H9N2 influenza viruses from Bangladesh: Transmission in chicken and New World quail

Abstract The H9N2 influenza viruses that have become established in Bangladeshi live poultry markets possess five gene segments of the highly pathogenic H7N3 avian influenza virus. We assessed the replication, transmission, and disease potential of three H9N2 viruses in chickens and New World quail. Each virus replicated to high titers and transmitted by the airborne route to contacts in both species. Infected chickens showed no disease signs, and the viruses differed in their disease potential in New World quail. New World quail were more susceptible than chickens to H9N2 viruses and shed virus after airborne transmission for 10 days. Consequently, New World quail are a potential threat in the maintenance and spread of influenza virus in live poultry markets.

(GenBank KC757840), and A/quail/Bangladesh/19462/2013 (H9N2) (GenBank KJ643700). The viruses were propagated and titrated in the allantoic cavities of 10-day-old embryonated chicken eggs at 35°C for 48 hours as described previously. 5 The birds used in the study were northern bobwhite quail (Colinus virginianus) aged 5-6 weeks for Old World quail were not available to us. Previous studies have shown that bobwhite quail support influenza virus replication with receptors in their respiratory tract. 6,7 White leghorn specific pathogen-free (SPF) chickens (Gallus domesticus) aged 6 weeks were also used. The number of birds and the sampling times differed between species due to the space limitations in our BSL3 facilities and not handling two different species on the same day.
For each virus, five donor quail and five donor chickens were infected by the natural route with 10 6 EID 50 in 0.5 mL total volume phosphate-buffered saline (0.1 mL intraocularly, 0.1 mL intranasally, 0.2 mL intraorally, and 0.1 mL intratracheally) as others have done. 8 Twenty-four hours after infection, donor birds were mixed with five direct-contact birds of the same species in the same cage, five airborne-contact quail were placed in an adjacent cage approxi- Each of the H9N2 influenza viruses from chickens, quail, and the environment replicated to a high titer (EID 50 5.4-6.4 log 10 /mL) in the upper respiratory tract of both chickens and quail (Table 1, Figure 1).
Virus was also shed in the feces but with much lower titers (0.5-2.7 log 10 /mL). In donor chickens, peak shedding of virus occurred at 3 dpi and the titers fell to background levels by Day 7. Similar virus titers were measured for each of the three viruses at 3, 5, and 7 dpi (data not shown). Shedding in quail donors peaked at 4 dpi, and shedding was sustained beyond 8 dpi in three of five donor birds for each virus.
At least one donor quail in each group shed virus orally for 10 days.
We determined the direct and airborne transmissibility of the

| CON CLUS IONS
This study shows that the New World quail (Bobwhite species) is In this study, quail were more severely affected by the H9N2 viruses containing five gene segments from HPAI H7N3 than were chickens. H9N2 viruses from Pakistan containing four gene segments (PB2, PB1, PA, and NS) from HPAI H7N3-affected quail more severely than chickens and caused sporadic deaths (one donor, one direct contact, and two airborne contacts). 10  in the retail markets, quail and other minor poultry tend to remain in the markets longer than chickens, which are sold out daily. 12 We do not know whether quail containing a gene constellation that differs from those found in birds in Hong Kong are the source of the H9N2 viruses in chickens in Bangladeshi live poultry markets. We do know that chickens in the Bangladeshi markets are more often infected than chickens sampled on the farms in that region, which suggests that poultry in the markets are the primary source of H9N2 viruses and that quail are a major contributor thereof. 12

ACK N OWLED G EM ENTS
We thank Dr. Sajeda Begum and Ashis Kumar Datta of the Department of Zoology, Jahangirnagar University, for their support during sample collection in Bangladesh. We also thank Dr. Keith A.

Laycock of the St. Jude Department of Scientific Editing and James
Knowles for their assistance with manuscript preparation. This work was funded by the National Institute of Allergy and Infectious Diseases (HHSN272201400006C) and ALSAC.