enteric disease. A collection of 48 samples from bovine enteric disease diagnostic submissions were assayed by PCR to investigate statovirus F prevalence and 6 of 48 (12.5 %) were positive. An ELISA to detect antibodies to the coat protein found that antibodies to statovirus F were almost ubiquitous in bovine serum. Combined, the PCR and ELISA results suggest that statovirus F commonly infects cattle. Further research is needed to elucidate the aetiological significance of statovirus infection.

A Functional K+ Channel from Tetraselmis Virus 1, a Member of the Mimiviridae.

Potassium ion (K+) channels have been observed in diverse viruses that infect eukaryotic marine and freshwater algae. However, experimental evidence for functional K+ channels among these alga-infecting viruses has thus far been restricted to members of the family Phycodnaviridae, which are large, double-stranded DNA viruses within the phylum Nucleocytoviricota. Recent sequencing projects revealed that alga-infecting members of Mimiviridae, another family within this phylum, may also contain genes encoding K+ channels. Here we examine the structural features and the functional properties of putative K+ channels from four cultivated members of Mimiviridae. While all four proteins contain variations of the conserved selectivity filter sequence of K+ channels, structural prediction algorithms suggest that only two of them have the required number and position of two transmembrane domains that are present in all K+ channels. After in vitro translation and reconstitution of the four proteins in planar lipid bilayers, we confirmed that one of them, a 79 amino acid protein from the virus Tetraselmis virus 1 (TetV-1), forms a functional ion channel with a distinct selectivity for K+ over Na+ and a sensitivity to Ba2+. Thus, virus-encoded K+ channels are not limited to Phycodnaviridae but also occur in the members of Mimiviridae. The large sequence diversity among the viral K+ channels implies multiple events of lateral gene transfer.

Trapping the Enemy: Vermamoeba vermiformis Circumvents Faustovirus Mariensis Dissemination by Enclosing Viral Progeny inside Cysts.

Viruses depend on cells to replicate and can cause considerable damage to their hosts. However, hosts have developed a plethora of antiviral mechanisms to counterattack or prevent viral replication and to maintain homeostasis. Advantageous features are constantly being selected, affecting host-virus interactions and constituting a harsh race for supremacy in nature. Here, we describe a new antiviral mechanism unveiled by the interaction between a giant virus and its amoebal host. Faustovirus mariensis infects Vermamoeba vermiformis, a free-living amoeba, and induces cell lysis to disseminate into the environment. Once infected, the cells release a soluble factor that triggers the encystment of neighbor cells, preventing their infection. Remarkably, infected cells stimulated by the factor encyst and trap the viruses and viral factories inside cyst walls, which are no longer viable and cannot excyst. This unprecedented mechanism illustrates that a plethora of antiviral strategies remains to be discovered in nature.IMPORTANCE Understanding how viruses of microbes interact with its hosts is not only important from a basic scientific point of view but also for a better comprehension of the evolution of life. Studies involving large and giant viruses have revealed original and outstanding mechanisms concerning virus-host relationships. Here, we report a mechanism developed by Vermamoeba vermiformis, a free-living amoeba, to reduce Faustovirus mariensis dissemination. Once infected, V. vermiformis cells release a factor that induces the encystment of neighbor cells, preventing infection of further cells and/or trapping the viruses and viral factories inside the cyst walls. This phenomenon reinforces the need for more studies regarding large/giant viruses and their hosts.

Complex Membrane Remodeling during Virion Assembly of the 30,000-Year-Old Mollivirus Sibericum.

Cellular membranes ensure functional compartmentalization by dynamic fusion-fission remodeling and are often targeted by viruses during entry, replication, assembly, and egress. Nucleocytoplasmic large DNA viruses (NCLDVs) can recruit host-derived open membrane precursors to form their inner viral membrane. Using complementary three-dimensional (3D)-electron microscopy techniques, including focused-ion beam scanning electron microscopy and electron tomography, we show that the giant Mollivirus sibericum utilizes the same strategy but also displays unique features. Indeed, assembly is specifically triggered by an open cisterna with a flat pole in its center and open curling ends that grow by recruitment of vesicles never reported for NCLDVs. These vesicles, abundant in the viral factory (VF), are initially closed but open once in close proximity to the open curling ends of the growing viral membrane. The flat pole appears to play a central role during the entire virus assembly process. While additional capsid layers are assembled from it, it also shapes the growing cisterna into immature crescent-like virions and is located opposite to the membrane elongation and closure sites, thereby providing virions with a polarity. In the VF, DNA-associated filaments are abundant, and DNA is packed within virions prior to particle closure. Altogether, our results highlight the complexity of the interaction between giant viruses and their host. Mollivirus assembly relies on the general strategy of vesicle recruitment, opening, and shaping by capsid layers similar to all NCLDVs studied until now. However, the specific features of its assembly suggest that the molecular mechanisms for cellular membrane remodeling and persistence are unique.IMPORTANCE Since the first giant virus Mimivirus was identified, other giant representatives are isolated regularly around the world and appear to be unique in several aspects. They belong to at least four viral families, and the ways they interact with their hosts remain poorly understood. We focused on Mollivirus sibericum, the sole representative of "Molliviridae," which was isolated from a 30,000-year-old permafrost sample and exhibits spherical virions of complex composition. In particular, we show that (i) assembly is initiated by a unique structure containing a flat pole positioned at the center of an open cisterna, (ii) core packing involves another cisterna-like element seemingly pushing core proteins into particles being assembled, and (iii) specific filamentous structures contain the viral genome before packaging. Altogether, our findings increase our understanding of how complex giant viruses interact with their host and provide the foundation for future studies to elucidate the molecular mechanisms of Mollivirus assembly.

Host behavior alters spiny lobster-viral disease dynamics: a simulation study.

Social behavior confers numerous benefits to animals but also risks, among them an increase in the spread of pathogenic diseases. We examined the trade-off between risk of predation and disease transmission under different scenarios of host spatial structure and disease avoidance behavior using a spatially explicit, individual-based model of the host pathogen interaction between juvenile Caribbean spiny lobster (Panulirus argus) and Panulirus argus Virus 1 (PaV1). Spiny lobsters are normally social but modify their behavior to avoid diseased conspecifics, a potentially effective means of reducing transmission but one rarely observed in the wild. We found that without lobster avoidance of diseased conspecifics, viral outbreaks grew in intensity and duration in simulations until the virus was maintained continuously at unrealistically high levels. However, when we invoked disease avoidance at empirically observed levels, the intensity and duration of outbreaks was reduced and the disease extirpated within five years. Increased lobster (host) spatial aggregation mimicking that which occurs when sponge shelters for lobsters are diminished by harmful algal blooms, did not significantly increase PaV1 transmission or persistence in lobster populations. On the contrary, behavioral aversion of diseased conspecifics effectively reduced viral prevalence, even when shelters were limited, which reduced shelter availability for all lobsters but increased predation, especially of infected lobsters. Therefore, avoidance of diseased conspecifics selects against transmission by contact, promotes alternative modes of transmission, and results in a more resilient host-pathogen system.

Prevalence of Panulirus argus Virus 1 (PaV1) and habitation patterns of healthy and diseased Caribbean spiny lobsters in shelter-limited habitats.

Caribbean spiny lobsters Panulirus argus are socially gregarious, preferring shelters harboring conspecifics over empty shelters. In laboratory trials, however, healthy lobsters strongly avoided shelters harboring lobsters infected with the highly pathogenic Panulirus argus Virus 1 (PaV1). Because PaV1 is transmitted by contact, this behavior may thwart its spread in wild lobsters. In a field experiment conducted from 1998 to 2002 in a shelter-poor reef lagoon (Puerto Morelos, Mexico), densities of juvenile P. argus increased significantly on sites enhanced with artificial shelters (casitas) but not on control sites. Because PaV1 emerged in this location during 2000, we reexamined these data to assess whether casitas could potentially increase transmission of PaV1. In 2001, PaV1 prevalence was 2.5% and the cohabitation level (percentage of healthy lobsters cohabiting with diseased lobsters) was similar between natural shelters (3.5%) and casitas (2.4 %). The relative lobster densities in casita sites and control sites did not change significantly before (1998-1999) or after (2001-2002) the disease emergence. In late 2006, data from casita sites showed a significant increase in prevalence (10.9%) and cohabitation level (29.4%), but no significant changes in lobster density. In May 2006, casitas were deployed on shelter-poor sites within Chinchorro Bank, 260 km south of Puerto Morelos. In late 2006, prevalence and cohabitation level were 7.4 and 21.7%, respectively. Our results are inconclusive as to whether or not casitas increase PaV1 transmission, but suggest that across shelter-poor habitats, lobsters make a trade-off between avoiding diseased conspecifics and avoiding predation risk.

Molecular characterisation and phylogenetic analysis of Chronic bee paralysis virus, a honey bee virus.

The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.

Preliminary studies on the biology of Borna disease virus.

Borna disease virus (BDV) is an unclassified agent that causes neurological disease in a wide range of animal species and possibly in humans. The infectious nature of BDV has been long established but, despite extensive progress on the pathogenesis of the infection, the aetiological agent is still uncharacterized. Recent studies have shown that BDV replicates productively in cultures of foetal rabbit glial cells (FRG) which produce a virus-specific protein that is easily detected immunocytochemically. This provides a marker for BDV infectivity. This cell culture system was used to investigate the replication cycle of BDV. The agent required at least 1 h to bind to and penetrate the cells and the antigen was detected 24 h later. Cycloheximide and actinomycin D inhibited production of the antigen in inoculated cells, indicating that both protein synthesis and a DNA-dependent function were required for the production of viral antigen. Cocultivation of BDV-infected FRG cells with Vero cells resulted in a persistent productive infection in the latter. Use of these cells showed that the infectious agent matured exclusively in the cytoplasm and within the plasma membrane of the cell. Antigen-laden nuclei did not have infectivity. These studies showed that BDV has the physical and replicative properties typical of conventional viruses but its mechanism of replication and site of morphogenesis may be unique.

Temporal distribution of transmissible mink encephalopathy virus in mink inoculated subcutaneously.

Information was sought on the temporal distribution of transmissible mink encephalopathy virus in royal pastel mink inoculated subcutaneously with 10(3.0) 50% intracerebral lethal doses of the Idaho strain. As determined by intracerebral assay in mink, extremely little replication of the virus occurred during the preclinical stage of infection. It seemed largely limited to lymph nodes draining the site of inoculation. Virus first appeared in the central nervous system (CNS) at 20 weeks, when all mink were still clinically normal. Early spongiform degeneration, limited to the posterior sigmoid gyrus of the frontal cortex, was first found at 28 weeks, or a few weeks before onset of clinical disease in most of the mink. Once virus reached the CNS, where greater concentrations occurred than elsewhere, it appeared in many extraneural sites (spleen, liver, kidney, intestine, mesenteric lymph node, and submandibular salivary gland). These seemingly anomalous findings, especially the limited extraneural replication of virus as a prelude to infection of the CNS, suggest that mink are not natural hosts of the virus. The results of this study support the generally held view that transmissible mink encephalopathy arises from chance or inadvertent infection of ranch mink with an exogenous virus, most likely feed-borne wild scrapie virus.

Creutzfeldt-Jakob disease in mice: persistent viremia and preferential replication of virus in low-density lymphocytes.

The mode of replication of the "unconventional virus" of Creutzfeldt-Jakob disease was studied in BALB/c mice infected intracerebrally. Virus was detected in the brain, spleen, lung, thymus, liver, kidney, and blood, but not in urine, at various time intervals after inoculation. The highest infectivity was present in the spleen from the second through the ninth weeks postinfection. Density gradient separation of spleen cells with colloidal silica (Percoll) revealed that the highest concentration of virus was present in blastoid cells from lower-density (1.05 to 1.07 g/ml) fractions. These results suggest that blastoid cells play an important role as the initial replication site of virus in the pathogenesis of Creutzfeldt-Jakob disease in mice.

[Creutzfeld-Jakob disease: recent advances; biology of unconventional viruses (author's transl)]. / Maladie de Creutzfeldt-Jakob: nouvelles acquisitions sur la biologie des cirus non conventionnels.

The authors update their earlier review (1973) of the unconventional viruses responsible for the subacute spongiform encephalopathies of man and animals. Newer data about the viruses of scrapie and Creutzfeldt-Jakob disease are summarized. The biology and experimental pathology of the diseases are discussed.

Observation on the remarkable stability of transmissible mink encephalopathy virus.

Transmissible mink encephalopathy (TME) virus retained biological activity after prolonged storage in formalin (about three and a half years) and in paraffin tissue blocks used for histologic preparations (about six and a half years). Thus, TME tissues in formalin and in paraffin blocks should be handled as contaminated materials.

Biophysical properties of frog virus and its deoxyribonucleic acid: fate of radioactive virus in the early stage of infection.

Frog virus (FV-3) was banded by isopycnic centrifugation in cesium chloride, sucrose, or potassium tartrate. Two bands of infectivity were regularly found at positions in cesium chloride corresponding to densities of 1.26 and 1.30 g/cm(3), respectively. Deoxyribonucleic acid from either band had the following characteristics: double-stranded; a T(m) of 76.3 C in 0.1 SSC (0.015 m NaCl plus 0.015 m sodium citrate) and a buoyant density of 1.720 g/cm(3) in cesium chloride, corresponding to a guanine plus cytosine content of 56 to 58% and a molecular weight of 130 x 10(6) daltons, determined by velocity sedimentation. These data, together with electron micrographs of sections of cells infected with material from either band suggest that two types of infectious frog virus particles exists, rather than a second virus in the frog virus stocks. The composition of frog virus was determined. It was found that highly purified preparations of frog virus were composed of 55.8% protein, 30.1% deoxyribonucleic acid, and 14.2% lipid. The kinetics of adsorption and uncoating of FV-3 was studied with radioactive virus. Uncoating is comparatively rapid and in contrast to poxvirus is unaffected by inhibitors of protein synthesis.