key: cord-0001305-qtbrelfv authors: Wang, Jinbao; Zhao, Pengwei; Guo, Lihui; Liu, Yueyue; Du, Yijun; Ren, Sufang; Li, Jun; Zhang, Yuyu; Fan, Yufeng; Huang, Baohua; Liu, Sidang; Wu, Jiaqiang title: Porcine Epidemic Diarrhea Virus Variants with High Pathogenicity, China date: 2013-12-03 journal: Emerg Infect Dis DOI: 10.3201/eid1912.121088 sha: 5df6a629545e9d444c3ee76b24095606a25e48e1 doc_id: 1305 cord_uid: qtbrelfv nan of 3 isolates were amplified by reverse transcription PCR with 2 pairs of primers (5) and sequenced to identify the genetic variation of the isolates. Sequences for the S gene were submitted to GenBank (accession nos. for YS: JQ771753; SH: JQ771751; and ZB: JQ771752). The S gene nucleotide sequences and deduced amino acid sequences of the 3 new isolates were aligned with the sequences of published isolates by using MEGA 4.0 (www.megasoftware. net). S protein identity among the 3 new isolates was 99.4%-99.6% and shared 93.6%-93.7% identity with classical CV777 strain. We identified numerous sequence variations in S protein of the 3 isolates (online Technical Appendix Table, wwwnc. cdc.gov/EID/article/19/12/12-1088-Techapp1.pdf). Two separate insertions were discovered: a 1-aa (N) insertion at position 140 and a 4-aa (QGVN) insertion at positions 59-62. A 2-aa (NI) deletion was identified at positions 163-164. A total of 34 separate substitutions were identified, and the number(s) of replaced amino acids ranged from 1 through 5. These sequence variations were similar to those in CH/FJND-3/2011, CH1, CH8, and CHGD-01 isolates recently reported in China (6) . To determine the virulence of the PEDV isolates, we experimentally infected fifteen 4-day-old Duroc crossbred piglets with the YS and ZB isolates. The piglets were randomly allotted to 3 groups, each group consisting of 5 pigs. These piglets were shown by serologic analysis to be negative for antibodies against PEDV, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, and pseudorabies virus. In the piglets inoculated orally with 1.5 mL of YS isolate (10 3.0 50% tissue culture infectious doses/mL), diarrhea was observed at 3-6 days postinfection (dpi). One piglet died at 5 dpi, and 4 piglets died at 6 dpi. The dead piglets showed hemorrhage and shedding in the gastric mucosa, swelling and congestion in the mesenteric lymph nodes, and hemorrhage in the intestinal wall ( Figure, panel A) . Histopathologic changes included epithelial cell shedding; intrinsic layer hemorrhage and excessive infiltration of lymphocytes in the stomach; and congestion, edema, and epithelial cell shedding in the intestinal mucosa (Figure, panel B) . PEDV was recovered from the dead piglets, and the full-length S gene was amplified by reverse transcription PCR and sequenced. In the S genes of YS and ZB isolates, 3 and 2 single nucleotides were replaced, respectively, but no mutated amino acid was introduced between the inoculated and recovered viruses. For the piglets orally infected with 1.5 mL of ZB isolate (10 3.1 50% tissue culture infectious doses/mL), diarrhea was observed at 3-5 dpi, and all 5 piglets died at 5 dpi. The dead piglets showed similar lesions to those of the piglets infected with YS isolate in the stomach, intestines, and mesenteric lymph nodes. Piglets in the control group orally inoculated with Dulbecco minimal essential medium remained healthy during the experiment, and no obvious pathologic changes were observed. Our investigation indicated that the recent diarrhea outbreaks were mainly caused by PEDV variants with novel genetic markers that distinguish them from classical strains. The YS and ZB isolates were highly virulent in piglets. Unlike CV777, the PEDV variants remained almost unchanged in the epitope at positions 499-638; however, a 2-aa deletion, a 1-aa insertion, and 18 separate substitutions were identified in the epitope at positions 83-276 (7, 8) . These variations of amino acid sequences probably changed the immunogenicity of S protein and led to immunization failure of current commercial vaccines made from classical PEDV strains. However, how PEDV has evolved and varies in pig herds are not clear. Further studies, including extensive genomic sequence analyses and serologic cross-neutralization tests, should be conducted. Chinese-like strain of porcine epidemic diarrhea virus, Thailand Molecular epidemiology of porcine epidemic diarrhea virus in China Sequence of the spike protein of the porcine epidemic diarrhea virus Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea Cloning and characterization analysis of spike protein gene of porcine epidemic diarrhea virus Complete genome sequence of a porcine epidemic diarrhea virus variant Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus Cloning and sequence analysis of the spike protein gene of porcine epidemic diarrhea virus CH/JL strain and identification of its antigenic region containing linear epitopes To the Editor: Carbapenem resistance in Enterobacteriaceae can occur through the production of carbapenem-hydrolyzing enzymes such as New Delhi metallo-β-lactamase-1 (NDM-1) (1). In recent years, plasmid-mediated NDM-1 has spread rapidly worldwide and into multiple Enterobacteriaceae species, such as Klebsiella pneumoniae and Escherichia coli (2) .NDM-1 has been reported in 2 strains of Salmonella spp., which were isolated from feces and urine specimens during screening for multidrugresistant bacteria in patients from India (3, 4) . We report the isolation of 1 community-acquired NDM-1-bearing Salmonella strain isolated from a child with acute diarrhea.The Salmonella strain was isolated from the feces of an 11-monthold girl at Lishui Central Hospital, Zhejiang Province, China, on July 25, 2012. Six days before admission, a fever <40°C, accompanied by a cough, developed in the patient. Four days before admission, physical examination showed fine rales in both lungs. The leukocyte count was 8,900 cells/ µL, with 80% neutrophils. No obvious abnormalities were found on a chest radiograph.The patient was given a diagnosis of acute bronchitis, and the condition was treated with parenteral cefoxitin for 3 days and parenteral piperacillin/ tazobactam for 1 day, but fever persisted. Two days before admission, diarrhea (4-5 times/day with loose feces containing mucus and blood) developed. On admission day, fecal analysis showed 3-4 leukocytes and 1-3 erythrocytes per high-power field.