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HPAI H5nx Situation in Bulgaria

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HPAI H5Nx situation in Bulgaria Assoc. Prof. Gabriela Goujgoulova, DVM, PhD, NDRVMI, Sofia, Bulgaria, Bianca Zecchin, Alice Fusaro, Isabella Monne, Calogero Terregino EU/OIE/National Reference Laboratory for Avian Influenza and Newcastle Disease IZSVe, Legnaro (Italy) POULTRY SECTOR IN BULGARIA Distribution of farms for ducks, broilers, laying hens and breeding flocks HIGH RISK AREAS FOR HPAI IN BULGARIA Main migration routes across the country • Via Pontica • Via Aritotelis HIGH RISK AREAS FOR HPAI IN BULGARIA Outbreaks of HPAI for 2019 OUTBREAKS 2020 First outbreak is on 17.02.2020 Six of the outbreaks are in a 10 km zone of the first outbreak. In a 3 km zone there are 25 poultry farms (136,669 mule ducks), and in a 10-km zone - 24 poultry farms (167,349 mule ducks and 127,550 hens). Transport is cited as a major risk factor, as both hens and waterfowl are transported by the same vehicles. OUTBREAKS 2020 The sixth outbreak is in Kardzhali The car owned by a Plovdiv company, which supplies the farm in Kardzhali with cardboard egg boxes, circulates between other farms in the region of town Rakovski. OUTBREAKS 2020 OUTBREAKS 2020 ADNS code Date Region Town/village Birds Technology Number of Subtype birds ADNS 1 17.02.2020 Plovdiv Rakovski Mule Closed cycle 15729 H5N2/ duck H5N8 ADNS 2 24.02.2020 Plovdiv Padarsko Mule Closed cycle 11 600 H5N8 duck ADNS 3 24.02.2020 Plovdiv Trilistnik Laying Adult laying 55 437 H5N2 hens hens ADNS 5 02.03.2020 Plovdiv Bolyarino Mule Closed cycle 3620 H5N8 duck ADNS 6 05.03.2020 Plovdiv Trilistnik Laying Adult laying 31 322 H5N2 hens hens ADNS 7 12.03.2020 Kardzhali Perperek Laying Adult laying 16 800 H5N8 hens hens ADNS 8 12.03.2020 Plovdiv Manole Laying Rearing 39 070 H5N2 hens laying hens ADNS 9 04.06.2020 Plovdiv Asenovgrad Laying Adult laying 75 000 H5N8 hens hens Phylogenetic analysis The genetic analysis of the HA gene showed that the 2020 H5N8/N2 Bulgarian viruses are related to HPAI H5N8 viruses of clade 2.3.4.4b identified in Bulgaria in 2017-2019 (blue viruses) and cluster separately from the HPAI H5N8 viruses circulating in East- Central Europe (Poland, Germany, Hungary, Czech Republic, Slovakia) in 2019-2020. H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses H5N8 viruses collected in East-Central Europe in 2019-2020 Maximum Likelihood phylogenetic tree of the HA gene (IQ-TREE v.1.6.8) H5N8 The H5N8 viruses from outbreaks 2020/2 (ducks), 2020/5 (mule ducks), 2020/7 (hens) and 2020/9 (hens) cluster together with H5N8 viruses, that were circulating in Bulgaria from 2017 to 2019, for all the eight gene segments. In particular, the H5N8 virus from outbreak 2020/2 is highly related to the virus from outbreak 2020/5, while the virus from outbreak 2020/7 clusters with the virus from outbreak 2020/9 for all the eight gene segments. Maximum Likelihood phylogenetic tree of the PB2 gene (IQ-TREE v.1.6.8) H5N2 The H5N2 viruses from outbreaks 2020/1 (mule ducks), 2020/3 (hens), 2020/6 (hens) and 2020/8 (hens) were generated after the occurrence of multiple reassortment events. In the H5N2 reassortant strain: ➢ the HA and M genes cluster with H5N8 viruses circulating in Bulgaria in 2017-2020; ➢ the N2 gene clusters with a H4N2 virus identified in duck in Bulgaria in 2018 and with H6N2 viruses identified in mule ducks in Bulgaria in 2009-2010. ➢ the other gene segments cluster with LPAI viruses identified in wild and domestic birds in Eurasia. Of note, the NS gene segment of the Bulgarian H5N2 viruses belongs to allele B, while the H5N8 viruses possess the allele A. H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the PB2 gene (IQ-TREE v.1.6.8). H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the PB1 gene (IQ-TREE v.1.6.8). H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the PA gene (IQ-TREE v.1.6.8). H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the NP gene (IQ-TREE v.1.6.8). H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the NA (N8) gene (IQ-TREE v.1.6.8). H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the NA (N2) gene (IQ-TREE v.1.6.8). H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the M gene (IQ-TREE v.1.6.8). H5N8 viruses collected in Bulgaria in 2017-2019 H5N8/H5N2 viruses collected in Bulgaria in 2020 Cluster of 2020 Bulgarian H5N2 viruses Cluster of Bulgarian H5N8 viruses including the 2020 H5N8 viruses Maximum Likelihood phylogenetic tree of the NS gene (IQ-TREE v.1.6.8). Molecular analysis • Molecular analysis of the HA protein revealed that the Bulgarian H5N2 viruses, obtained from outbreak 2020/1 and 2020/3, possess a potential additional glycosylation site at position 70-72 (D70N) (numbering starting form the first methionine) which indicates an adaptation of the virus to domestic birds (Matrosovich et al., 1999; Banks and Plowright, 2003). • For the NA protein, potential additional glycosylation sites were identified in the H5N8 viruses from outbreak 2020/7 and 2020/9 at position 411-413 (E411N). • No evidence of mutations associated with mammalian adaptation in all the analysed viruses was recorded. References • Matrosovich M, Zhou N, Kawaoka Y, Webster R. 1999. The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 73: 1146– 55 • Banks J, Plowright L. 2003. Additional glycosylation at the receptor binding site of the hemagglutinin (HA) for H5 and H7 viruses may be an adaptation to poultry hosts, but does it influence pathogenicity? Avian Dis 47: 942– 50 Evolutionary analysis H5N8 To estimate the time of emergence of the H5N8 2020.5 and H5N2 viruses in Bulgaria were performed a 2020 Bayesian evolutionary analysis of all the eight gene 2019.5 segments and were calculated the time to the most 2019 2018.5 common recent ancestor (tMRCA) in BEAST 1.10.4. 2018 The H5N8 viruses collected in Bulgaria in 2020 2017.5 2017 seem to have emerged in the first half of 2017 2016.5 (December 2016-August 2017). 2016 M_H5N8 PA_H5N8 N8_H5N8 NP_H5N8 HA_H5N8 PB2_H5N8 PB1_H5N8 The gene segments of the reassortant H5N2 viruses NSA_H5N8 collected in Bulgaria in 2020 show TMRCAs ranging between May 2019 and January 2020, with the exception of the NS gene segment, for which the H5N2 limited number of allele B viruses has widened the 2020.5 time range of the possible origin of this segment 2020 between October 2017 and July 2019. 2019.5 2019 To conclude: 2018.5 2018 • H5N8 viruses with the same gene constellation 2017.5 have been circulating since 2017. 2017 2016.5 • The results of the evolutionary analyses suggest 2016 that the reassortment events which gave origin M_H5N2 PA_H5N2 N2_H5N2 to the H5N2 viruses may have occurred within NP_H5N2 HA_H5N2 PB2_H5N2 PB1_H5N2 the country in late 2019. NSB_H5N2 THANK YOU FOR YOUR ATTENTION!.
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