Sánchez‑González et al. Vet Res (2020) 51:113 https://doi.org/10.1186/s13567-020-00835-4 RESEARCH ARTICLE Open Access Pathobiology of the highly pathogenic avian influenza viruses H7N1 and H5N8 in different chicken breeds and role of Mx 2032 G/A polymorphism in infection outcome Raúl Sánchez‑González1,2* , Antonio Ramis1,2, Miquel Nofrarías1, Nabil Wali1, Rosa Valle1, Mónica Pérez1, Albert Perlas1,2 and Natàlia Majó1,2 Abstract Chickens are highly susceptible to highly pathogenic avian influenza viruses (HPAIVs). However, the severity of infec‑ tion varies depending of the viral strain and the genetic background of the host. In this study, we evaluated the pathogenesis of two HPAIVs (H7N1 and H5N8) and assessed the susceptibility to the infection of local and com‑ mercial chicken breeds from Spain. Eight chicken breeds were intranasally inoculated with 1­ 05 ­ELD50 of A/Chicken/ Italy/5093/1999 (H7N1) or A/Goose/Spain/IA17CR02699/2017 (H5N8 clade 2.3.4.4. B) and monitored during 10 days. Chickens were highly susceptible to both HPAIVs, but H7N1 was considerably more virulent than H5N8 as demon‑ strated by the highest mortality rates and shortest mean death times (MDT). Both HPAIVs produced severe necrosis and intense viral replication in the central nervous system, heart and pancreas; however, the lesions and replication in other tissues were virus-dependent. High levels of viral RNA were detected by the oral route with both viruses. In contrast, a low number of H5N8-inoculated chickens shed by the cloacal route, demonstrating a different pattern of viral shedding dependent of the HPAIV. We found a high variation in the susceptibility to HPAIVs between the different chicken breeds. The birds carrying the genotype AA and AG at position 2032 in chicken Mx gene presented a slightly higher, but not significant, percentage of survival and a statistically significant longer MDT than GG individuals. Our study demonstrated that the severity of HPAI infection is largely dependent of the viral isolate and host factors, under‑ lining the complexity of HPAI infections. Keywords: highly pathogenic avian influenza, Gs/GD lineage, classical strain, chickens, breed, pathogenicity Highlights • Huge differences in susceptibility to HPAIVs exist between chicken breeds. • H7N1 HPAIV is more virulent to chickens than H5N8 HPAIV. • The lower cloacal excretion of H5N8 suggests a lower Introduction adaptation to chickens. Chickens are highly susceptible to highly pathogenic avian influenza viruses (HPAIVs); however, the sever- ity of infection varies depending on the viral strain. The *Correspondence: raul.sanchezgo@e‑campus.uab.cat inoculation of most HPAIVs in chickens causes evi- 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA‑UAB), Campus dent clinical signs (e.g. apathy, nervous signs) and gross de la Universitat Autònoma de Barcelona (UAB), Bellaterra, España lesions (e.g. cutaneous edema, cyanosis of the comb and Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​ mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Sánchez‑González et al. Vet Res (2020) 51:113 Page 2 of 13 wattles, haemorrhages in skin), and chickens usually die influence the infection outcome, the aims of this study in a period ranging from 3 to 6 days post-inoculation were to (1) evaluate the pathobiology of a recent H5N8 (dpi). However, some HPAIVs produce a peracute infec- HPAIV isolated in Spain (Gs/GD lineage, clade 2.3.4.4, tion that kills birds in a shorter period of time (within 3 Group B) in comparison with a classical H7N1 HPAIV dpi) [1, 2]. Differences can be observed even in closely in different local, commercial and experimental chicken related isolates of the same subtype, indicating that lines from Spain with diverse genetic backgrounds; and point or few mutations in viral proteins may have a piv- (2) determine the role of virus factors (differences in the otal effect in their virulence [3]. In 2016/2017, the H5N8 sequence of amino acids in viral proteins between both HPAIV belonging to clade 2.3.4.4 B of Goose/Guang- HPAIVs) and host factors (allele at position 2032 of dong (Gs/GD) H5 lineage was responsible for the fourth chicken Mx gene) in the infection outcome. intercontinental wave of this lineage. Despite Europe has been affected by the four waves of Gs/GD H5 line- Materials and methods age, the one caused by H5N8 B HPAIV was the largest Viruses in magnitude (reported poultry outbreaks and deaths), The viruses used in this study were: A/Chicken/ geographic spread and rapidity of incidents [4, 5]. Since Italy/5093/1999 (H7N1), isolated in 1999–2000 dur- Gs/GD H5N8 B HPAIVs may present altered biological ing an Italian epidemic that mainly affected Veneto and properties in chickens, the pathobiological evaluation of Lombardia regions (kindly provided by Dr. Ana Moreno this new viral strain is needed. from the Instituto Zooprofilattico Sperimentale della The pathogenicity of HPAIVs is also influenced by Lombardia e dell’Emilia Romagna), and A/Goose/ numerous host factors, including species, age at infection Spain/IA17CR02699/2017 (H5N8 clade 2.3.4.4. group and immune response. Several reports demonstrate that B), isolated in Catalonia (Northern Spain) during the a wide range of susceptibility to HPAIV infection is pre- 2016/2017 European epizootics. Both viruses are highly sent between chicken breeds/lines. Some breeds display a pathogenic based on the amino acid sequences at the comparatively high resistance, whereas other breeds are HA0 cleavage site: PEIPKGSRVRR↓GLF (H7N1) and particularly susceptible [3, 6–10]. Local chicken breeds PLREKRRKR↓GLF (H5N8). Virus stocks were produced are generally raised in small-scale farms and in backyards in 10 days-old specific-pathogen free (SPF) embryonated that allow the direct contact with wild and synanthropic eggs. The allantoic fluid was obtained at 24–48 h post- birds and their droppings. Consequently, these birds are inoculation (hpi), filtered and aliquoted at − 75 °C until likely more exposed to AIVs than commercial chicken use. Serial ten-fold dilutions of the filtered viruses in PBS breeds, which are raised under high biosecurity stand- were used for titration in 10 days-old SPF embryonated ards. However, there is a general belief that local chicken eggs. The mean embryo lethal doses (­ ELD50) were deter- breeds are naturally resistant to disease, which is associ- mined by Reed and Muench method [17]. The consensus ated with the natural selection over the years by autoch- full genome sequences corresponding to the eight seg- thonous pathogens, food availability and harmful climate ments of the local H5N8 are available in Genbank under [11]. This assumption is usually a result of empiric expe- accession numbers MK494920 to MK494927 (H5N8). rience at the field, and the results of experimental stud- ies addressing the susceptibility of local chicken breeds to Animals and facilities HPAIVs do not always support this theory [10]. To date, Fifteen day-old chickens (Gallus gallus domestica) of six there is no information regarding the susceptibility of different local breeds from Spain (Empordanesa, Pened- local Spanish chicken breeds to HPAIVs. esenca, Catalana del Prat, Flor d’Ametller, Castellana Despite the genetic background that confers higher negra, and Euskal oiloa), a commercial breed (Ross 308 resistance to HPAIVs in chickens remains unknown, Broiler) and a commercial-experimental line (SPF White it was reported that the G/A polymorphism at posi- Leghorns) were used. The breeds included in this study tion 2032 in chicken Mx gene (substitution of serine to were not vaccinated. All local breeds were obtained from asparagine at position 631 in the protein) conferred an local breeders. The local breeds included in this study are antiviral effect against AIV infection in vitro [12]. How- common in non-commercial, small-scale flocks, usually ever, several in vitro and in vivo experiments have failed in backyards alone or mixed with other species in dif- to demonstrate a clear correlation between this polymor- ferent regions of Spain. For their characteristics, these phism and inhibition of AIVs or survival after infection, breeds are common in chicken contests, and their meat respectively [6, 13–16]. Therefore, the role of Mx in AIVs and sub-products are used for self-consumption or sold infections in chickens is still under debate. in local markets because of their added value in the mar- Since the pathobiology of HPAIVs in chickens is multi- ket chain. Specific programs have been established in all factorial and numerous viral and host factors can largely included breeds to ensure their conservation [18]. Sánchez‑González et al. Vet Res (2020) 51:113 Page 3 of 13 At arrival, the animals were individually identified and with H7N1 and H5N8 HPAIVs, and from negative con- placed in negative-pressured high efficiency particulate trol animals, at 1, 3, 6 and 10 dpi. The same birds were air (HEPA)-filtered isolators present in Biosecurity Level sampled through the experiment. 3 (BSL-3) facilities of Centre de Recerca en Sanitat Ani- mal (Programa de Sanitat Animal, IRTA). During the Pathological examination and immunohistochemical 5 days-acclimation period, serum samples were obtained testing from all birds to ensure that they were seronegative to Tissue samples collected from the chickens necropsied AIV by an ELISA competition test (ID-VET, Montpellier, at 3 dpi were immersed in 10% formalin for fixation dur- France). Furthermore, OS and CS were collected from 5 ing 72 h and embedded in paraffin wax. Samples included randomly selected chickens of each group and confirmed skin, thymus, pectoral muscle, nasal cavity, trachea, to be negative to AIV RNA by one-step qRT-PCR. lung, central nervous system, heart, spleen, liver, kidney, proventriculus, gizzard, pancreas, small intestine, large Experimental design and sampling intestine and bursa of Fabricius. After acclimation, 15 chickens of each breed (except for Microtome sections of 3 µm of thickness (Leica Castellana negra and Broilers that consisted in groups of RM2255, Nussloch, Germany) from formalin-fixed, 13 birds, and Euskal oiola inoculated with H7N1 that was paraffin-embedded tissues collected at 3 dpi were pro- a group of 10 animals) were intranasally challenged with cessed, stained with haematoxylin and eosin (H/E) and H7N1 or H5N8 HPAIV diluted in PBS in order to inoc- then examined under light microscopy. An immuno- ulate ­105 ­ELD50 in a final volume of 0.05 mL (0.025 mL histochemical (IHC) technique was performed in the inoculated in each nostril). Animals belonging to nega- same tissues. Briefly, samples were pretreated with 0.1% tive control group (2–5 animals/breed) were intranasally protease at 37 °C during 8 min. A mouse-derived mono- inoculated with 0.05 mL of sterile phosphate-buffered clonal commercial antibody against the nucleoprotein saline (PBS). (NP) of IAVs (ATCC, HB-65, H16L-10-4R5) was used All birds were monitored daily for clinical signs until 10 as a primary antibody. The slides were incubated over- dpi. A standardized World Organization for the Animal night at 4 °C. The samples were then incubated with an Health (OIE) clinical scoring system was used [19]. Ani- anti-mouse secondary antibody conjugated to an HRP- mals with absence of clinical signs were classified as 0. Labelled Polymer (Dako, immunoglobulins As, Den- Birds presenting one of the following clinical signs were mark). The antigen–antibody reaction was visualized considered sick (1) and those showing more than one using the chromogen 3,3′-diaminobenzidine tetrahy- were considered severely sick (2): respiratory involve- drochloride. Sections were counterstained with Mayer’s ment, depression, diarrhea, cyanosis of the exposed haematoxylin and examined under light microscopy. The skin or wattles, edema of the face and/or head and nerv- positivity in the tissues was semi-quantitatively assessed ous signs. Birds found dead were scored as 3. For ethi- taking into consideration the percentage of NP-positive cal reasons, moribund chickens were anesthetized using and negative cells in the tissue. The samples were classi- the combination of ketamine/xylazine (20 mg/kg body fied as follows: no positive cells (−), < 10% positive cells weight, Imalgene 100 and 5 mg/kg body weight, Rompun (+), 10–40% positive cells (++), > 40% positive cells 20 mg/mL) via the intramuscular route, euthanized by (+++) in a tissue section. Positive and negative con- intracardiac injection of pentobarbital overdose (140 mg/ trols were used. The positive control was a central nerv- kg body weight, Euthasol 400 mg/mL) and scored as ous system from a chicken experimentally infected with dead. The percentage of mortality and mean death time H7N1 HPAIV [20], and the negative control consisted (MDT) were calculated for each virus in all the breeds. in the same tissue incubated with PBS instead of the pri- All birds presenting severe clinical signs or found dead mary antibody and also the tissues collected from nega- were subjected to macroscopic examination. In addition, tive control chickens. three chickens of each breed inoculated with H7N1 and H5N8 HPAIVs were killed at 3 dpi using the combina- Viral RNA quantitation in swabs tion of drugs reported above to collect tissue samples for Swabs were placed in 0.5 mL of sterile PBS enriched pathological studies. The selection of birds was biased with Penicillin–Streptomycin (Thermo Fisher Scientific, towards those found dead or presenting evident clinical Waltham, Massachusetts, USA) and Nystatin (Sigma- signs of disease. Two birds of each breed belonging to Aldrich, Missouri, USA) at a final concentration of 6%. mock-infected groups were also necropsied at 3 dpi. In Swabs were conserved at − 75 °C until further use. Viral order to evaluate viral shedding, oral swabs (OS) and clo- RNA was extracted using Nucleospin RNA virus kit acal swabs (CS) were collected from 9 chickens of each (Macherey–Nagel, Düren, Germany), following manufac- breed (selected previously to the inoculation) challenged turer’s instructions. A highly conserved region of 99 bp Sánchez‑González et al. Vet Res (2020) 51:113 Page 4 of 13 present in IAV M1 gene was amplified and detected by The restriction enzyme (5′-GTN|NAC-3′) cleaves the one-step Taqman RT-PCR technique in Fast7500 equip- sequence 2 pb downstream of the Mx polymorphism in ment (Applied Biosystems, Foster City, CA, USA), using presence of guanine (G), whereas the product is not cut the same primers and probe as well as conditions of in case of an adenine (A) at this position. Digestion prod- amplification previously described [21, 22]. To extrapo- ucts were visualized in a 2% agarose gel in 1X TAE buffer late the genome equivalent copies (GEC) present in the with EtBr. Animals were classified in homozygous-resist- swabs, a standard curve obtained by amplification of the ant genotype (AA), heterozygous-intermediate genotype same region of M1 gene was used. Briefly, the amplified (AG) and homozygous-susceptible genotype (GG). region was ligated in pGEM-T vector (Promega, Madi- The proportion of birds dead at the end of the study son, Wisconsin, USA). The ligation product was purified by genotype groups were compared using the Pearson’s using MinElute Reaction Cleanup Kit (Qiagen, Valencia, Chi square test. Then, post hoc pairwise comparisons CA, USA) and transfected into electrocompetent E.coli with Bonferroni corrections were carried out [24]. Also, cells (Thermo Fisher Scientific, Waltham, Massachusetts, for the animals that succumbed to infection, the MDT USA) by electroporation. The recombinant plasmid was by genotypes were compared. First, the normality of the purified from transformed colonies using NucleoSpin data was assessed using the Shapiro–Wilk test. Then Plasmid (Macherey–Nagel, Düren, Germany) and quan- groups were compared using either the Anova test (in tified in Biodrop (Biodrop µLite, Cambridge, England). case of normally distributed data), or the Kruskal–Wal- GEC were calculated using DNA Copy Number Calcula- lis test (in case of non-normally distributed data). Finally, tion (Thermo Fisher Scientific, Waltham, Massachusetts, post hoc comparisons were carried out using the Tukey USA). Serial ten-fold dilutions were used to obtain the test (for normally distributed data), or Dunn’s test with standard curve. The limit of detection of the technique Bonferroni correction (in case of non-normally distrib- was 1.89Log10 GEC in both OS and CS. uted data). All calculations were carried out using R sta- tistical software (http://cran.r-proje​ct.org/). RFLP‑PCR Mx Prior to infection, total blood in a 1:1 ratio with antico- Results agulant (Alsever’s solution, Sigma-Aldrich, Missouri, Clinical signs and mortality USA) was obtained from all chickens belonging to H7N1 The mortality and percentage of clinical signs after exper- and H5N8 HPAIV-inoculated groups. Genomic DNA imental infection with either H7N1 or H5N8 HPAIVs was isolated from 10 µL anticoagulated blood using a in the different chicken breeds are presented in Figure 1 standard DNA purification kit (DNeasy Mini Kit, Qia- and Table 1. Severe clinical signs were observed in H7N1 gen, Valencia, CA, USA), following manufacturer’s and H5N8 HPAIVs-inoculated chickens in all breeds, instructions. To avoid RNA contamination, samples were but the frequency varied depending on the viral isolate treated with RNase (RNase A, Qiagen, CA, USA). As and the chicken breed. At 2 dpi, several chickens of dif- described by Sironi et al. [23], the following primers were ferent breeds inoculated with H7N1 HPAIV presented used to amplify a 299 pb region in exon 14 of chicken Mx severe apathy, were prostrated or found dead without gene: forward 5′-GCA​CTG​TCA​CCT​CTT​AAT​AGA-3′ previous evident clinical signs. Few chickens inoculated and reverse 5′-GTA​TTG​GTA​GGC​TTT​GTT​GA-3′. PCR with H5N8 HPAIV also presented severe apathy at 2 dpi reaction mixture included 60 ng genomic DNA, 10 µmol and were consequently euthanized. From 2 dpi and last- of each primer, 10 × buffer, 1.5 mM M ­ gCl2, 0.2 mM ing until 9 (H7N1) or 10 dpi (H5N8), severe clinical signs of each dNTP and native Taq DNA polymerase (5 U/ were detected in several chickens at different times post- µL) (Taq DNA Polymerase, native, ThermoFisher Sci- inoculation. The main clinical signs observed in both entific, Massachusetts, USA) in a final volume of 25 µL. HPAIV infections were moderate apathy that progressed Mx region was amplified in GeneAmp PCR System 9700 to prostration, and less frequently subcutaneous oedema, equipment (Applied Byosistems, CA, USA) as follows: cyanosis of the comb and wattles and nervous signs 95 °C for 10 min, 35 cycles of 94 °C for 1 min, annealing (ataxia, circling, tremor and head shaking). The percent- at 53 °C for 1 min, and 72 °C for 1 min, and a final exten- age of animals presenting prostration and nervous signs sion step at 72 °C for 10 min. was higher in chickens challenged with H7N1 HPAIV Five μL of PCR products were run in a 2% agarose gel than in those inoculated with H5N8 HPAIV (55% and 8% in 1X TAE buffer with ethidium bromide to confirm the for H7N1 versus 35% and 4% for H5N8). Similarly, the presence of a specific band at 299 pb. PCR products were onset of nervous signs was earlier in H7N1 HPAIV-inoc- incubated at 37 °C during 16 h with a restriction enzyme ulated groups than in the inoculated with H5N8 HPAIV (Hpy8I-MjaIV, 10 U/µL, Thermo Fisher Scientific, Mas- (3 dpi versus 5 dpi). Moreover, H7N1 HPAIV produced a sachusetts, USA), following manufacturer’s instructions. higher mortality rate in chickens than H5N8 HPAIV (70 Sánchez‑González et al. Vet Res (2020) 51:113 Page 5 of 13 Figure 1 Survival curves of the different chicken breeds experimentally inoculated with H7N1 (A) or H5N8 (B) HPAIVs at a dose of 1­ 05 ­ELD50. Table 1 Clinical signs, mortality and MDT of the different chicken breeds challenged with H7N1 or H5N8 HPAIVs. Virus Parameter Empordanesa Penedesenca C. Prat F. Ametller C. Negra E. Oiloa 308 Ross SPF White Mean Broiler Leghorn H7N1 Percentage of dead birds 93 100 100 80 25 73 46 40 70 Mean death time (dpi) 3.3 4.2 3.3 3.3 2.7 3 3 3.8 3.3 Clinical signs (%) Severe apathy 93 73 67 40 15 67 31 40 55 Cutaneous edema/cyanosis 7 20 7 0 0 0 0 7 5 Nervous signs 0 7 20 0 8 20 8 0 8 H5N8 Percentage of dead birds 33 76 85 73 25 50 25 8 47 Mean death time (dpi) 4.2 7.9 5.8 5.7 2.7 2.8 3.7 6 4.9 Clinical signs (%) Severe apathy 7 40 40 53 23 40 8 67 35 Cutaneous edema/cyanosis 0 7 7 7 0 0 0 8 4 Nervous signs 0 7 0 0 0 0 0 0 4 versus 47%, respectively), as well as a shorter MDT (3,3 inoculated with H5N8 HPAIV. From 3 dpi to the end versus 4,9 dpi, respectively). of the study, the most common findings in the chickens Regarding breeds, Castellana negra, Broiler and SPF inoculated with H7N1 and H5N8 HPAIVs were multi- chicken breeds presented a lower frequency of clini- focal petechiae and necrotic areas in pancreas, and/or cal signs and considerably lower mortality rates (≤ 50%) multifocal petechiae in proventriculus, gizzard and in than Penedesenca, Catalana del Prat, Flor d’Ametller and the proventriculus-gizzard junction. Congestion in cen- Euskal Oiloa (≥ 50%) breeds in both H7N1 and H5N8 tral nervous system was also a common finding. Less HPAIVs-inoculations. Only Empordanesa breed pre- frequently, several chickens exhibited hemorrhages of sented differing susceptibility depending on the virus variable intensity in skin (e.g. legs), subcutaneous edema, tested (93 and 33% mortality after challenge with H7N1 lung consolidation and diffuse congestion in internal and H5N8, respectively). Catalana del Prat and Pened- organs. At 10 dpi, one chicken inoculated with H5N8 esenca presented the highest incidence of nervous signs HPAIV presented multifocal petechias in bursa of Fab- and cutaneous edema among all the tested breeds, ricius. No macroscopic lesions were observed in negative respectively (Table 1). control birds. Gross lesions Histopathological findings Macroscopic examination of the chickens inoculated Microscopic examination of the tissues collected from with H7N1 or H5N8 HPAIVs revealed similar lesions dead or severely-affected chickens at 3 dpi revealed evi- with both viruses in all breeds. At 2 dpi, few chick- dent lesions of variable intensity in all breeds in mostly ens inoculated with H7N1 HPAIV exhibited multifocal all the collected organs. However, we detected differences haemorrhages in proventriculus and gizzard, whereas in the severity and viral replication in the different tissues non-evident lesions were present in the chickens between H7N1 and H5N8 HPAIV-inoculated chickens Sánchez‑González et al. Vet Res (2020) 51:113 Page 6 of 13 (Additional file 1, Additional file 2, respectively). In both intestines) presented mild multifocal necrotic and/or viral infections, the predominant microscopic lesions inflammatory lesions and few positive cells. in the tissues were areas of necrosis and haemorrhages with mixed inflammatory infiltrate (macrophages, lym- Viral shedding phoplasmacytic cells and heterophils). The extension and Differences in the viral shedding between H7N1 and severity of microscopic lesions correlated well with the H5N8 HPAIV-inoculated chickens were detected (Fig- intensity of IAV antigen (IHC techniques) in the tissues. ures 3A–D, respectively). High viral excretion by both The main organs affected were similar in all the chicken oropharyngeal and cloacal routes was detected in chick- breeds inoculated with the same virus. ens inoculated with H7N1 HPAIV. At 1 dpi, viral RNA In H7N1 HPAIV-inoculated chickens, the most rel- was detected in several OS but not in CS. The peak of evant microscopic lesions and viral replication were shedding occurred at 3 dpi. By 6 dpi, few birds presented observed in heart, followed by central nervous system detectable levels of virus RNA in OS and CS, but the lev- and pancreas. Viral replication in the heart was associ- els detected were similar with those collected at 3 dpi. ated with fiber degeneration/necrosis and hyalinization No viral RNA was present in the OS and CS collected of myocardiocytes mixed with mild inflammatory cell at 10 dpi. Regarding H5N8 HPAIV-inoculated groups, infiltration (mainly macrophages) (Figures 2A, B). In a low number of OS presented detectable levels of viral the central nervous system, non-suppurative encephali- RNA at 1 dpi. The proportion of positive OS and levels tis consisting in multifocal areas of necrosis in cerebral of viral RNA peaked at 3 dpi. In contrast, a low number hemispheres, intense spongiosis, neuronal chromatoly- of birds inoculated with H5N8 HPAIV presented cloacal sis and gliosis were commonly observed (Figures 2C, D). shedding at the different dpi tested. However, the levels In cerebellum, chromatolysis of Purkinje neurons was a of viral RNA in the positive CS at 3 dpi were similar to common finding. The lesions observed in the pancreas those present in OS. By 6 and 10 dpi, viral shedding was were multifocal areas of lytic necrosis of exocrine gland still detected in several birds by both the oral and cloacal cells (Figure 2E, F). The remaining tissues generally pre- routes and in some samples, the levels were high. sented mild necrotic and/or inflammatory lesions and Both H7N1 and H5N8 HPAIV-inoculated chickens few positive cells, such as lung and spleen (Figures 2G–J, presented similar oral shedding, as demonstrated by a respectively). similar number of chickens shedding virus (70% and 58% The chickens inoculated with H5N8 HPAIV presented by 3 dpi, respectively) and mean levels of viral RNA in OS the most severe lesions and intense viral replication in (5.1 and 4.6 L­ og10 GEC by 3 dpi, respectively). The mean the lung and heart, but evident lesions and high viral levels of viral RNA in CS were also similar between H7N1 replication were also detected in spleen, thymus, central and H5N8-HPAIV inoculated groups (5.3 and 5.2 ­Log10 nervous system, nasal cavity, gizzard, pancreas and liver. GEC by 3 dpi, respectively). However, a higher number of The lesions in the heart, central nervous system and pan- chickens inoculated with H7N1 HPAIV presented cloa- creas were similar to those described in H7N1 HPAIV- cal excretion in comparison with those inoculated with inoculated chickens (Figures 2K–P, respectively). In the H5N8 HPAIV (56% and 21% by 3 dpi, respectively). lung, interstitial pneumonia consisting on moderate to Despite the levels of viral RNA in OS and CS obtained severe increase of the cellularity (macrophages and lym- through the experiment were quantitatively similar in all phoid cells) in air capillaries and focal areas of necrosis the chicken breeds included in the study, a higher pro- associated with intense viral replication was commonly portion of chickens of the Empordanesa, Penedesenca, observed (Figures 2Q, R). In lymphoid tissues, including Catalana del Prat, Flor d’Ametller and Euskal Oiloa spleen, thymus and bursa of Fabricius, multifocal areas breeds excreted virus by oral and cloacal routes in both of necrosis/apoptosis of variable intensity in mononu- viral infections than those belonging to Castellana negra, clear cells were present. Particularly, diffuse necrotic Broiler and SPF breeds. areas and widespread viral replication were present in the spleen of one chicken (Figures 2S, T). Several ani- Polymorphism at position 2032 of Mx gene and association mals also presented multifocal areas of necrosis in res- with infection outcome piratory and olfactory epithelial cells in the nasal cavity. We found marked differences regarding genotype and Multifocal areas of necrosis in glandular cells with mixed allele distribution at position 2032 of chicken Mx gene inflammatory cell infiltration, muscular cell degeneration in the breeds included in this study (Table 2). The three and necrosis of lymphoid tissue were detected in gizzard genotypes AA, AG, GG were present in Empordanesa, and proventriculus. In the liver, we detected focal areas Penedesenca, Catalana del Prat and Castellana negra of necrosis with mild distention of hepatic sinusoids. breeds with variable frequency. The heterozygous geno- The remaining tissues (skin, pectoral muscle, kidney and type (AG) was the predominant in Empordanesa and Sánchez‑González et al. Vet Res (2020) 51:113 Page 7 of 13 Figure 2 Microscopic lesions (HE staining) and viral replication (IHC staining) at 3 dpi in several organs obtained from chicken breeds experimentally inoculated with H7N1 and H5N8 HPAIVs. Myocardium A, B (H7N1 infected), K, L (H5N8 infected): multifocal necrosis of myocardiocytes with inflammatory infiltrate (A, K) and NP-positive myocardiocytes and inflammatory cells (B, L). CNS C, D (H7N1 infected) M, N (H5N8 infected): multifocal areas of necrosis in cerebral hemispheres (C, M), widespread NP-positive neurons and glial cells (D, N). Pancreas E, F (H7N1 infected), O, P (H5N8 infected): diffuse area of necrosis in pancreatic acinar cells (E, O) associated with widespread NP-positive cells in necrotic areas and surrounding acinar pancreatic cells (F, P). Lung G, H (H7N1 infected), Q, R (H5N8 infected): mild increase of cellularity (mixed inflammatory cells) in air capillaries interstitium (G) and few NP-positive inflammatory cells (H). Severe increase of increase of cellularity in air capillaries interstitium, focal areas of necrosis in pneumocytes, microthrombi and diffuse oedema (Q), widespread NP-positive cells in inflammatory cells, endothelial cells and air capillary cells (R). Spleen I, J (H7N1 infected), S, T (H5N8 infected): non-apparent lesions (I) and few NP-positive lymphoid-cells (J). Areas of necrosis with mixed inflammatory cell infiltration (S), widespread NP-cells in inflammatory and endothelial cells (T). Sánchez‑González et al. Vet Res (2020) 51:113 Page 8 of 13 Figure 3 Viral titers expressed as ­Log10 GEC in OS and CS obtained from chickens inoculated with H7N1 (A, B) or H5N8 (C, D) HPAIVs at different time points. Viral titers are represented as the mean values ± SEM. The numbers above the columns represent the number of chickens shedding virus out of the total sampled. GEC: Genome equivalent copies; Dpi: day post-inoculation. Table 2 Genotype AA, AG and GG and allele frequencies all Flor d’Ametller and Euskal Oiloa chickens presented of A and G alleles in exon 14, position 2032 of chicken Mx the homozygous-susceptible genotype (0.939 and 0.923, gene in different chicken breeds. respectively), and any the resistant genotype. SPF chick- Breed Genotype frequency Allele ens presented predominantly the homozygous-resistant frequency genotype (AA), and in a minor amount the heterozygous AA AG GG A G one, but not the homozygous-susceptible one. Broiler chickens were fixed for the homozygous-susceptible gen- Empordanesa 0.129 0.452 0.419 0.355 0.645 otype (GG). Overall, the average genotype frequency of Penedesenca 0.485 0.364 0.152 0.667 0.333 the susceptible genotype GG (0.50) was higher than AG Catalana del Prat 0.156 0.750 0.094 0.531 0.469 (0.29) and AA (0.21) genotypes (data not shown). The Flor d’Ametller 0.000 0.061 0.939 0.030 0.970 A allele was present in all the chicken breeds except in Castellana negra 0.100 0.400 0.500 0.300 0.700 Broilers, but the frequency varied from 0.030 and 0.038 Euskal oiloa 0.000 0.077 0.923 0.038 0.962 in Flor d’Ametller and Euskal oiloa breeds, respectively, 308 Ross Broiler 0.000 0.000 1.000 0.000 1.000 to 0.903 in SPF chickens. G allele was present in all the SPF White Leghorn 0.806 0.194 0.000 0.903 0.097 chicken breeds. In this case, the range of frequencies var- AA resistant genotype, AG heterozygous genotype, GG susceptible genotype ied in the interval of 1.00 present in Broiler chickens to 0.097 in SPF chickens. G allele (0.65) was more predomi- nant in the chicken population tested than A allele (0.36) Catalana del Prat breeds. Penedesenca presented a (data not shown). higher frequency of the homozygous-resistant genotype In order to study the association of Mx genotypes at (AA), whereas Castellana negra had more frequently position 2032 with percentage of survival at the end the homozygous-susceptible genotype (GG). Almost of the study and mean days of death in the birds that Sánchez‑González et al. Vet Res (2020) 51:113 Page 9 of 13 succumbed to infection, statistical analyses were per- The lower frequency of clinical signs and mortality, formed. The differences in the proportion of dead birds longer MDT and reduced excretion in H5N8 HPAIV- (containing both HPAIVs) were not statistically signifi- inoculated chickens in comparison with those inoculated cant among genotypes. However, the Kruskal–Wallis with H7N1 HPAIV suggest a lower affinity and/or adap- test indicated statistically significant differences in the tation of H5N8 HPAIV to chickens. This could have par- MDTs among genotype groups (p value < 0.0001). Post- tially contributed to the limited number of H5N8 HPAIV hoc pairwise using the Dunn’s test with Bonferroni cor- outbreaks in chicken holdings during the 2016–2017 epi- rection indicates differences were statistically significant demics in Europe (12% of the total reported outbreaks), between groups AA and GG (5 versus 3.6 dpi, respec- in comparison with its detections in waterfowl holdings, tively; p = 0.0015), and between groups AG and GG (4.7 including in Spain [5]. However, different genotypes versus 3.6 dpi, respectively; p = 0.0006). of H5N8 HPAIV circulated in Europe at that time [30]. Therefore, important differences in the biological prop- erties and virulence of H5N8 HPAIV between European Discussion strains may exist. The production characteristics of this Available data demonstrates that the susceptibility to species could also be an important factor of the com- HPAIVs varies largely depending of the viral isolate and paratively lower incidence of H5N8 outbreaks. Migratory the genetic background of the host. In order to evalu- wild birds are thought to have played a pivotal role in the ate the existence of viral-and host-dependent differences worldwide dissemination of Gs/GD H5N8 HPAIVs [31]. in HPAIV infections in chickens, we selected a classical Since chicken production in Europe in mostly intensive HPAIV (H7N1 isolated from a chicken in Italy) and a farms presents high biosecurity standards, the low detec- recent HPAIV of the Gs/GD H5 lineage (H5N8 Gs/GD tion in chicken holdings during the 2016/2017 epidemics clade 2.3.4.4 group B isolated from a domestic goose in may also be due to a low exposure to the virus. Spain) and assessed their pathobiology in a broad spec- Despite the different course of infection caused by trum of chicken breeds from Spain (local and commer- H7N1 and H5N8 HPAIVs, the clinical signs and mac- cial breeds). roscopic lesions in the severely affected chickens were Both HPAIVs used in this study were highly virulent to similar and consistent with HPAIV infection. The viral chickens, as expected based on the presence of a multi- antigen detected in mostly all collected organs in H7N1 basic cleavage site in the HA protein and demonstrated and H5N8 HPAIVs-inoculated chickens that succumbed experimentally by the severe clinical signs and fatal to infection demonstrates the widespread dissemination outcomes observed through the experiment. However, of both HPAIVs. However, the intensity of replication H7N1 and H5N8 HPAIVs differed in the progression of and associated microscopic lesions in the different tissues the disease they caused in chickens. With the highest were virus-dependent, indicating differences in their tis- frequency of prostration and neurological signs, high- sue tropism. Chickens inoculated with H7N1 presented est mortality rates and shortest MDT, H7N1 HPAIV is severe lesions and high viral replication in heart, cen- more virulent to chickens than H5N8 HPAIV. The viral tral nervous system and pancreas. In addition to those shedding pattern also varied between H7N1 and H5N8 observed in H7N1-inoculated birds, inflammatory and HPAIVs. The differences in oral excretion between H7N1 necrotizing lesions associated to intense viral replication and H5N8-inoculated chickens were minor. However, a were detected in the lungs and primary lymphoid organs low number of chickens inoculated with H5N8 HPAIV collected from chickens inoculated with H5N8 HPAIV. presented cloacal excretion. Despite some birds were still Widespread staining was generally detected in lympho- shedding at the end of the study, our findings suggest the cytes in these tissues, indicating a high avidity of H5N8 potential for decreased horizontal transmission efficiency HPAIV for lymphoid cell populations. The cause of death of Gs/GD H5N8 clade 2.3.4.4 B HPAIV among chickens. in the chickens that succumbed to infection after H7N1 In concordance with our results, previous studies dem- and H5N8 HPAIV inoculation appears to be the result onstrate that Italian H7N1 HPAIV exhibit high virulence of the multi-organ replication of these viruses. However, and transmissibility in several galliformes species [20, 25, H5N8 HPAIV presents a reduced neurotropism, a hall- 26], whereas several Gs/GD clade 2.3.4.4 H5Nx reassor- mark of HPAIV pathogenesis, based on the compara- tants (including H5N8 HPAIVs) cause lower mortalities, tively lower amounts of viral antigen and lesions detected longer MDTs and present lower transmissibility in chick- in the brain of H5N8 HPAIV-inoculated chickens in ens compared to their ancestral Gs/GD H5N1 HPAIVs [8, comparison with those inoculated with H7N1 HPAIV. 27–29]. These results confirm the variable pathogenicity The reduced neurotropism could be a reason of the lower and potential transmissibility of HPAIVs of different line- mortalities caused by H5N8 HPAIV. Other mechanisms ages and host-origin in the chicken species. of pathogenicity not evaluated in the present study may Sánchez‑González et al. Vet Res (2020) 51:113 Page 10 of 13 also impact the differences in virulence between H7N1 GD H5N1, H5N6, H5N8 HPAIVs [3, 7–10, 45, 46] and and H5N8 HPAIVs in chickens, including an aberrant the Italian HPAIV H7N1 [6] have been reported. We innate immune response in H7N1 HPAIV-inoculated then evaluated the existence of breed-related differences chickens after infection [32]. in the susceptibility to HPAIVs in a broad range of local Particular amino acids in specific positions of PB2, PB1, chicken breeds, and in two commercial breeds. In the PA, NP, MP, NS1 and NEP/NS2 proteins sequence have present study, four breeds (Empordanesa, Penedesenca, been associated with increased pathogenicity and trans- Catalana del Prat, Flor d’Ametller, Euskal Oiloa) were missibility of HPAIVs in chickens [33–35]. Even closely highly susceptible to HPAIV infection, whereas three related HPAIV isolates differ in their virulence in chick- breeds (Castellana negra, Broiler and SPF chickens) were ens, indicating that few mutations in the viral genome considerably more resistant. The breeds that were more may produce significant biological effects [3]. Although resistant exhibited less frequency of severe clinical signs, there is no evidence of sustained circulation of Spanish lower mortality rates and lower number of animals shed- H5N8 HPAIV in galliformes species, the Italian H7N1 ding virus to both HPAIVs infections than susceptible HPAIV emerged from a LPAIV precursor that had been breeds, demonstrating that the genetic background of circulating in gallinaceous poultry for several months particular chicken breeds confer a higher natural resist- [36]. Therefore, H7N1 HPAIV may present markers of ance to diverse HPAIVs subtypes. Moreover, two of the adaptation and/or virulence in internal and non-struc- highly susceptible breeds presented a higher incidence of tural proteins that are lacking in H5N8 HPAIV. With the cutaneous edema and nervous signs, suggesting that the exception of NS protein, the amino acid identity in inter- clinical presentation may vary to some extent dependent nal and non-structural proteins was high (˃98%). Sev- of the breed. eral amino acid substitutions associated with increased Local chicken breeds are believed to be more resistant virulence of HPAIVs in chickens or chicken-derived cells to disease as a result of the natural selection by autoch- were present in PB2 (123E), PB1 (3 V, 38Y), PA (672L), thonous pathogens and minor artificial selection towards NP (105 V, 184 K), M1 (43 M) and NS1 (106 M, 125D) productive-associated genes [11]. However, almost all the proteins in both H7N1 and H5N8 HPAIVs [34, 35, 37– local chicken breeds included in our study were highly 43]. Only two differing amino acids in the sequence of susceptible to both HPAIVs. This is in concordance with the proteins between H7N1 and H5N8 HPAIVs have previous reports [10], demonstrating that local breeds do been reported to have a biological effect in the chicken not necessarily present an improved resistance to infec- species: 103F and 114S that are present in NS1 protein tious diseases. Since these breeds are usually raised in of H5N8 HPAIV. These mutations are associated with backyards in the absence or few biosecurity measures and inhibition of host gene expression [42]. However, they Spain is located within natural migratory routes between were reported in a LPAIV strain and their effect in vivo Eurasia and Africa, these particular breeds are expected was not evaluated. Therefore, the differences in infection to be highly vulnerable to infection with HPAIVs carried outcome between the two HPAIVs may be due to a sin- by migratory birds. Because of that, local chicken breeds gle or a combination of amino acid substitutions whose could act as sentinels for HPAIV environmental con- effects have not been yet characterized. Since they belong tamination. Despite the susceptibility of local breeds to to different subtypes, the HA and NA surface glycopro- HPAIVs, their role in the global epidemiology of HPAIVs teins are also expected to have played a critical role in the is much less evident. For instance, the role of backyard differential outcomes. Actually, we detected that H7N1 poultry on H5N8 epidemiology was suggested to be lim- HPAIV presented an amino acid substitution in HA ited [47]. (388T, H5 numbering) which is associated with increased The genetics of resistance to HPAIVs remains pathogenicity in chickens [37]. However, the biological unknown. However, particular alleles present in immune implications of the amino acid substitutions reported response-related genes have shown a positive correlation here require further evaluation. H7N1 HPAIV presents with antiviral activity [48, 49]. Mx proteins are induced a stalk deletion in the NA protein, commonly observed by type 1 interferons and interfere with viral functions after a transmission of HPAIV from waterfowls to poul- by inhibiting viral polymerases [50]. The substitution try and a known major virulence determinant of HPAIVs of serine with asparagine at position 631 of Mx protein, in chickens [44]. which is produced by a particular non-synonymous G/A The outcome after infection with HPAIVs is also largely polymorphism in exon 14 of chicken Mx gene, was asso- influenced by host factors. Several reports demonstrate ciated with higher antiviral activity in vitro [48]. In vitro a wide range of susceptibilities to AIVs among breeds and in vivo studies do not always show a clear correla- and lines of chickens. Specifically, significant variations tion between this allele and inhibition of AIV replication in mortalities after experimental inoculation with Gs/ and/or survival after HPAIV infection, respectively [6, Sánchez‑González et al. Vet Res (2020) 51:113 Page 11 of 13 13–16]. Therefore, the impact of this particular amino Empordanesa, P: Penedesenca, C: Catalana del Prat, F: Flor d’Ametller, N: acid substitution is still unclear. In the present study, we Castellana Negra, O: Euskal Oiloa, B: 308 Ross Broiler, S: SPF White Leghorn. evaluated the genotype frequencies of that particular Additional file 2. Average distribution of NP-positive cells and associated polymorphism in the different chicken breeds and evalu- lesions in tissues collected at 3 dpi from different chicken breeds inocu‑ ated their association with infection outcome. Similar to lated with H5N8 HPAIV. n=3/group. -: no positive cells, +: <10% positive cells, ++: 10-40% positive cells, +++: >40% positive cells. E: Empor- in other studies reporting high diversity in genotype and danesa, P: Penedesenca, C: Catalana del Prat, F: Flor d’Ametller, N: Castellana allele frequency in that position between breeds [51], Negra, O: Euskal Oiloa, B: 308 Ross Broiler, S: SPF White Leghorn. we detected huge differences in the frequency of the three genotypes among the spectrum of local, commer- Abbreviations cial and experimental breeds included in our study. As AIVs: avian influenza viruses; BSL-3: biosecurity level 3 containment; c-ELISA: competitive ELISA; CS: cloacal swab; DAB: 3,3′-diaminobenzidine tetrahydro‑ reported previously, resistant and susceptible genotypes chloride; Dpi: day post-infection; ELD50: median embryo lethal dose; FFPE: appear to be the predominant in White Leghorns and formalin-fixed, paraffin-embedded; Gs/GD: Asian-origin goose/Guangdong Broilers, respectively [52, 53]. In contrast, the results in H5 lineage; HA: hemagglutinin; H/E: hematoxilin-eosin; HEPA: high efficiency particulate air; HPAI: highly pathogenic avian influenza; Hpi: hours post- local breeds were more variable, which could be associ- inoculation; HPAIVs: highly pathogenic avian influenza viruses; IHC: immuno‑ ated with the higher genetic diversity generally present in histochemistry; M: matrix protein; MDT: mean death time; NA: neuraminidase; unselected breeds. Overall, the susceptible G allele pre- NP: nucleoprotein; NS: non-structural protein; OIE: World Organization for the Animal Health; OS: oral swab; PBS: phosphate buffered saline; PB1: polymerase vails in the Spanish chickens tested, while the resistant A basic protein 1; PB2: polymerase basic protein 1; PA: polymerase acidic protein; allele was the predominant in two Indian native chicken qRT-PCR: quantitative reverse-transcription PCR; SPF: specific-pathogen free. breeds [54], a native chicken breed from Indonesia [55] Acknowledgements and 2 chicken breeds and 7 strains from Egypt [56]. The Authors thank all the staff of the BSL3 animal facilities for their support in the statistical analyses showed that the different genotypes in vivo experiment and Sebastian Napp from Centre de Recerca en Sanitat in the target Mx region were not associated with signifi- Animal (IRTA-CReSA) for the statistical analyses. cant differences in mortality ratios. However, the birds Authors’ contributions carrying the AA and AG presented a statistically signifi- RSG, ARS and NMM planned, designed the study and interpreted the results. cant longer MDT than those carrying the GG genotype. RSG wrote the manuscript. ARS and NMM revised the manuscript. RSG prepared the inocula and performed the experimental inoculation. RASG, ARS, In concordance with our results, the study carried out by NMM, MNE, NWA, RVG and MPM sampled and performed the necropsies and Ewald et al. [57] observed that chickens homozygous for laboratory analysis. RASG and MPM performed the immunohistochemistries. AA allele presented a delayed MDT. These suggest that All authors read and approved the final manuscript. the presence of an asparagine at position 631 in Mx pro- Funding tein may result in a higher antiviral effect response of Mx This work was supported by the coordinated project RTA2015-00088-C03-03 protein against HPAIVs, but, as shown in our studies, the of the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and FI-DGR Pre-Doctoral Grant from the Agencia de Gestión de Ayudas Universi- biological implications of this change in vivo are probably tarias y de Investigación (AGAUR). limited. This study represents an exhaustive characterization Availability of data and materials Not applicable. of the pathobiology of two HPAIVs in a broad range of chicken breeds. Our results demonstrate that the out- Ethics approval and consent to participate come after infection with HPAIVs is influenced by The experimental design was approved by the ethical Committee of Institut de Recerca i Tecnologia Agroalimentàries (IRTA) and the Government of Cata‑ numerous tightly interconnected factors, including the lonia (Departament de Territori i Sostenibilitat, Direcció General de Polítiques viral isolate, the genetic background of the breed and Ambientals i Medi Natural) under reference code CEEA 18/2017-9457. particular alleles in genes encoding antiviral proteins, Consent for publication underlining the complexity of HPAIV infections. A Not applicable. proper surveillance, education of caretakers and bios- ecurity measures in commercial but also in local chicken Competing interests The authors declare that they have no competing interests. holdings are required to early detect the circulation of HPAIVs in the territory. Author details 1 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA‑UAB), Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, España. 2 Departament Supplementary information de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, Campus Supplementary information accompanies this paper at https​://doi. de la Universitat Autònoma de Barcelona (UAB), Bellaterra, España. org/10.1186/s1356​7-020-00835​-4. Received: 15 June 2020 Accepted: 27 August 2020 Additional file 1. Average distribution of NP-positive cells and associ‑ ated lesions in tissues collected at 3 dpi from different chicken breeds inoculated with H7N1 HPAIV. n=3/group. -: no positive cells, +: <10% positive cells, ++: 10-40% positive cells, +++: >40% positive cells. E: Sánchez‑González et al. Vet Res (2020) 51:113 Page 12 of 13 References influenza A virus (H7N1) infection in chickens inoculated with three dif‑ 1. Swayne DE (2007) Understanding the complex pathobiology of high ferent doses. 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