The Kaumoebavirus LCC10 Genome Reveals a Unique Gene Strand Bias among "Extended Asfarviridae".

Kaumoebavirus infects the amoeba Vermamoeba vermiformis and has recently been described as a distant relative of the African swine fever virus. To characterize the diversity and evolution of this novel viral genus, we report here on the isolation and genome sequencing of a second strain of Kaumoebavirus, namely LCC10. Detailed analysis of the sequencing data suggested that its 362-Kb genome is linear with covalently closed hairpin termini, so that DNA forms a single continuous polynucleotide chain. Comparative genomic analysis indicated that although the two sequenced Kaumoebavirus strains share extensive gene collinearity, 180 predicted genes were either gained or lost in only one genome. As already observed in another distant relative, i.e., Faustovirus, which infects the same host, the center and extremities of the Kaumoebavirus genome exhibited a higher rate of sequence divergence and the major capsid protein gene was colonized by type-I introns. A possible role of the Vermamoeba host in the genesis of these evolutionary traits is hypothesized. The Kaumoebavirus genome exhibited a significant gene strand bias over the two-third of genome length, a feature not seen in the other members of the "extended Asfarviridae" clade. We suggest that this gene strand bias was induced by a putative single origin of DNA replication located near the genome extremity that imparted a selective force favoring the genes positioned on the leading strand.

Different tertiary interactions create the same important 3D features in a distinct flavivirus xrRNA.

During infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3' untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific three-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5' end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3' UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3' UTR of the Tamana bat virus (TABV) and solved its structure by X-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

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.

Kaumoebavirus, a New Virus That Clusters with Faustoviruses and Asfarviridae.

In this study, we report the isolation of a new giant virus found in sewage water from the southern area of Jeddah (Saudi Arabia), with morphological and genomic resemblance to Faustoviruses. This new giant virus, named Kaumoebavirus, was obtained from co-culture with Vermamoeba vermiformis, an amoeboid protozoa considered to be of special interest to human health and the environment. This new virus has ~250 nm icosahedral capsids and a 350,731 bp DNA genome length. The genome of Kaumoebavirus has a coding density of 86%, corresponding to 465 genes. Most of these genes (59%) are closely related to genes from members of the proposed order Megavirales, and the best matches to its proteins with other members of the Megavirales are Faustoviruses (43%) and Asfarviruses (23%). Unsurprisingly, phylogenetic reconstruction places Kaumoebavirus as a distant relative of Faustoviruses and Asfarviruses.

Diversity of viruses of the hyperthermophilic archaeal genus Aeropyrum, and isolation of the Aeropyrum pernix bacilliform virus 1, APBV1, the first representative of the family Clavaviridae.

We have surveyed the morphological diversity of viruses infecting the archaeon Aeropyrum pernix, the most thermophilic species among aerobic organisms, growing optimally at 90 degrees C, and isolated and characterized a novel virus, Aeropyrum pernix bacilliform virus 1, APBV1. This is the first virus to be described of the genus Aeropyrum and the archaeal order Desulfurococcales. The virion of APBV1 has rigid bacilliform morphology, about 140x20nm, with one end pointed and the other rounded. It contains highly glycosylated single major protein and three minor proteins. The circular, double-stranded DNA genome comprising 5278bp is the smallest for known archaeal viruses. None of the 14 putative genes, all on the same DNA strand, shows significant similarity to sequences in the public databases. The APBV1 infection caused neither retardation of host growth nor lysis of host cells, and integration of the viral genome into the host chromosome was not detected. On the basis of unusual morphological and genomic properties, we propose to consider APBV1 as the first representative of a new viral family, the Clavaviridae.

Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales.

Here, we report the sequencing and classification of Nyamanini virus (NYMV) and Midway virus (MIDWV), two antigenically related viruses that were first isolated in 1957 and 1966, respectively. Although these viruses have been cultured multiple times from cattle egrets, seabirds, and their ticks, efforts to classify them taxonomically using conventional serological and electron microscopic approaches have failed completely. We used a random shotgun sequencing strategy to define the genomes of NYMV and MIDWV. Contigs of 11,631 and 11,752 nucleotides, representing the complete genome of NYMV and the near-complete genome of MIDWV, respectively, were assembled. Each virus genome was predicted to carry six open reading frames (ORFs). BLAST analysis indicated that only two of the ORF proteins of each virus, the putative nucleocapsid and polymerase, had detectable sequence similarity to known viral proteins. Phylogenetic analysis of these ORF proteins demonstrated that the closest relatives of NYNV and MIDWV are negative-stranded-RNA viruses in the order Mononegavirales. On the basis of their very limited sequence similarity to known viruses, we propose that NYMV and MIDWV define a novel genus, Nyavirus, in this order.

Isolation and characterization of a single-stranded RNA virus infecting the bloom-forming diatom Chaetoceros socialis.

Diatoms are very significant primary producers in the world's oceans. Various environmental factors affect the depletion of diatom populations. The importance of viruses as a potential mortality source has recently been recognized. We isolated and characterized a new diatom virus (Chaetoceros socialis f. radians RNA virus [CsfrRNAV]) causing the lysis of the bloom-forming species Chaetoceros socialis Lauder f. radians (Schütt) Proschkina-Lavrenko. The virus infectious to C. socialis f. radians was isolated from water samples collected in Hiroshima Bay. Here we show the physiology, morphology, and genome characteristics of the virus clone. Virions were 22 nm in diameter and accumulated in the cytoplasm of the host cells. The latent period and the burst size were estimated to be <48 h and 66 infectious units per host cell, respectively. CsfrRNAV harbors a single-stranded RNA (ssRNA) genome and encodes at least three polypeptides of 32.0, 28.5, and 25.0 kDa. Sequencing analysis shows the length of the genome is 9,467 bases, excluding a poly(A) tail. The monophyly of CsfrRNAV and other diatom-infecting RNA viruses, Rhizosolenia setigera RNA virus and Chaetoceros tenuissimus RNA virus, was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA-dependent RNA polymerase domains. This suggested a new ssRNA virus family, Bacillariornaviridae. This discovery of CsfrRNAV may aid in further understanding the ecological dynamics of the C. socialis f. radians population in nature and the relationships between ssRNA diatom viruses and their hosts.

Systematic morphogenesis of the viral haemorrhagic disease virus in infected rabbits and in adapted cell culture.

The morphogenesis of viral haemorrhagic disease virus (VHDV) of rabbits in infected cell cultures and in tissues and cells from infected animals has been observed systematically by electron microscopy. Viral particles have been found to assemble through a condensation process within the nucleus of infected cells, having passed partially into the cytoplasm through disrupted nuclear membranes, enlarged nuclear pores or perinuclear space. Late in infection, both mature and immature virions are observed in the cytoplasm; these are often aggregated or interspersed and remain cell-associated long after infection. Release of the virus occurs when the cell finally lyses. In studies of the localisation and development of VHDV antigens, the viral antigens have been observed to appear first in the nucleus of infected cells and often thereafter in the cytoplasm. This finding is very similar to observations made by electron microscopy.

Application of control measures against viral haemorrhagic disease of rabbits in the Czech and Slovak Federal Republic.

The first outbreaks of viral haemorrhagic disease (VHD) of rabbits were reported from eastern Slovakia in 1987. In 1988, the infection spread throughout the Czech and Slovak Federal Republic. Electron microscopy was used by the Veterinary Research Institute in Brno to diagnose the disease during the early stage of infection. At present, the regional laboratories of the veterinary investigation services use the haemagglutination and the direct immunofluorescence tests as the principal methods to demonstrate the causal agent. Indirect immunofluorescence and immunoperoxidase techniques have been developed to demonstrate VHD virus, while the enzyme-linked immunosorbent assay (ELISA) has been used to detect antibodies. Diagnostic kits, allowing a wide use of these methods, are now available commercially. Two types of inactivate vaccines were developed and produced in 1988 and 1989. VHD is controlled by vaccination of exposed rabbit colonies. This is accompanied by other preventive and protective measures, directed by district veterinary officers following instructions from federal authorities.

Electron microscopic reporting of gastrointestinal viruses in the United Kingdom, 1985-1987.

We examined some epidemiological features of the viruses associated with gastrointestinal illness, using national data reported by electron microscopists in the United Kingdom. During the 3 years analyzed (1985-1987), a total of 1,993 positive detections of astroviruses, caliciviruses, coronaviruses, and small round structured viruses (SRSVs) were reported. In 1 year of this period, 8,210 rotaviruses were reported. More than 90% of the astroviruses and caliciviruses were detected in children under 5 years of age, while coronaviruses and SRSVs were detected in adults as well as children. Detections of astroviruses increased in the winter and were infrequent during the summer, a seasonal pattern similar to that observed for rotaviruses. There was some variability between reporting regions in rates of detection of fecal viruses. We have attempted to identify the reasons for this. We make suggestions for improving the detection of human fecal viruses, and we recognize the need for continued surveillance of these agents.

Six-year retrospective surveillance of gastroenteritis viruses identified at ten electron microscopy centers in the United States and Canada.

To identify the prevalence, seasonality and demographic characteristics of patients with viral gastroenteritis, we reviewed 6 years of retrospective data on viral agents of gastroenteritis screened by electron microscopy at 10 centers in the United States and Canada. From 52,691 individual electron microscopic observations, a virus was detected in 16% of specimens, and the yearly positive detection rate among centers ranged from 8 to 34%. Rotavirus was the agent most commonly observed (26 to 83%), followed by adenoviruses (8 to 27%, respiratory and enteric combined), and small round viruses (SRVs) (0 to 40%) which were second most common at two of the centers. Rotavirus and astrovirus detections occurred more often in the winter but seasonal trends in detection were not apparent for the other viruses. Of all astroviruses detected 64% were found in infants (less than 1 year); unlike the other agents studied SRVs were detected in a large percentage of infants (48%) and older children and adults (20%). Among hospitalized patients a majority of all astroviruses, caliciviruses and SRVs were detected 7 days or more after admission in contrast to both rotaviruses and adenoviruses which were more likely to be detected earlier. The data suggest that SRVs are common agents of gastroenteritis and may be important causes of nosocomial infections. Because of the relative insensitivity of direct electron microscopy as a screening method for SRVs, astroviruses and caliciviruses, these data probably underestimate the true prevalence of disease caused by these agents.

Western blot (immunoblot) assay of small, round-structured virus associated with an acute gastroenteritis outbreak in Tokyo.

Small, round-structured virus (SRSV) was detected in a stool specimen of a patient during an acute gastroenteritis outbreak in Tokyo and was tentatively named SRSV-9. SRSV-9 was purified by sucrose velocity gradient centrifugation after CsCl density gradient centrifugation. The buoyant density of SRSV-9 appeared to be 1.36 g/ml in CsCl. A Western blot (immunoblot) assay using the biotin-avidin system revealed that SRSV-9 was antigenically related to the Hawaii agent but distinct from the Norwalk agent and contained a single major structural protein with a molecular size of 63.0 +/- 0.6 kilodaltons. The prevalence of SRSV-9 infection in Tokyo was surveyed by the Western blot antibody assay by using a crude virus preparation as the antigen. Seroconversion was observed in 56.5% of the patients involved in the outbreaks from which SRSV was detected by electron microscopy.

Physical properties of the Creutzfeldt-Jakob disease agent.

In this report, we present the first physical characterization of the Creutzfeld-Jakob disease agent. Preparations with high yields of infectivity (assayed infectious units) were obtained by a novel, gentle procedure in which initially sedimenting Gp34 ("prion" protein) was disaggregated by a variety of criteria with no subsequent loss of infectivity. Studies with this preparation indicate that most of the Creutzfeld-Jakob disease agent has both a viruslike size and density. In velocity sedimentation and isopycnic sucrose gradients, infectivity comigrated with nucleic acid-protein complexes of appreciable size.

Preliminary characterisation of torovirus-like particles of humans: comparison with Berne virus of horses and Breda virus of calves.

Pleomorphic virus-like particles have been observed by electron microscopy in the faeces of children and adults with diarrhoea. Some of these particles were approximately 100 nm in diameter and had a "fringe" of closely applied peplomers approximately 10 nm long; they closely resembled Berne virus of horses and Breda virus of calves, the two representatives of a newly proposed family called the Toroviridae. In one sample a toroidal nucleoprotein-like structure was observed within the particles. For two samples a buoyant density of 1.14 g/ml was determined by centrifugation through a sucrose density gradient. One sample possessed a haemagglutinin for rat erythrocytes. The serological relationship between these different viruses was observed by immune electron microscopy, haemagglutination inhibition, and serum neutralisation. The role of these virus-like particles as candidate pathogens of humans is discussed.

Comparative studies on three isolates of Breda virus of calves.

Three isolates of Breda virus of calves were compared morphologically and antigenically. The isolates demonstrated similar morphology and shared common antigens, as determined by enzyme-linked immunosorbent assay and immunoelectron microscopy. On the basis of results of the hemagglutination-inhibition test, enzyme-linked immunosorbent assay, and immunoelectron microscopy, the 3 isolates were further subdivided into 2 serotypes: serotype 1 (Breda virus 1) represented by the Iowa isolate 1; and serotype 2 (Breda virus 2), by the Ohio isolate and the Iowa isolate 2. The 3 isolates caused diarrhea in gnotobiotic calves.

Cellular lesions in intestinal mucosa of gnotobiotic calves experimentally infected with a new unclassified bovine virus (Breda virus).

Four gnotobiotic calves were inoculated intranasally with Breda virus within two hours after cesarean section. Three calves developed diarrhea between 48 and 60 hours after inoculation; one calf was killed at 36 hours. Consistent microscopic findings in the intestinal mucosae of calves with diarrhea were cytopathologic changes in enterocytes of the lower small intestine, large intestine, and dome epithelial cells and an acute inflammatory response with cellular infiltration and subtle changes in capillaries. Virions with average dimensions of 35 x 80 nm were seen in rounded cells that had severe cell swelling, dilatation of the cytocavitary network, and hydropic degeneration. Virions were most often in multiple, large autophagolysosomes. There was a good correlation in detection of virus in cells with electron microscopy and indirect immunofluorescence. No lesions were detectable in two control calves killed five days after inoculation.