Complete genomic sequences, a key residue in the spike protein and deletions in nonstructural protein 3b of US strains of the virulent and attenuated coronaviruses, transmissible gastroenteritis virus and porcine respiratory coronavirus

Abstract

Transmissible gastroenteritis virus (TGEV) isolates that have been adapted to passage in cell culture maintain their infectivity in vitro but may lose their pathogenicity in vivo. To better understand the genomic mechanisms for viral attenuation, we sequenced the complete genomes of two virulent TGEV strains and their attenuated counterparts: virulent TGEV Miller M6 and attenuated TGEV Miller M60 and virulent TGEV Purdue and attenuated TGEV Purdue P115, together with the ISU-1 strain of porcine respiratory coronavirus (PRCV-ISU-1), a naturally occurring TGEV deletion mutant with an altered respiratory tropism and reduced virulence. Pairwise comparison at both the nucleotide (nt) and amino acid (aa) levels between virulent and attenuated TGEV strains identified a common change in nt 1753 of the spike gene, resulting in a serine to alanine mutation at aa position 585 of the spike proteins of the attenuated TGEV strains. Alanine was also present in this protein in PRCV-ISU-1. Particularly noteworthy, the serine to alanine mutation resides in the region of the major antigenic site A/B (aa 506-706) that elicits neutralizing antibodies and within the domain mediating the cell surface receptor aminopeptidase N binding (aa 522-744). Comparison of the predicted polypeptide products of ORF3b showed significant deletions in the naturally attenuated PRCV-ISU-1 and TGEV Miller M60; these deletions occurred at a common break point, suggesting a related mechanism of recombination that may affect viral virulence or tropism. Sequence comparisons at both genomic and protein levels indicated that PRCV-ISU-1 had a closer relationship with TGEV Miller strains than Purdue strains. Phylogenetic analyses showed that virulence is an evolutionarily labile trait in TGEV and that TGEV strains as a group share a common ancestor with PRCV.

Fig. 1

Schematic representation of the spike gene of the four TGEV strains and PRCV-ISU-1 showing deletions and mutations. △, 3 nt (nt 2385 to 2387) deletion in M60 spike gene and 6 nt (nt 1122 to 1127) deletion in P115 spike gene. , 681 nt (nt 65 to 745) deletion in PRCV spike gene. The T to G (bold) mutation at nt 1753 in M60, P115 and PRCV leading to an S to A substitution at aa 585 in the spike protein is shown. The approximate antigenic region of site A/B (nt 1518–2118) and the aminopeptidase N (APN) binding site (nt 1566–2232) are indicated. The potential enteric tropism determining residue at nt position 655 identified by Ballesteros et. al. (1997) is shown.

Fig. 2

(a) Schematic representation of ORF3a/3b gene (nt 1 to 1043) and the 100 nt preceding the gene (nt − 100 to 0): Gene start codon ATG for nonstructural protein 3a starts from nt 1 and ends at nt 247 for M6 and M60 and nt 215 for virulent Purdue and Purdue P115. No nonstructural protein 3a is encoded for PRCV-ISU-1 due to the 184 nt deletion that includes the ATG start codon. Nonstructural protein 3b starts from nt 310 and ends at nt 1043. △, 3 nt (nt − 100 to − 98) deletion at 5′ end and 5 nt (nt 163 to 167) deletion downstream in ORF3a of PRCV-ISU-1. In TGEV M6 and M60, △ represents 16 nt (nt − 76 to − 61) deletions. □, 29 nt (nt 195 to 223) deletion in PRCV, M6 and M60. , 531 nt (nt 405 to 935) deletion in M60 3b and 184 nt (nt − 87 to 97) and 117 nt (nt 407 to 523) deletions in PRCV 3b respectively. (b) ClustalW alignment of ORF3b predicted polypeptides of TGEV strains and PRCV-ISU-1 showing the deletion start point in Miller M60 and PRCV. (c) ClustalW alignment of the nucleotide sequence of TGEV strains and PRCV-ISU-1 in the region of ORF3b deletion start point.

Fig. 3

Phylogenetic analysis of genomic nucleotide sequences of the four TGEV strains and PRCV-ISU-1 with the reference TGEV genomes available in GenBank, and a Feline Coronavirus outgroup. The tree is based on muscle alignment of whole genomes. The tree search was conduced with TNT (Goloboff et al., 2005) with equally weighted parsimony counting gaps as a fifth state. Tree search was conducted using new technology parameters until a stable consensus was discovered. This search procedure produced a single tree of 7360 steps. Bootstrap values for 1000 resampling replicates are shown at nodes.