Highly Pathogenic Reassortant Avian Influenza A(H5N1) Virus Clade 2.3.2.1a in Poultry, Bhutan

Highly pathogenic avian influenza A(H5N1), clade 2.3.2.1a, with an H9-like polymerase basic protein 1 gene, isolated in Bhutan in 2012, replicated faster in vitro than its H5N1 parental genotype and was transmitted more efficiently in a chicken model. These properties likely help limit/eradicate outbreaks, combined with strict control measures.

In Bhutan, the poultry sector consists of free-range backyard chickens, a rising number of commercial chicken farms, and domestic waterfowl in the south (8,9). Live-bird markets do not exist, but live birds are imported from India (8,9).
Bhutan's poultry sector was severely affected by outbreaks of HPAI A(H5N1) clade 2.3.2.1 virus infection during 2012-2013 (10). Veterinary authorities enforced strict control measures, including depopulation of poultry in affected regions and burning of related housing and equipment (11). Illegal movement of poultry was the major source of outbreaks (11). Although the introduction of HPAI A(H5N1) from neighboring H5N1-endemic countries is a constant threat, the subtype is not yet entrenched in poultry in Bhutan.

The Study
We isolated HPAI A(H5N1) viruses from samples from 36 chickens and 9 wild birds in Bhutan, all from affected backyard farms adjacent to the highway connecting India with the capital, Thimphu (Figure 1; online Technical Appendix 1  Figure 2). Antigenic analysis of selected H5N1 isolates from Bhutan (online Technical Appendix 1) showed homogeneity and a reactivity pattern similar to that of H5N1 reference viruses from Bangladesh (Table). Amino acid differences were observed between strains A/chicken/ Bhutan/346/2012 (Ck/Bh/346) (rH5N1) and A/chicken/ Bangladesh/22478/2014 (Ck/BD/22478), representing the parental H5N1 clade 2.3.2.1a genotype (pH5N1) (online Technical Appendix 1 Table 2).
We placed Ck/Bh/346 (rH5N1) and Ck/BD/22478 (pH5N1) in direct competition by co-housing chickens inoculated with each virus with naive contacts (online Technical Appendix 1). All donors shed virus oropharyngeally and cloacally, starting at 1 day postinoculation (dpi). By day 3 after contact, real-time reverse transcription PCR to detect PB1 (online Technical Appendix 1) revealed that 7 of 8 naive contacts simultaneously exposed to both viruses were infected with Ck/Bh/346 (rH5N1) alone, none was infected with Ck/BD/22478 (pH5N1) alone, and 1 was co-infected with both viruses. Thus, despite the lower infectious dose used for 30 LD 50 , Ck/Bh/346 (rH5N1) killed inoculated chickens faster than did Ck/BD/22478 (pH5N1) and was transmitted faster and more efficiently to naive contacts.  We assessed the risk for human infection with rH5N1 by investigating its pathogenicity and transmissibility in ferrets (online Technical Appendix 1). Donors shed 4.5 log 10 EID 50 /mL and 3.4 log 10 EID 50 /mL in nasal wash samples at 2 dpi and 4 dpi, respectively, but cleared the virus by 6 dpi. No direct or aerosol contacts shed virus, suggesting that Ck/Bh/346 (rH5N1) was not transmitted (data not shown). No Ck/Bh/346 (rH5N1)-inoculated ferrets lost >5% of their body weight or showed elevated body temperature (data not shown). Histopathologic analysis showed that 1 donor, who was lethargic at 3-10 dpi, had mild meningoencephalitis at 14 dpi (online Technical Appendix 2, http://wwwnc.cdc.gov/EID/ article/22/12/16-0611-Techapp2.pdf). A nucleocapsid protein-positive cell was detected in the brain, suggesting that Ck/Bh/346 (rH5N1) is neurotropic. The other ferrets showed no clinical signs of disease. Virus replication was detected in the lung at 4 dpi (log 10 2.75 EID 50 /g) (online Technical Appendix 2).

Conclusions
Our study revealed that the viruses that caused the 2012 outbreaks in Bhutan belonged to the rH5N1 genotype (2.3.2.1a/H9-like PB1 [7:1]), whereas neither H9N2 nor the pH5N1 genotype have been detected there. rH5N1 has been isolated sporadically at live-bird markets and from chickens on farms where outbreaks occurred in Bangladesh (5,6), India (12), and Nepal (7) in 2011-2013. The lack of data on the effect of the H9-like PB1 gene on the virulence of rH5N1 makes determining its pathogenicity and transmissibility a critical public-health goal for Bhutan and neighboring countries.
Ck/Bh/346 (rH5N1) killed inoculated chickens faster than did Ck/BD/22478 (pH5N1), despite the lower infectious dose used for Ck/Bh/346. In CEFs, Ck/ Bh/346replicated with greater efficiency during the first 36 hpi than did Ck/BD/22478, which possibly explains why rH5N1 transmits more efficiently to naive chickens when directly competing with pH5N1. How faster replication contributes to the increased mortality rate of naive chickens might be crucial to eradicating the disease in Bhutan. In a mountainous region with widely separated villages, small-scale poultry farming, and no live-bird markets, the severity and rapid onset of the infection could lead to hostresource exhaustion and self-limitation.
To determine whether the reassortant PB1 gene accounts for the observed phenotypic properties of rH5N1, reverse genetics experiments are required. Despite its enhanced transmissibility, rH5N1 did not supplant pH5N1 in India or Bangladesh after undergoing multiple reassortment events. Possible reasons for this include the involvement of other influenza subtypes, which would complicate the competition/transmission model, especially at live-bird markets, as well as the large duck population, which is prone to inapparent HPAI infection (indicating possible underreporting). Our ferret model results suggest that avian-to-human transmission of rH5N1 is possible. However, further adaptation to the host is necessary for rH5N1 to become transmissible among mammals. Similar results have been reported for H5N1 clade 2.3.2.1 (13), H5N1 clade 2.3.4 (14), and H5Nx clade 2.3.4.4 (15). rH5N1 is potentially neurotropic, manifesting clinically as mild meningoencephalitis with no obvious respiratory involvement. This finding has implications on early diagnosis and use of antiviral drugs during the first 48 hours after clinical diagnosis for optimal therapeutic effect. pH5N1 and H9N2 virus strains will likely continue to co-circulate on the Indian subcontinent, enabling further reassortment events. Our results highlight the need for active surveillance and full-genome sequencing of all H5N1 virus isolates. Dr. Marinova-Petkova was a postdoctoral research associate at St. Jude Children's Research Hospital, Memphis, Tennessee, USA, while this research was conducted. She is now affiliated with the Centers for Disease Control and Prevention, Atlanta, Georgia, USA, where her research interests include emerging influenza viruses at the animal-human interface, the evolution of influenza A viruses, and animal models for studying influenza pathogenesis and transmission.