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. 2011 Mar 24;6(3):e18106.
doi: 10.1371/journal.pone.0018106.

Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related flavivirus

Jingliang Su  1 , Shuang Li, Xudong Hu, Xiuling Yu, Yongyue Wang, Peipei Liu, Xishan Lu, Guozhong Zhang, Xueying Hu, Di Liu, Xiaoxia Li, Wenliang Su, Hao Lu, Ngai Shing Mok, Peiyi Wang, Ming Wang, Kegong Tian, George F Gao
Affiliations

Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related flavivirus

Jingliang Su et al. PLoS One. .

Abstract

Since April 2010, a severe outbreak of duck viral infection, with egg drop, feed uptake decline and ovary-oviduct disease, has spread around the major duck-producing regions in China. A new virus, named BYD virus, was isolated in different areas, and a similar disease was reproduced in healthy egg-producing ducks, infecting with the isolated virus. The virus was re-isolated from the affected ducks and replicated well in primary duck embryo fibroblasts and Vero cells, causing the cytopathic effect. The virus was identified as an enveloped positive-stranded RNA virus with a size of approximately 55 nm in diameter. Genomic sequencing of the isolated virus revealed that it is closely related to Tembusu virus (a mosquito-borne Ntaya group flavivirus), with 87-91% nucleotide identity of the partial E (envelope) proteins to that of Tembusu virus and 72% of the entire genome coding sequence with Bagaza virus, the most closely related flavivirus with an entirely sequenced genome. Collectively our systematic studies fulfill Koch's postulates, and therefore, the causative agent of the duck egg drop syndrome occurring in China is a new flavivirus. Flavivirus is an emerging and re-emerging zoonotic pathogen and BYD virus that causes severe egg-drop, could be disastrous for the duck industry. More importantly its public health concerns should also be evaluated, and its epidemiology should be closely watched due to the zoonotic nature of flaviviruses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Regions of BYDV infection outbreaks in China.
The provinces or autonomous cities (regions) affected are indicated in gray. Regions from which viruses were isolated and comfirmed by RT-PCR/sequencing are labeled with triangles.
Figure 2
Figure 2. The course of natural disease.
Representative of the average feed uptake of the infected flock at Farm 1 (A); Daily egg production rate represented by two infected duck farms (B). The flocks at Farm 1 were 35 weeks old, and Farm 2 flocks 1–4 (Farm 2-F1, -F2, F3, -F4) are four different flocks (76 weeks old) at another representative farm.
Figure 3
Figure 3. Gross lesions and histopathology of the clinical samples.
Mild hemorrhage of ovarian follicles (arrow) in the early stage of infection (A); Severe hamorrhage and regression of ovarian follicles (B); HE stained ovary section, the follicles ruptured and filled with round or granular eosinophilic bodies (C). HE-stained brain section showing focal gliosis (arrow) (D).
Figure 4
Figure 4. CPE in BYDV infected DEFs and electron micrographs of virus particles.
HE-stained, non-infected DEF control (A); HE stained infected cells, 52 hrs post-infection, showing cell disruption with a large number of red-stained particles (B); Non-infected Vero cell monolayer (C); Infected Vero cells rounded up and focal detachment 60 hrs post-infection (D); Thin section of the infected cell, 24 hrs post-infection, showing dense particles (white arrow head) and virions (arrow) within the vesicles in the cytoplasm (N, nucleus). Insets are magnified virions (E); Negative staining of the purified BYDV from the cell culture supernatants (arrow indicates the typical enveloped flavivirus) (F).
Figure 5
Figure 5. Daily egg production rate before and after experimental infection.
The decline of egg production rate was evident, similar to the natural infection.
Figure 6
Figure 6. Pathological changes of the experimentally-infected ducks.
Mock-infected control (A); Severe hemorrhage and regression of ovarian follicles (B); Spleens from mock-infected control (left) and enlarged spleens from the infected ducks (C); HE-stained ovary section showing hemorrhage and follicle rupture with red stained bodies (D); HE-stained brain section revealing lymphocytes and mononuclear cell infiltration under cranial arachnoid (E) and focal gliosis (arrow) (F).
Figure 7
Figure 7. Phylogenetic relationships of the isolated BYDV with other flaviviruses.
Nucleotide sequence comparisons of the E protein (A), NS5 protein (B) and the genome coding polyprotein (C). BYDV is labeled in bold.
Figure 8
Figure 8. Detection of the viral RNA from clinically infected duck samples.
Infection in duck brain tissues collected from different regions was assessed using RT-PCR with BYDV E gene-specific primers. -: negative control; 1,2,3,4 and 5: samples from Hebei, Jiangxi, Jiangsu, Beijing and Shandong provinces. These PCR products were sequenced and their identities were confirmed.
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