Transgenic Chickens Expressing the 3D8 Single Chain Variable Fragment Protein Suppress Avian Influenza Transmission.

The 3D8 single chain variable fragment (scFv) is a mini-antibody that causes unusual sequence-independent nuclease activity against all types of nucleic acids. We used recombinant lentiviruses to generate transgenic chickens expressing the 3D8 scFv gene under the control of the chicken ß-actin promoter. From 420 injected embryos, 200 chicks (G0) hatched and were screened for the 3D8 scFv using PCR, and 15 chicks were identified as transgenic birds expressing the transgene in their semen. The G0 founder birds were mated with wild-type hens to produce seven transgenic chicks (G1). 3D8 scFv expression in the chicken embryonic fibroblasts (CEFs) was verified by RT-PCR and Western blot analysis. Immunofluorescence staining for 3D8 scFv in the CEFs revealed that the 3D8 scFv protein was primarily cytosolic. To identify 3D8 scFv anti-viral activity, wild-type and two transgenic CEF lines were infected with H9N2 avian influenza virus (AIV). We selected one line of transgenic chickens that exhibited the lowest number of plaque-forming units to be challenged with H9N2 virus. The challenge experiment revealed that contact exposed transgenic chickens expressing 3D8 scFv exhibited suppressed viral shedding. This results suggest that the transgenic chickens developed in this study could be useful for controlling potential within-flock AIV transmission.

Efficacy of clade 2.3.2 H5 commercial vaccines in protecting chickens from clade 2.3.4.4 H5N8 highly pathogenic avian influenza infection.

Emerging clade 2.3.4.4 of the highly pathogenic avian influenza (HPAI) virus strain H5N8, which had been detected sporadically in domestic poultry in China, started to affect wild birds and poultry in South Korea in 2014. The virus was spread to Germany, Italy, the Netherlands, United Kingdom, and even United States by migratory birds. Here, we tested currently used commercial clade 2.3.2 H5 vaccines to evaluate mortality, clinical signs, virus shedding, and histological damage after experimental infection of chickens with the clade 2.3.4.4 HPAI H5N8 virus. Although the vaccination protected chickens from death, it failed to prevent chickens from shedding the virus and from tissue damage according to histological examination. These results suggest that the use of appropriate vaccines that match the currently epidemic HPAI virus is recommended, and continuous HPAI surveillance and testing of currently used commercial vaccines should be performed.

Genetic analyses of avian influenza viruses in Mongolia, 2007 to 2009, and their relationships with Korean isolates from domestic poultry and wild birds.

The present study was conducted to monitor wild birds based on the concern that they could disseminate avian influenza virus (AIV) between Mongolia and Korea, which shares the same migratory flyway. Of 1,528 fecal samples analyzed, 21 low-pathogenic AIV were isolated from 2007 to 2009. Nineteen AIV-positive fecal samples were identified as Anseriformes by DNA bar coding. The most frequently isolated subtype was H3 (61.9%), and the most prevalent hemagglutinin/neuraminidase combination was H3N8 (52.4%). Phylogenetic analysis was performed to assess their genetic relationships with those of domestic poultry and wild birds in Korea. The H3 and H7 surface genes belonged to the Eurasian lineage and clustered together in a group with Korean wild birds and poultry. Most N8 genes clustered phylogenetically with viruses isolated in Eurasia, whereas 1 of the Mongolian viruses and some Korean viruses belonged to the North American lineage. The polymerase acidic protein of the internal gene was not distinguishable from the H5N1 highly pathogenic AIV of the goose/Guangdong/1/1996 (Gs/Gd)-like virus. Our study suggests that Mongolian AIV isolates have evolved with genetically multiple genotypes and are closely related to those of AIV in poultry as well as in wild birds in Korea.