Molecular characterization of a bovine enteric calicivirus: relationship to the Norwalk-like viruses

Abstract

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5' terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the Caliciviridae JV is most closely related to the group 1 NLVs.

FIG. 1

Diagrammatic representation of reading frame usage in JV. The nucleotide coordinates of the translation products are numbered on the open boxes. The repeat motifs at the 5′ genomic terminus and the predicted 5′ terminus of the subgenomic RNA are aligned beneath the genomic map. Shaded boxes indicate the genomic locations of these motifs.

FIG. 2

Translation products encoded by the three major ORFs of JV. The conserved motifs defining the 2C helicase (GPPGIGKT), 3C protease (GDCG), and 3D RNA-dependent RNA polymerase (GLPSG.....YGDD) are underlined with asterisks. The putative cleavage sites surrounding the 2C helicase are indicated by shaded boxes. The amino acid sequence coordinates for each of the three reading frames are on the left.

FIG. 3

RIPA of the JV capsid protein produced by in vitro transcription/translation. Lane A, preinfection serum from a colostrum-deprived, newborn calf. Lane B, serum taken at 4 weeks postinfection with JV. The position of the immunoprecipitated capsid protein is marked (arrow). Molecular size markers (in kilodaltons) are on the left.

FIG. 4

Unrooted phylogenetic tree constructed for a region of the 3D RNA-dependent RNA polymerase gene showing the relationship of JV to other caliciviruses. Shaded ellipses have been added to highlight the distinction between the group 1 and group 2 NLVs. Accession numbers (in parentheses) for caliciviruses are as follows; swine (AB009412); Mexico (U22498); Toronto (U02030); SMA (L23831); Lordsdale (X86557); Hawaii (U07611); Melksham (X81879); Norwalk (M87661); Southampton (L07418); Desert Shield virus (DSV) (U04469); bovine Jena (AJ011099); Sapporo (S77903); human calicivirus (HuCV) DCC (U67856); Manchester (X86559); Parkville (U73124); HuCV 27 (U67859); HuCV Lon (U67858); rabbit hemorrhagic disease virus (RHDV) (M67473); European brown hare syndrome virus (EBHSV) (Z69620); Pan 1 (U52086); cetacean (U52091); reptile (U52092); San Miguel sea lion virus (SMSV 1) (M87481); feline calicivirus (FCV) Urbana (L40021); FCV F9 (M86379).