Molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms

Abstract

Massive numbers of palm civets were culled to remove sources for the reemergence of severe acute respiratory syndrome (SARS) in Guangdong Province, China, in January 2004, following SARS coronavirus detection in market animals. The virus was identified in all 91 palm civets and 15 raccoon dogs of animal market origin sampled prior to culling, but not in 1,107 palm civets later sampled at 25 farms, spread over 12 provinces, which were claimed to be the source of traded animals. Twenty-seven novel signature variation residues (SNVs) were identified on the spike gene and were analyzed for their phylogenetic relationships, based on 17 sequences obtained from animals in our study and from other published studies. Analysis indicated that the virus in palm civets at the live-animal market had evolved to infect humans. The evolutionary starting point was a prototype group consisting of three viral sequences of animal origin. Initially, seven SNV sites caused six amino acid changes, at positions 147, 228, 240, 479, 821, and 1080 of the spike protein, to generate low-pathogenicity viruses. One of these was linked to the first SARS patient in the 2003-2004 period. A further 14 SNVs caused 11 amino acid residue changes, at positions 360, 462, 472, 480, 487, 609, 613, 665, 743, 765, and 1163. The resulting high-pathogenicity groups were responsible for infections during the so-called early-phase epidemic of 2003. Finally, the remaining six SNVs caused four amino acid changes, at positions 227, 244, 344, and 778, which resulted in the group of viruses responsible for the global epidemic.

FIG. 1.

Geographic locations and numbers of palm civets sampled in China in 2004.

FIG. 2.

Quantitative measurement of SARS-CoV-like virus in rectal swabs taken from palm civets after their arrival at an animal market, determined by fluorescent real-time RT-PCR. Bars are labeled with the mean viral load for each time point. Error bars represent standard deviations for triplicate determinations.

FIG. 3.

Signature nucleotide and amino acid variation analysis of spike protein.

FIG. 4.

Phylogenetic analysis of SARS-CoV-like virus based on S gene sequences using the maximum likelihood methods of PAUP. A bootstrap analysis with 1,000 replicates was used to measure the support of the resulting topologies. The prototype, low-pathogenicity, high-pathogenicity, and epidemic groups of viruses are shaded with various colors. The years and host species of the determined virus sequences are provided. Detailed information regarding those sequences is listed in Table 1.

FIG. 5.

The evolution process and signature nucleotide variation of SARS-CoV-like virus, based on spike gene sequences. The phylogenetic tree was constructed using the maximum likelihood methods of PAUP, with the raccoon dog sequence A031G used as the root. The viruses of the prototype group, the low-pathogenicity group, the high-pathogenicity group, and the epidemic group and their SNV patterns are unshaded or shaded in yellow, red, or pink, respectively. Information on various sequences is detailed in Table 1.