Thursday, September 27, 2012

CDC: Spread of Influenza Virus A (H5N1) Clade to Bulgaria in Common Buzzards

October 2012
Atanaska Marinova-Petkova, Georgi Georgiev, Patrick Seiler, Daniel Darnell, John Franks, Scott Krauss, Richard J. Webby, and Robert G. WebsterComments to Author 
Author affiliations: Regional Diagnostic Laboratory on Avian Influenza and Newcastle Disease in Birds, Varna, Bulgaria (A. Marinova-Petkova); National Diagnostic and Research Veterinary Medical Institute, Sofia, Bulgaria (A. Marinova-Petkova, G. Georgiev); and St. Jude Children’s Research Hospital, Memphis, TN, USA (P. Seiler, D. Darnell, J. Franks, S. Krauss. R.J. Webby, R.G. Webster)


On March 15, 2010, a highly pathogenic avian influenza virus was isolated from the carcass of a common buzzard (Buteo buteo) in Bulgaria. Phylogenetic analyses of the virus showed a close genetic relationship with influenza virus A (H5N1) clade viruses isolated from wild birds in the Tyva Republic and Mongolia during 2009–2010. Designated A/common buzzard/Bulgaria/38WB/2010, this strain was highly pathogenic in chickens but had low pathogenicity in mice and ferrets and no molecular markers of increased pathogenicity in mammals. The establishment of clade highly pathogenic avian influenza viruses of the H5N1 subtype in wild birds in Europe would increase the likelihood of health threats to humans and poultry in the region.


On March 15, 2010, the carcass of a common buzzard (Buteo buteo) containing HPAIV (H5N1) was found at St. Konstantin and Helena Black Sea Resort in Bulgaria and submitted to the Regional Diagnostic Laboratory on Avian Influenza (Varna, Bulgaria). The virus was characterized as clade

The 50% mouse lethal dose of A/common buzzard/Bulgaria/38WB/2010 was 30 EID50 (EID50 of the virus was 109/mL). All 3 donor ferrets were shedding virus by day 5 postinfection; however, only 1 was still shedding virus by day 7 postinfection (Figure). Virus titers were not detected in the nasal washes of contact ferrets, indicating that A/common buzzard/Bulgaria/38WB/2010 is not transmissible by direct contact or respiratory droplets. All ferrets used in this study were healthy during the experiment and showed no clinical signs of disease; they were alert, playful, and eating and drinking normally.

Compared with that of A/goose/Guangdong/1/96, the amino acid sequence of A/common buzzard/Bulgaria/38WB/2010s neuraminidase had a 20-residue deletion in the stalk region (residues 49–68), which was thought to be required for influenza viruses to adapt from wild aquatic birds to domestic chickens (24). This deletion causes a loss of the N terminal NQS glycosylation site (positions 50–52). Residues E119, H275, or N295 (N1 numbering) were not mutated, which suggests sensitivity to oseltamivir and zanamivir (25,26).

The M gene–encoded ORF of matrix (M) 1 protein consists of 252 aa, and that of M2 consists of 97 aa. Residues 26L, 27V, 30A, 31S, and 34G of M2’s transmembrane region indicate that A/common buzzard/Bulgaria/38WB/2010 is an amantadine-sensitive strain (29).

To assess possible reassortment in the A/common buzzard/Bulgaria/38WB/2010 genome, we performed phylogenetic analysis of the remaining genes using the same group of viruses that had been used to make the HA tree. In the N1, PB1, PB2, PA, NS, and NP phylogenetic trees, A/common buzzard/Bulgaria/38WB/2010 clustered with the other subtype H5N1 viruses from clade (data not shown). In the M gene tree, the Bulgarian subtype H5N1 virus clustered with the clade 2.3.4 subtypes from Guangxi, Hunan, Fujian, Shantou, and Hong Kong that were isolated in 2005, 2006, and 2008; all other subtype H5N1 viruses from clade clustered in a separate group (Technical Appendix Figure Adobe PDF file [PDF - 838 KB - 3 pages]). The evolutionary distance between the 2 groups of isolates in the M tree is long, indicating that the M gene of A/common buzzard/Bulgaria/38WB/2010 originates from a non–clade 2.3.2 ancestor.


The day after we received the HPAIV (H5N1)–containing common buzzard carcass, officials in Romania notified the OIE of an outbreak of an HPAIV (H5N1) in Letea Village (Danube Delta); 47 backyard chickens were found dead. These reports are considered to be the introduction of HPAIV (H5N1) clade into Europe. Furthermore, the European Reference Laboratory on Avian Influenza and Newcastle Disease (Weybridge, UK) found that the HA gene sequence of the Bulgarian isolate is 99.9% similar to that of the Romanian isolates from Letea Village (34), confirming that both viruses are derived from a common source, which is most likely wild birds.

Map of Buzzard/Bulgaria/38WB/2010 & OIE Report of Letea Village Backyard Chickens 

Our results show that chickens are highly susceptible to influenza virus A/common buzzard/Bulgaria/38WB/2010 (H5N1) and that the virus is highly pathogenic in them. Mammals appear not to be susceptible. Although buzzards can serve as intermediate hosts of HPAIV (H5N1) between migratory birds and poultry, the lack of gross pathologic findings in the buzzard carcass we examined indicates that the bird died shortly after infection. Thus, in this case, the buzzard could not have served as a reservoir of infection to spread the virus over a long distance. Additionally, the lack of poultry farms within 10 km of the area where the buzzard carcass was found may partially explain why no outbreak occurred. As part of a regular avian influenza surveillance plan, we tested 1,709 cloacal and fecal samples from mule ducks that were collected monthly during January 1, 2010–April 30, 2010, from 64 farms in 5 regions of Bulgaria (Plovdiv, Pazardjik, Stara Zagora, Haskovo, and Dobrich). No notifiable avian influenza viruses were isolated from any sample.

The potential of clade HPAIV (H5N1) to cause an outbreak is heightened because vaccines currently in use do not efficiently protect poultry flocks from a strain of this clade that was recently identified in Vietnam (40). Now that clade has spread to Europe, implementing active surveillance plans in all high-risk areas and monitoring the wild birds in the region will play key roles in early detection of incidences of HPAIV (H5N1) infection and in prevention of outbreaks. The expansion of the geographic distribution of HPAIV (H5N1) in wild birds and poultry and the virus’s repeated interspecies transmission to humans make this virus a substantial pandemic threat.

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