A Single Amino Acid at the Hemagglutinin Cleavage Site Contributes to the Pathogenicity but Not the Transmission of Egyptian Highly Pathogenic H5N1 Influenza Virus in Chickens Sun-Woo Yoon,a Ghazi Kayali,a Mohamed A. Ali,b Robert G. Webster,a Richard J. Webby,a Mariette F. Ducateza,c Division of Virology, Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, USAa; Environmental Research Division, National Research Centre, Dokki, Giza, Egyptb; INRA UMR1225 IHAP Interactions Hôtes-Agents Pathogènes, ENVT, Toulouse, Francec
Highly pathogenic avian influenza (HPAI) virus H5N1 has been enzootic in Egypt since 2008. Virus-associated mortality (but not the number of cases) in humans and poultry seems to have decreased over time, but the reason for this remains unknown. We investigated the role of a single amino acid substitution in the hemagglutinin cleavage site on virus pathogenicity and transmission in chickens. The R325G substitution significantly reduced pathogenicity without altering the transmission efficiency of HPAI H5N1 virus.
S
ince their emergence in geese in China in 1996 (1, 2), highly pathogenic avian influenza (HPAI) H5N1 viruses have crossed the species barrier and infected humans (610 cases, including 360 fatalities, reported to the World Health Organization as of December 2012) (3). Egypt reported the first occurrence of HPAI H5N1 in 2006 (4, 5) and declared the disease enzootic in July 2008 after 1,084 poultry outbreaks were reported to the World Organization for Animal Health (6). Despite implementing countermeasures to reduce the virus burden in Egypt (7, 8), the pathogen continues to circulate and has had more than 6 years to evolve, most likely from a single initial introduction (9, 10). In Egypt, HPAI H5N1-related mortality in poultry and humans decreased between 2006-2008 and 2009-2012, due to a single consistent genetic change between sequences from Egyptian HPAI H5N1 virus: the hemagglutinin (HA) cleavage site sequence changed from PQGERRRK/RKR*GLF in 2006-2008 to PQGEGRRK/RKR*GLF in 2009-2012 (3, 7). The pathogenicity of H5 viruses in poultry largely depends on the number of basic amino acids in their HA cleavage site: the higher the number of basic amino acids, the more readily proteins are cleaved and the higher the tissue tropism (11, 12). Because the HA cleavage amino acid residues (-RRRKK-) can be cleaved by furin-like protease (13), which is expressed in most organs of hosts, in HPAI H5N1 virus this site plays important roles in host pathogenicity and tissue tropism in hosts (14). However, the role of specific amino acids in the HA cleavage site of avian influenza viruses with regard to protease cleavage remains unknown (15–18). Therefore, to understand the mechanism by which virus-related mortality was reduced in poultry over time in Egypt, we investigated the role of the HAR325G substitution (substitution in the HA cleavage site by H3 numbering) on the pathogenicity and transmission of Egyptian HPAI H5N1 viruses. To determine the functionality of the HA protein after substitution of the multibasic cleavage site (MBCS) at position 325, we successfully generated reverse genetics (rg) viruses containing the 8 gene segments of A/chicken/Qalubia/1/2006 (A/ck/Eg/06) and A/turkey/Egypt/7/2007 (A/tk/Eg/07) (clade 2.2.1) and their counterparts with the same 6 internal gene and neuraminidase (NA) segments, as well as HA with a unique amino acid mutation in the MBCS R325G (H3 numbering): rg-A/ck/Eg/06MBCS and rg-A/tk/ Eg/07MBCS (Table 1) (19). Comparison of growth kinetics for the 4
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TABLE 1 Characterization of Egyptian HPAI H5N1 influenza viruses depends on the amino acid on HA position 325
Virus
Amino acid sequence at multibasic cleavage site (MBCS)a
Endpoint virus titer in eggs (log10 EID50/ml)
CLD50 (log10)b
Rg-A/ck/Eg/06 Rg-A/ck/Eg/06MBCS Rg-A/tk/Eg/07 Rg-A/tk/Eg/07MBCS
-PQGERRRKKRGLF-PQGEGRRKKRGLF-PQGERRRKKRGLF-PQGEGRRKKRGLF-
8.5 9.75 8.5 9.5
1.5 3.25 3.5 5.25
a HA positions 321 to 333 (H3 numbering); amino acid at position 325 is in bold and underlined. b CLD50 as calculated by the Reed and Muench method (n ⫽ 3 per group).
viruses in eggs and MDCK cells (American Type Culture Collection, Manassas, VA) revealed that a single amino acid substitution at position 325 in the HA MBCS caused minimal differences in replication in eggs but decreased growth in MDCK cells (Table 1 and data not shown). We tested the pathogenicity and transmissibility of Egyptian HPAI H5N1 viruses in chickens. To compare the pathogenicity of the 4 rg viruses in vivo in an avian host, we calculated their 50% chicken lethal dose (CLD50) in 6-week-old White Leghorn chickens (Charles River, Wilmington, MA) in enhanced biosafety level 3 (BSL3⫹) facilities. All animal experiments were approved by the Animal Care and Use Committee of St. Jude Children’s Research Hospital, Memphis, TN, and complied with institutional, National Institutes of Health, and Animal Welfare Act policies and regulations. All birds were inoculated by intranasal, intraocular, and intratracheal instillation. Interestingly, the single HAR325G substitution was sufficient to cause an increase of almost 2 log10 in the CLD50 of the 2 types of influenza viruses (Table 1). We next
Received 26 December 2012 Accepted 1 February 2013 Published ahead of print 13 February 2013 Address correspondence to Richard J. Webby,
[email protected], or Mariette F. Ducatez,
[email protected]. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03551-12
p. 4786 – 4788
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Pathogenicity and Transmission of Egyptian H5N1 Virus
TABLE 2 Transmissibility of Egyptian HPAI H5N1 viruses in chickens and subsequent mortality
Virus and groupa
No. of dead birds/ total no. of birds
Number of seroconverted birds/total no. of birds (HI titer range)b
Rg-A/ck/Eg/06 I DC
2/2 5/5
ND ND
Rg-A/ck/Eg/06MBCS I DC
2/2 0/5
ND 5/5 (20–80)
Rg-A/tk/Eg/07 I DC
2/2 5/5
ND ND
Rg-A/tk/Eg/07MBCS I DC
2/2 0/5
ND 5/5 (20–80)
a
I, inoculated; DC, direct contact. Sera were collected 14 days postinoculation (13 days postcontact). Shown are hemagglutination inhibition (HI) titers to homologous virus (the reciprocal titers are indicated); limit of detection, HI titer of 1:10. ND, not done.
b
investigated whether the HAR325G substitution also altered the transmission efficiency of HPAI H5N1 influenza viruses in chickens. We infected 2 donor birds per virus with 3 to 5.6 times their CLD50 to ensure infection and disease of the 2 chickens while extending their time to death and hence their chances of transmitting the pathogen to naive birds (direct-contact [DC] chickens) via contact 24 h postinoculation. As expected, all inoculated donors died within 4 days, irrespective of the virus strain (Table 2). All DC chickens in the presence of rg-A/ck/Eg/06 and rg-A/tk/ Eg/07 succumbed to infection 6 to 8 days postcontact. In contrast, all DC birds in the presence of HAR325G-mutated viruses survived. However, all DC chickens (n ⫽ 10) seroconverted 14 days postinfection (as observed by hemagglutination inhibition assay as described in reference 20), indicating that they had all been infected. Therefore, the HAR325G mutation did not markedly alter the transmission efficiency of Egyptian HPAI H5N1 viruses but reduced pathogenicity in contact birds. We checked the HA sequence of virus from swabs of inoculated and contact birds, and the HAR325G
substitution had not reverted after replication and transmission in chickens (data not shown). Zhang et al. recently performed a comprehensive genetic analysis of HPAI H5N1 HA cleavage site sequences from data available on public databases. As they found residues at amino acid position 325 to be the most polymorphic, they mutated sequences at position 325 in several strains to compare their replication in vitro and their pathogenicity in mice. They observed an increase in pathogenicity of the A/chicken/Sheny/0606/2008 (H5N1) (clade 7) virus in mice (50% mouse lethal dose, 106.2 and ⬎106.7 for 325R and 325G variants, respectively) (21), which was associated with unrestricted organ tropism and neurovirulence. The 2 viruses also had slightly different intravenous pathogenicity indexes in chickens (2.80 for the 325G and 2.91 for the 325R variants). The arginine makes the loop more basic, which may allow proteases to cut the cleavage site at a pH lower than what glycine would allow (21). We next compared virus shedding in rg-A/tk/Eg/07- and rg-A/ tk/Eg/07MBCS-infected chickens. Chickens were infected with equivalent lethal doses or equivalent infectious doses, and virus titers were determined in oropharyngeal and cloacal swabs. Although there were differences in the relative amounts of virus shed from cloacal and oropharyngeal routes in infected chickens, there were no substantial differences between virus shedding patterns in rg-A/tk/Eg/07- and rg-A/tk/Eg/07MBCS-infected birds (Fig. 1). Although we could not detect transmission differences between wild-type and mutant viruses, rg-A/ck/Eg/06MBCS and rgA/tk/Eg/07MBCS were not lethal in contact birds, suggesting that the transmitted pathogenicity of virus with the HAR325G substitution is lower than that for parental counterparts. When infected with equivalent lethal doses of rg-A/tk/Eg/07 or rg-A/tk/Eg/ 07MBCS, chickens shed virus to similar levels, independent of the infecting strain. These data highlight the fact that unless active surveillance is conducted, the lower pathogenicity of HAR325Gcontaining viruses could foster their undetected spread and may have contributed to their enzootic nature in Egypt. Nevertheless, these viruses are still widely spread in multiple domestic avian species in Egypt. Our study suggests that HAR325G viruses may circulate unnoticed and therefore spread more efficiently than their HA325R counterparts. However, it is not reasonable to speculate that HAR325G viruses are responsible for the lower case fatality rate in humans in 2009 than in previous years, especially be-
FIG 1 Replication of Egyptian HPAI H5N1 viruses in chickens. Chickens (n ⫽ 5 per group) were infected with equivalent lethal doses (A) or equivalent infection doses (B) of Rg-A/tk/Eg/07 and the HAG325 (mutation introduced in the multibasic cleavage site [MBCS])-mutated virus. After infection, cloacal (open symbols) and oropharyngeal (closed symbols) swabs were collected on days 3, 5, 7, and 9 postinfection. Virus titers were determined by the 50% tissue culture infective dose (TCID50) assay. The limit of virus detection was ⬍1 log10 TCID50/ml (dotted line). An asterisk indicates significant difference in replication kinetics between oropharyngeal and cloacal swabs (P ⬍ 0.05).
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cause no human H5N1 Egyptian isolate with HA325G has yet been sequenced. ACKNOWLEDGMENTS We thank Jeri Carol Crumpton, Jon Seiler, and Thomas P. Fabrizio for excellent technical assistance and Lisa Kercher, Beth Little, and David Carey for the assistance with animal work in the ABSL3⫹ laboratory. We thank Vani Shanker for editorial assistance and the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children’s Research Hospital for assistance with the sequencing. This study was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract no. HHSN266200700005C and by ALSAC.
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