Competing co-infections of LP and HP AIV H7N7

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1 Competing co-infections of LP and HP AIV H7N7 Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health Suedufer 10, Greifswald-Island of Riems, Germany Annika Graaf, Timm Harder

evidence of previous LP infection) Source and mode of virus introduction not clear Dietze & Graaf et al.

2 Epidemiological background Detection of H7N7 LP/HPAIV in Germany Outbreaks of LPAIV H7N7 in two farms, 2015 LPAIV outbreak at begin of June in farm A HPAIV outbreak at end of July in farm B (B tested negative during June/July 2015, but had serological evidence of previous LP infection) Source and mode of virus introduction not clear Dietze & Graaf et al. (2018), Transbound Emerg Dis doi: /tbed PMID: HPAIV AR1385/2015 (H7N7) arose from LPAIV AR915/2015 (H7N7) by spontaneous mutation

3 Emergence, competition and co-circulation of LP/HPAIV in chickens How does LPAIV mutate to HPAIV to induce disease? How is HPAIV able to spread in an individual and in a population in which (antigenically identical) LPAIV is already circulating? Problem: Underlying LPAIV infection functions like a live vaccine and effectively reduces susceptibility of the host to (antigenically identical) HPAIV infection and reduces transmission dynamics of HPAIV (Nickbakhsh et al., 2016). Experimental approach (in vivo and in ovo): Competing co-infections of LP AR915 and HP AIV AR1385: Pathogenesis in subadult chickens and in embryonating eggs. Determination of transmission kinetics of HPAIV in a mixture with LPAIV in chickens

In vivo trial experimental design Parent strains: LPAIV

A/chicken/Germany/AR1385/2015 + Oculonasal inoculation

groups; 14 animals per group - 10 animals inoculated, 4

4 In vivo trial experimental design Parent strains: LPAIV H7N7 A/chicken/Germany/AR915/2015 HPAIV H7N7 A/chicken/Germany/AR1385/ Oculonasal inoculation Infection with variable doses of H7N7 virus(es) 12 groups; 14 animals per group - 10 animals inoculated, 4 contact/sentinel chickens 14 days of observation (clinical scoring and sampling)

5 Experimental design group definition group C1 group C3 group C4 group C5 C = co-infection 0,001% HP + 99,99 % LP 10 1 EID 50 /0.5 ml 0,1% HP + 99,9 % LP 10 3 EID 50 /0.5 ml 1% HP + 99 % LP 10 4 EID 50 /0.5 ml 10% HP + 90 % LP 10 5 EID 50 /0.5 ml group M1 Day 21 group M3 group M4 group M5 M = mono-infection 0,001% HP + 99,99 % PBS 10 1 EID 50 /0.5 ml 0,1% HP + 99,9 % 10 3 EID 50 /0.5 ml 1% HP + 99 % 10 4 EID 50 /0.5 ml 10% HP + 90 % 10 5 EID 50 /0.5 ml group C % HP + 50 % LP 10 5,7 EID 50 /0.5 ml group M % HP + 50 % 10 5,7 EID 50 /0.5 ml

6 Experimental design control groups group B group M6 100 % LP (IVPI: 0) 10 6 EID 50 /0.5 ml 100 % HP (IVPI: 2.8) 10 6 EID 50 /0.5 ml Group EID50/0.5 ml C (+10 6 LP) C1 C3 C4 C5 C5.7 - M M1 M3 M4 M5 M5.7 M6 B B

7 Results Increased survival rate of co-infected chickens Solid lines: Inoculated animals Dashed lines: Sentinels / HPmono-infection group / co-infection / LP mono-infection group B

8 Results Increased survival rate of co-infected chickens Neither morbidity nor mortality was observed in any bird of group B, C1, C3 & M1 Dose-dependent morbidity and mortality was evident in groups that received an inoculum of HPAIV H7N7 AR1385, the HP successor of LPAIV AR915, as a mono-infection (groups M1-M6) As a mono-infection, at least 10 3 EID 50 of the HPAIV AR1385 inoculum (group M3) per animal was required to induce infection, clinical signs and mortality When compared to HPAIV mono-infections, an attenuated course of disease was statistically evident when less than 10 5 EID 50 of the HPAIV where mixed to an inoculum of 10 6 EID 50 of the LPAIV

(data not shown); not HP infected Group C3: no HP shedding Group C4: singular HP excretion peak at 6 dpi receding to zero (chickens

9 Results Impaired HP viral shedding in co-infected chickens For virus excretion kinetics, area under the curve (AUC) graphs were compared LPAIV-group B: no HP shedding HPAIV mono-infection group M6: steady increase until death Groups C1 and M1: no HP shedding (data not shown); not HP infected Group C3: no HP shedding Group C4: singular HP excretion peak at 6 dpi receding to zero (chickens survived) Groups C5 and C5.7: No differences With increasing amounts of HPAIV in the inoculation mixture less LP H7-specific RNA was excreted

10 Results Histopathological findings in chickens at 2 dpi HPAIV mono-infection or LP/HPAIV co-infection, sacrificed at 2 dpi: viral antigen detectable in various tissues including brain, heart and intestine, endothelial cells (rare)

11 In ovo trial Experimental design Parent strains: LPAIV H7N7 A/chicken/Germany/AR915/2015 HPAIV H7N7 A/chicken/Germany/AR1385/2015 Inoculation through the allantoic cavity 10-and 14-day-old SPF embryonating chicken eggs (ECEs) Infection with H7N7 virus(es), 5 eggs/group 4 days of observation (death) and harvesting of allantoic fluids Group EID 50 /0.2 ml C (+10 6 LP) C1 C3 C4 C5 C5.7 - M M1 M3 M4 M5 M5.7 M6 B B

12 Results Survival rates in ovo Solid lines: 10-day-old ECE Dashed lines: 14-day-old ECE / mono-infection group / co-infection / LP mono-infection group B

13 Results Survival rates in ovo No dose-dependent mortality in HPAIV mono-infection groups (except M1) No correlation of HP dose with MDT Delayed embryonic death in coinfected ECE of groups M3-M5 Delayed death of older ECE (10 vs 14 days-old)

14 Results HP virus yields in ovo are reduced in co-infected ECE HP Constant yield of LP virus in all ECE (no influence of age of ECE) Constant yield of HP virus in all ECE (independent of virus inoculum dose and ECE age) LP Grossly reduced HP yield in coinfected ECEs up to C5.7 (more pronounced in 10 day-old ECE)

15 Results Histopathological findings in ovo (CAM) Trilamellar structure of CAM LPAIV remained localized in the allantoic membrane (A+B) HPAIV spread into endothelial and parenchymal cells as well as into the allantoic and the chorionic membrane (C+D)

16 Conclusions High-titred co-infection with LPAIV significantly hampers HP virus replication and spread in ovo and in vivo Co-transmission of HPAIV to sentinel animals is hampered at higher ratios of LP : HP AIV How does HP virus succeed after de novo generation in a bird that is already LP infected? Early mutation event? Systemic sequestration and spread via eggs, feathers, hematophagous ectoparasites? Death and decomposed carcass? How does HP spread in an index population where LP infection isongoing? Is LP-to-HP mutation more frequent than we see?

17 Acknowledgements Thanks to: Martin Beer Aline Maksimov Diana Parlow David Scheibner Lab. Reiner Ulrich Susanne Köthe Pavlo Maksimov Lab. Höper/Pohlmann animal caretakers Thank you for your attention! Funded by an intramural grant at FLI, Riems (HR-0008)

18 LP and HP AIV H7N7, phylogenesis LPAI poultry farm 2013_05 LPAI poultry farm 2015_06 HPAI poultry farm 2015_07

Phylogenetic analysis Figure: Phylogenetic tree of the hemagglutinin (HA) gene (full-length coding sequence) of low and highly pathogenic avian influenza viruses (LP/HPAIV) of subtype H7 viruses

19 Phylogenetic analysis Figure: Phylogenetic tree of the hemagglutinin (HA) gene (full-length coding sequence) of low and highly pathogenic avian influenza viruses (LP/HPAIV) of subtype H7 viruses derived from poultry and wild birds in Eurasia in the period from (A). The zoomed section (B) depicts the topology of viruses detected in two chicken layer farms in Germany, 2015 (red virus designations). Phylogenetic analysis was conducted using a maximum likelihood approach (IQtree) based on the TIM+I+G4 model (Nguyen et al., 2014).

20 Table: Mutations identified outside the hemagglutinin cleavage site in the highly pathogenic H7N7 avian influenza virus AR1385/15 in comparison to its low pathogenic AIV AR915/15 precursor isolated from outbreaks in the district of Emsland, Lower Saxony, Germany, in Gene Mutations in AR1385/15 Mutations as minor variants in AR915/15 segment silent coding silent coding PB2 2 E123K, I147V, K355R 1 (21.3%) E123K (22.6%), K355R (42.2%) PB1 2 F254C PA 3 K185R 0 F254C (18.8%) 0 K185R (18.2%) HA 1 I13S 0 0 NP 1 S478F 0 0 NA 3 V439A 1 (23.8%) 0 M 2 V68L (M2) 1 (19.7%) 0 NS 0 N92D (NS-1) 0 N92D (25.1%)

Endemic non-notifiable avian influenza virus infections in poultry. NRL Avian Influenza. Friedrich-Loeffler Institute, Germany. Foto: C.

Endemic non-notifiable avian influenza virus infections in poultry NRL Avian Influenza Friedrich-Loeffler Institute, Germany Foto: C. Illing Avian influenza virus subtype H9N2 H9N2 became a wider problem