T embusu Virus in Ducks, China Zhenzhen Cao,1 Cun Zhang,1 Yuehuan Liu,1 Weicheng Ye, Jingwen Han, Guoming Ma, Dongdong Zhang, Feng Xu, Xuhui Gao, Yi Tang, Shaohua Shi, Chunhe Wan, Chen Zhang, Bin He,
Mengjie Yang, Xinhao Lu, Yu Huang, Youxiang Diao, Xuejun Ma, and Dabing Zhang
In China in 2010, a dia outbreak in egg-laying ducks was associated with a fl avivirus. The virus was isolated and partially quenced. The isolate exhibited 87%–91% identity with strains of Tembusu virus, a mosquito-borne fl avivirus of the Ntaya virus group. The fi ndings demonstrate emergence of Tembusu virus in ducks.
鲜肉馄饨F
rom June through November 2010 in the People’s Republic of China, a dia characterized by a sudden ont was obrved on many egg-laying and breeder duck farms. Egg production in affected ducks dropped verely within 1–2 weeks after dia ont. Other consistent signs included acute anorexia, antisocial behavior, rhinorrhea, diarrhea, ataxia, and paralysis. Rate of illness was usually high (up to 90%), and mortality rates varied from 5% to 30%. From affected ducks we isolated and identi fi ed a Tembusu virus (TMUV).
The Study
During the outbreak, we examined 11 diad ducks (7 Pekin ducks, 3 Cherry Valley Pekin ducks, and 1 Shaoxing duck) from 5 duck farms in 4 provinces. At necropsy, viscera samples (e.g., brain, heart, liver, spleen, lung, theca folliculi) were collected and placed in 10% buffered formalin. Sections were embedded in paraf fi n and stained
with hematoxylin and eosin. The theca folliculi from each duck were also ud for virus isolation or detection by PCR.
The main pathologic changes obrved consistently
in almost all diad ducks were found in the ovaries:
猴子山下hyperemia, hemorrhage, degeneration, distortion, macrophage and lymphocyte in fi ltration, and h yperplasia; in the liver, interstitial in fl ammation was found in the portal area (Figure l, panels A–C). On the basis of the changes, the dia was designated duck hemorrhagic ovaritis.
After samples underwent veral rounds of screening and identi fi cation attempts, we concluded that a new virus infection was the most likely cau of the dia. One virus strain, designated YY5,
was isolated from the Shaoxing duck after inoculation of clari fi ed theca folliculus suspension into the allantoic cavities of 9-day-old speci fi c pathogen–free chicken embryos. The embryos died 72–120 hours after inoculation, and vere cutaneous hemorrhages were obrved.
Becau eastern equine encephalitis (EEE) virus has been shown to cau paralysis in Pekin ducks (1), we performed EEE virus–speci fi c nested rever transcription PCR (RT-PCR) (2) to detect the isolate. RNA was extracted by using the TranZol RNA Extraction Kit (TransGen Biotech, Beijing, China). The primer pairs ud to amplify the E2 gene of EEE virus (EEE-4 and cEEE-7, EEE-5 and cEEE-6) have been described (2). A clear PCR product (268 bp) resulted and was then quenced; the deduced amino acid quence was compared with other quences by using a BLASTP (bi.i) arch in GenBank. Unexpectedly, a 221-nt quence (GenBank accession no. HQ641388) was shown to encode the nonstructural (NS) 1 protein of fl avivirus, which exhibited 73%–85% identity to fl avivirus in the Ntaya virus and Japane encephalitis virus groups, such as Bagaza virus (GenBank accession no. ACG60714, [3]) and St. Louis encephalitis virus (GenBank accession no. ABN11829 [4]). The result demonstrated the possible prence of a fl avivirus in ducks.
To further con fi rm fl avivirus as the causative agent of duck hemorrhagic ovaritis, we ud PCR to t
est the isolate and clinical samples with forward primer Usu5454f (5′-ATGGATGAAGCYCATTTCAC-3′) (5) and a newly designed rever primer 5861R (5′-CCAAAGTTGGCYCCCATCTC-3′). The primers were located in the conrved regions of the NS3 quences of Bagaza virus, St. Louis encephalitis virus, and Usutu virus (3–5) and were predicted to produce an ≈400-bp amplicon. The reaction conditions were as follows: 5 min at 94°C; followed by 38 cycles of denaturation at 94°C for 40 s, annealing at 47°C for 35 s, and extension at 72°C for 1 min; and a fi nal extension of 72°C for 10 min. RT-PCR
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1
The authors contributed equally to this article.
Author af fi liations: Key Laboratory of Zoonosis of Ministry of Agriculture, Beijing, People’s Republic of China (Z. Cao, G. Ma, Dongdong Zhang, Dabing Zhang); China Agricultural University, Beijing (Z. Cao, G. Ma, Dongdong Zhang, Dabing Zhang); Zhejiang Academy of Agricultural Sciences, Hangzh
ou, People’s Republic of China (Cun Zhang, W. Ye); Beijing Academy of Agriculture and Forestry Sciences, Beijing (Y . Liu, J. Han); Beijing University of Agriculture, Beijing (F. Xu); Shandong Agricultural University, Taian, People’s Republic of China (X. Gao, Y . Tang, Y . Diao); Fujian Academy of Agricultural Sciences, Fuzhou, People’s Republic of China (S. Shi, C. Wan, Y . Huang); Chine Center for Dia Control and Prevention, Beijing (Chen Zhang, B. He, M. Yang, X. Ma); and Yuyao Municipal Institute of Poultry Dia, Yuyao, People’s Republic of China (X. Lu)
DOI: /10.3201/eid1710.101890
was optimized by using the following controls, including nucleic acids extracted from theca folliculi of healthy Pekin ducks: avian in fl uenza virus, Newcastle dia virus, egg drop syndrome virus, anatid herpesvirus 1, Muscovy duck parvovirus, goo parvovirus, duck reovirus, goo reovirus, duck hepatitis A virus, duck astrovirus, duck circovirus, and goo hemorrhagic polyomavirus.
陈寺福All 11 theca folliculus samples and the isolate were positive for fl avivirus by RT-PCR, which was con fi rmed by amplicon quencing. The 367-nt quence (GenBank accession no. HQ641389) of part of the NS3 genomic region obtained from the isolate was 66%–77% identical to the corresponding quence of virus in the Ntaya virus and Japane encephalitis virus groups. The amplicon que
nces from the 11 theca folliculus samples shared 98%–100% identity with the YY5 isolate. PCR testing of another 52 samples from diad ducks from the 4 provinces detected fl avivirus-speci fi c RNA in 29 samples. Overall, 40 (63.5%) of 63 samples were positive for
fl avivirus. Flavivirus-speci fi c RNA was most frequently
detected in theca folliculi, followed by intestinal mucosa, uterus, spleen, trachea, cloaca (swab), and liver (Table).
Subquently, the YY5 isolate was injected intramuscularly into nine 55-week-old Pekin ducks and ten 30-week-old Shaoxing ducks. At day 4 postinoculation, the pathologic changes were reproduced in the experimentally infected ducks. The fl avivirus RNA was detected by NS3-bad RT-PCR, and the virus was again isolated from theca folliculi.
To investigate the genetic relationship of the isolate with fl avivirus, we obtained the genomic quence of a 1,035-bp gment at the 3′ terminus of the NS5 gene (GenBank accession no. HQ641390) from strain YY5 by RT-PCR and primers FU1 and cFD3 as described (6). Phylogenetic analysis showed that YY5 was more cloly related to TMUV than to other fl avivirus (Figure 2). Comparative quence analysis showed that YY5 was 87%–91% identical to different strains of TM
UV; therefore, we classi fi ed the fl avivirus isolated from ducks as a new genotype of TMUV, a mosquito-borne fl avivirus of the Ntaya virus group.
结膜炎的症状及治疗
Conclusions
We have demonstrated the prence of a mosquito-borne fl avivirus in ducks. On the basis of criteria for species of the members of the genus Flavivirus (6) and phylogenetic analysis, we consider the isolate to belong to a new genotype of TMUV.淘宝推广
DISPATCHES
感恩妈妈作文1874
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Figure 1. Pathologic changes in diad Pekin ducks. A) Ovary with hyperemia, hemorrhage, and distortion. B) Ovary with hemorrhage (gold arrow), macrophage and lymphocyte in fi ltration and hyperplasia (green arrow). C) Liver with interstitial in fl ammation in the portal area (gold arrow). D and E) Ovaries from healthy ducks. F) Liver from healthy duck. A, C) Original magni fi cation ×40; B, C, E, F) scale bars = 90 μm.
Table. Flavivirus detection in 63 samples from diad ducks, 4 provinces, People’s Republic of China, 2010
Sample
No. (%) flavivirus positive No. flavivirus
negative Theca folliculi, n = 15 14 (93.3) 1Intestinal mucosa, n = 4 3 (75.0) 1Uterus, n = 7 5 (71.4) 2Spleen, n = 9 6 (66.7) 3Trachea, n = 2 1 (50.0) 1Cloacal swab, n = 17 8 (47.1) 9Liver, n = 9 3 (33.3) 6
Tembusu Virus in Ducks, China
In this study, we found TMUV-speci fi c RNA in 63.5% samples from diad ducks in different provinces. In particular, it was found in 14 (93.3%) of 15 theca folliculus samples, suggesting that reproductive tissues may be a major site for viral persistence, replication, or both. Experimental infections further con fi rmed that TMUV can be reisolated from theca folliculi. The results suggested that TMUV may be the causative agent of duck hemorrhagic ovaritis.
Becau TMUV belongs to the mosquito-borne virus cluster of fl avivirus, mosquitoes might be involved in the
spread of this virus. Detection of the virus in cloacal swab
samples suggests probable horizontal transmission through ingestion or inhalation of feces-contaminated material.
TMUV was originally isolated from mosquitoes of the genus Culex , but the dia associated with TMUV infection was not known. However, a chick-origin TMUV isolate, originally named Sitiawan virus, can cau encephalitis and retarded growth in broiler chicks (8). In conclusion, this study shows that duck-origin TMUV is highly pathogenic for Pekin ducks, Cherry Valley Pekin ducks, and Shaoxing ducks.
温柔的老师
This work was supported by China Agriculture Rearch System (CARS-43) and the China Mega-Project for Infectious Dia (2008ZX10004-001).
Ms Cao is a graduate student at College of Veterinary Medicine, China Agricultural University in Beijing, in the Modern Agro-industry Technology Rearch System. Her rearch focus on the identi fi cation and characterization of novel virus found in waterfowl.References
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8. Kono Y , Tsukamoto K, Hamid MA, Darus A, Lian TC, Sam LS, et
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he genus Flavivirus. Am J Trop Med Hyg. 2000;63:94–101.Address for correspondence: Dabing Zhang, Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Yuanmingyuan West Rd, no. 2, Beijing 100193, People’s Republic of China; email: zdb@
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Figure 2. Phylogenetic analysis of isolate YY5 (in boldface ) from an ill Shaoxing duck in the People’s Republic of China and lected other fl avivirus obtained by using an ≈1-kb nt quence in the nonstructural 5 genomic region. The tree was constructed by the neighbor-joining method of MEGA (7). Numbers at nodes indicate bootstrap percentages obtained after 1,000 replicates; only bootstrap values >70% are shown. Scale bar indicates genetic distance. The quences ud in the phylogenetic analysis are listed online (v/EID/content/17/10/10-1890-F.htm). The nucleotide quence of isolate YY5 ud in the phylogenetic analysis has been deposited in GenBank under accession no. HQ641390.
