Detection and Phylogenetic Analysis of Caprine Arthritis Encephalitis Virus Using TaqMan-based qPCR in Eastern China

Abstract

Caprine arthritis encephalitis is an infectious disease caused by the caprine arthritis encephalitis virus that infects goats, sheep, and other small ruminants. An outbreak of CAEV could be extremely harmful to the goat farming industry and could cause severe economic losses. We designed specific primers and probes for the gag gene and established a TaqMan real-time quantitative polymerase chain reaction assay. This method's correlation coefficient (R²) was >0.999, and the sensitivity of the assay to the plasmid-carried partial gag gene was approximately 10 copies/µL, 1000 times higher than that of conventional PCR. No specific fluorescence was detected for other sheep viruses. Using this method, we tested 776 asymptomatic sheep blood samples and 4 neurodegenerative sheep brain samples from six farms in eastern China, and the positivity rate was 0.77% (6/780). The gag gene was partially sequenced in the three positive samples and compared with the sequences from other representative strains in GenBank. The results revealed that all three strains belonged to the B1 subtype and were most closely related to the strains from Shanxi and Gansu, previously isolated in China, with their homology ranging from 97.7% to 98.9%. These results suggest that the designed RT-qPCR assay can be used to detect subclinical CAEV in sheep and that the virus is still present in eastern China.

Keywords: TaqMan RT-qPCR; caprine arthritis encephalitis virus; detecting method; goat; infectious diseases; phylogenetic trees.

Figure 1

Comparison of the gag gene of different strains of caprine arthritis encephalitis virus (CAEV) and the positions of the primers and TaqMan probes in the viral genome. The GenBank accession number is shown in parentheses. The dots (∙) indicate identical bases.

Figure 2

(A) The amplification curves showing the sensitivity of the TaqMan-based real-time quantitative polymerase chain reaction (RT-qPCR) in detecting caprine arthritis encephalitis virus (CAEV). (B) The sensitivity of the conventional PCR toward CAEV (10⁹ copies/μL~10⁰ copies/μL). (C) The standard curve of the TaqMan RT-qPCR.

Figure 3

Specific amplification curves for CAEV. A specific fluorescent curve was observed in the TaqMan RT-qPCR assay for CAEV with RNA mixtures. ROX fluorescent signals specific for CAEV were detected only when CAEV isolates were used as templates. No ROX signal was observed in the samples containing other viruses.

Figure 4

Phylogenetic tree of caprine arthritis encephalitis virus (CAEV) based on the partial gag gene (about 571 bp). The three strains isolated in this study—JS-2023-1 (no. PP382771), JS-2023-2 (no. PP382772), and SH-2023 (no. PP382773)—are marked by red circles. The other 28 representative sequences were downloaded from GenBank and edited to obtain the corresponding segments using Editseq version 7.1.0 software (DNASTAR Inc., Madison, WI, USA; Lole et al., 1999) based on the sequencing primers used. Multiple sequence alignments were performed using the Clustal V method, and a phylogenetic analysis of the recombinants was performed using MegAlign version 7.1.0 software (DNASTAR Inc., Madison, WI, USA; Lole et al., 1999).