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Identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence
© Zhang et al. 2015
Received: 13 March 2015
Accepted: 27 May 2015
Published: 12 June 2015
Since 2010, a variant Porcine epidemic diarrhea virus (PEDV), which causes an acute, highly contagious, and devastating viral enteric disease with a high mortality rate in suckling pigs, broke out in China and spread rapidly to neighboring countries, even to the North America. This virus gradually became the main subtype of PEDV worldwide. However, there were no reports of mild pathogenicity of a variant porcine epidemic diarrhea virus in China.
In 2013, a PEDV-positive sample from a sow with very mild clinical sign was used to inoculate in Vero cells to isolate the virus. This PEDV field strain, designated FL2013 strain, was successfully propagated and genetically characterized. The phylogenetic trees based upon either the complete genome or S gene showed that the FL2013 strain belongs to the genogroup G2b. The S gene of FL2013 has a 7-aa deletion (FEKVHVQ) in the C-terminus comparison with the other G2 PEDV sequences. Further comparative pathology study indicated that the FL2013 strain had reduced virulence to newborn piglets.
A novel variant PEDV strain FL2013 with reduced virulence, as determined by the pathological study, was identified from east China. This strain is closely related to the genogroup- 2 PEDV strains prevalent in the U.S. and China currently, but had a short deletion at the 3′- end of the spike gene.
Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded, positive-sense RNA virus in the genus Alphacoronavirinae of the family Coronaviridae. PED, which is caused by PEDV, is characterized by severe diarrhea, dehydration, and high mortality rates in the affected swine . PEDV variants belonging to genogroup 2 (G2) have been emerging in China in a large-scale outbreak characterized by approximately 80–100 % morbidity rates and high mortality rates among suckling piglets since late 2010 . Highly virulent PEDV strains, phylogenetically related to the G2 Chinese PEDV variants (shared ≥99.5 % nt identity), suddenly emerged in the United States in May 2013 and rapidly spread throughout the country, causing severe economic losses [3, 4]. The second PEDV variant in the US, designated S INDEL PEDV, with insertions and deletions in the N terminal region (S1 subunit) of the spike (S) protein same as the G1 PEDV, was identified subsequently . The S INDEL strains caused reportedly milder disease in the field, indicating that the S gene contains major virulent determinants . The other large S-deletion PEDV strains were also identified, including one Korean field strain with a 204-aa deletion , and a US cell-adapted strain with a 197-aa deletion . Whether these S-deletion PEDV strains have distinct pathogenic characteristics, have not been described.
In July of 2013, fecal samples from diarrheal sows, collected in a pig farm in Jiangsu province of east China, were submitted to our lab for routine laboratory diagnostics. PEDV positivity was confirmed by reverse transcription PCR (RT-PCR) according to the method reported previously . One of the PEDV-positive samples, from a sow with very mild clinical sign, was used to inoculate into Vero cells to isolate the virus, as described previously . This PEDV strain, designated FL2013 strain, was successfully passaged and propagated, as characterized by typical PEDV-induced cytopathic effects (CPE), such as cell fusion and syncytia formation (data not shown), which was similar to what we observed for the CHGD-01 strains . The other field PEDV samples collected from pigs with severe clinical sign could not adapt to Vero cells. The complete genomic cDNA of the FL2013 strain was further determined using the 3rd cell culture passaging virus by amplification of twelve regions covering the PEDV genome as described previously . The sequences were assembled and analyzed using DNASTAR program and MEGA6.0 program .
Summary of representative PEDV sequences used in this study
In summary, we identified an apparently attenuated G2 PEDV field strain with 7-aa deletion at the 3′-end of the S gene. Genetic characterizations of various PEDV field strains are of importance for demonstration of PEDV outbreaks and development of efficacy vaccines. Findings from present study reveals that variant PEDV strains circulating in swine-producing areas in China, and provides new molecular epidemiological data for a better understanding of this disease. It is highly possible that the sequence deletions (especially the 7-aa deletion), and other unknown mutations found in the variant strain FL2013 might have contributed to the reduced severity of the clinical disease in the piglets. Whether the changes in the genomic sequence, insertions and deletion, could collectively alter the efficiency of viral replication and RNA synthesis in the FL2013 strain leading to reduced pathogenicity will be investigated in further. More studies will be conducted to test this hypothesis. In addition, the FL2013 isolate may serve as a potential vaccine candidate that could protect the Chinese piglets from the infection caused by the virulent strain of PEDV.
This work was supported by grants from the Wen’s research fund of Guangdong Wen’s Foodstuffs Group Co., Ltd (WSSC02-1-C-A-20120525-1327).
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