Figure 1 (Top left) Image reconstruction of frozen-hydrated recombinant Norwalk virus (NV)-like particles (rNV VLPs). A set of icosahedral 5- and 3-fold axes is marked. (Top right) Central cross-section of rNV VLPs. Negative contrast electron micrographs of (Bottom left) Norwalk virus (NV) in a human stool specimen and (Bottom right) Rabbit hemorrhagic disease virus (RHDV) in a rabbit liver specimen. The bars represent 100 nm. [Courtesy of Prasad, B.V.V. (cryo-images), Kapikian, A. (em NV), König, M. and Thiel, H.-J. (em RHDV).]
Figure 2 Genome organizations of viruses of the family Caliciviridae. The genomic organization and ORF usage are shown for representative species (with strain indication shown in brackets) in the following genera: Lagovirus; Rabbit hemorrhagic disease virus (RHDV) [Ra/LV/RHDV/GH/1988/GE], “Norwalk-like viruses”; Norwalk virus (NV) [Hu/NV/Nor/1968/US], “Sapporo-like viruses”; Sapporo virus (SV) [Hu/SV/Man/1993/UK], and Vesivirus; Feline calicivirus (FCV) [Fe/VV/FCV/F9/1958/US. Viruses in two genera (Lagovirus and “Sapporo-like viruses”) contain a large ORF1 in which the nonstructural polyprotein gene is continuous and in frame with the capsid protein (CP) coding sequence. Some strains in the genus “Sapporo-like viruses” encode a third predicted ORF that overlaps ORF1 (not shown). Viruses in the other two genera (“Norwalk-like viruses” and Vesivirus) encode the major structural CP in a separate reading frame (ORF2). The RNA helicase (HEL), protease (PRO), and polymerase (POL) regions of the genome are indicated. The linkage of VPg to the RNA of viruses in the genera “Norwalk-like viruses” and “Sapporo-like” viruses has not been confirmed. The designated VPg region of the genomes of representative viruses from the genera “Norwalk-like viruses”, “Sapporo-like viruses”, and Vesivirus is shown by homology with the mapped VPg of RHDV. The shaded region of the ORF2 of the representative member of the genus Vesivirus illustrates the leader sequence (approximately 125 amino acids in length) of the precursor CP. Studies of FCV and RHDV have identified two major positive sense RNA molecules in infected cells. One RNA molecule corresponds in size to the full-length genome and the other, a subgenomic-sized RNA, is co-terminal with the 3-end of the genome. The sgRNA is the template for translation of the major viral CP and the 3-terminal ORF product that has been identified as a minor structural protein in FCV.
Figure 3 Phylogenetic relationships within the family Caliciviridae and comparison with the family Picornaviridae. (Courtesy of Berke, T. and Matson D.) Full-length capsid gene sequences were used for the phylogenetic analysis and included representative strains from each genus in the family Caliciviridae and one strain each from genera in the family Picornaviridae. Clustal analysis was used to create a multiple alignment for the amino acid sequences, which were verified by alignment of known motifs in the region (e.g., PPG/N). The nucleic acid sequences were added and aligned by hand using the corresponding amino acid alignment as template resulting in a consensus length of 2,070 nts. The PHYLIP v3.5c package run on a UNIX system was used for the phylogenetic analyses of the aligned nucleic acid sequences. The multiple alignment data were bootstrapped (N = 1000) and submitted for the distance method. The consensus tree from the Fitch-Margoliash distance algorithm was then added as a user-defined tree to the non-bootstrapped sequence data and submitted for maximum likelihood algorithm. The unrooted phylogenetic tree resulting from the maximum likelihood method is presented. Branch points of the resulting trees had a confidence level of P<0.01. Scale bar represents units for expected number of substitutions per site. The following numbers represent the phylogenetic distances, in units of expected number of substitutions per site, among the calicivirus genera (representing distinct clades) as measured by the distances between the first branchpoints of: “Norwalk-like viruses” and “Sapporo-like viruses”, 1.59; “Norwalk-like viruses” and Lagovirus, 1.48; “Norwalk-like viruses” and Vesivirus, 1.46; “Sapporo-like viruses” and Lagovirus, 1.21; “Sapporo-like viruses” and Vesivirus, 0.84; and Lagovirus and Vesivirus, 1.07. For comparison, the shortest phylogenetic distance between two picornavirus genera is 2.10 (strains Polio 1 and HRV14) in this analysis. The hepatitis E virus (not shown), formerly classified in the Caliciviridae was recently placed into the “unassigned virus” status. Genbank accession numbers for the strains in this analysis were: X86557, U22498, U07611, L23831, U04469, L07418, M87661, M67473, Z69620, U95645, X86560, U95643, U65427, U95644, U73124, U13992, M86379, M87481, U52086, U52005, U76874, M87482, X00429, J02281, K02121, M22458, and K02990.
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