Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide each year. The causative agent of the disease, PRRS virus (PRRSV), is a member of the family Arteriviridae. Here we report our investigation of the unparalleled large-scale outbreaks of an originally unknown, but so-called "high fever" disease in China in 2006 with the essence of PRRS, which spread to more than 10 provinces (autonomous cities or regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. Different from the typical PRRS, numerous adult sows were also infected by the "high fever" disease. This atypical PRRS pandemic was initially identified as a hog cholera-like disease manifesting neurological symptoms (e.g., shivering), high fever (40-42 degrees C), erythematous blanching rash, etc. Autopsies combined with immunological analyses clearly showed that multiple organs were infected by highly pathogenic PRRSVs with severe pathological changes observed. Whole-genome analysis of the isolated viruses revealed that these PRRSV isolates are grouped into Type II and are highly homologous to HB-1, a Chinese strain of PRRSV (96.5% nucleotide identity). More importantly, we observed a unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2). Taken together, this is the first comprehensive report documenting the 2006 epidemic of atypical PRRS outbreak in China and identifying the 30 amino-acid deletion in NSP2, a novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, and will stimulate further study by using the infectious cDNA clone technique.

Figure 1. Outbreak sites of the 2006 PRRS epidemic in China.

The provinces or autonomous cities (regions) affected are indicated in white. The PRRSV isolates from the endemic sites (highlighted with red diamonds) have been sequenced at the whole genome level, while others from outbreak places (indicated in black triangles) have had selected genes sequenced, such as ORF5, and nsp2.

Figure 2. Clinical presentation of pigs with “high fever” disease.

(A) Sick pigs with a fat and healthy appearance. (B) Sick pigs with thin and debilitated features. (C) The shivering piglet. (D) The limping pig with erythematous blanching rash in its ears. (E) & (F) Pimples observed on the back of an infected pig. (G) & (H) Grown pigs killed during this epidemic.

Figure 3. Severe damage to multiple organs in dead pigs.

Lung haemorrhage (indicated at the arrow). (B) lung edema. (C) Spleen infarct and bladder dilatation filled with mahogany urine. (D) Kidney with many blood spots (at the arrows). (E) Heart with disorders. (F) Liver with yellow-white necrosis or haemorrhage. (G) Encephala softened slightly. (H) Brain putamen with blood egression emission. (I) Lymph node with haemorrhagic spots

Figure 4. Qualitative detection of the viral pathogen, PRRSV.

(A) PRRSV particles under the electron microscopy (EM). The PRRSV particles (indicated by arrows) are visualized in the cytoplasm of Marc-145 cells (in ultra-thin sections), and the PRRSVs are also observed in the TEM picture on the left bottom. (B) PCR assay of the infected tissues with specific primers for PRRSV. –: Negative control; 1, 2, 3, 4, 5 and 6: the supernatants from the homogenized tissues of lungs, spleen, kidney, heart, liver and brain, respectively. The target fragment of ∼0.6 kb is highlighted by the arrow.

Figure 5. Immunohistochemical analysis of various tissue specimens.

Figure 6. Phylogenetic relationship of PRRSVs.

(A) Phylogenetic tree of the isolates in this outbreak together with some other PRRSV strains with known genome sequences. (B) Phylogenetic analysis of PRRSV isolates from this study on the basis of amino acid sequences of ORF5. Both are unrooted trees. PRRSV isolates from this outbreak are highlighted in red; HB-1, a previously reported Chinese isolate is indicated in pink; VR2332, standard strain of Type II (Northern American PRRSV) is shown in blue; and prototype of Type I (European PRRSV) is in green.

Figure 7. Alignment of NSP2 at the amino acid level.

PRRSV isolates from this epidemic are highlighted in red, HB-1, a previously reported Chinese isolate is indicated in pink, and VR2332, the standard strain of Northern American PRRSV is shown in blue.