Isolation and Molecular Identication of Respiratory Diseases Viruses in Poultry During 2020

Poultry production has affected by multiple respiratory disease triggering serious economic losses in Egypt. In this study, the situation and genetic evolution of respiratory disease in Egypt during 2020 were studied. We collected 53 samples from infected ocks suffered from respiratory signs and variable mortality rate from nine governorates in Egypt during 2020. The collected samples were examined for detection of respiratory disease viruses (Avian inuenza virus (AIV), Infectious bronchitis virus (IBV), and Newcastle disease virus (NDV)) by rRT-PCR. The single infection was conrmed in 90.6% (37.7% I. B, 30.2% AIV (H5N8), 9.4% I. B and 5.7% NDV) and co-infection of HPAIV (H5N8) + I.BV and LPAIV (H9N2) +IBV were detected in 3.8% in nine governorates. The HA gene of HPAIV (H5N8) were cluster to clad 2.3.4.4.1b in new branch with characteristic specic mutations especially in T140A in antigenic site A and R72S in the receptor binding site when comparing with A/duck/Egypt/F446/2017 with low A.A identity percent with vaccinal strains (H5N1 and H5N2) reach to 91.9-94% and 84.6% respectively. The HA gene of AIV (H9N2) were belong to A/quail/Hong Kong/G1/97-like virus clustered with group B with specic mutation (212I) that may be effect on human transmission of the virus. The HVRs of S1 gene of IBV cluster to GI23 (Egy Var I) clad with multiple mutation in HVR1, HVR2 when comparing with IBV/CU/4/2014 and low identity percent (68.3%-78.8%) with vaccine strains (H120, M41, 4/91). In conclusion, the respiratory disease continues circulate and rapidly evolved in Egypt during 2020.


Introduction
The poultry industry is one of the most signi cant industries impacting the national economy in Egypt by promoting investment and jobs availability. Moreover, the primary sources of inexpensive animal protein for poor people in Egypt are poultry meat and eggs (  ). In addition, the co-infection with other bacterial infection as Escherichia coli and Haemophilus paragallinarum contribute to increase the virulence of H9N2 because it has endo proteases enzyme that cleavage precursor HA proteins (Haghighat-Jahromi et al. 2008;Pan et al. 2012). IBV that causing air saculitis in chickens is one of the major signi cant respiratory infections (S. Hofstad 1984). It is primarily recorded in Egypt in 1950s (Sheble et al. 1986

Materials And Methods
Collection and preparation of specimens In this study, AIV (H5N8 and H9N2), NDV and IBV were examined for 53 infected poultry ocks exhibiting severe respiratory manifestation and high mortality during 2020. The oropharyngeal swabs were obtained from disease or freshly dead birds in nine provinces of Egypt including Alexandria, Giza, Dakhalia, El-sharqia, Qualiobia, El-Mono a, El-Behira, Domiat and Cairo (Table 1

Virus isolation
The positive samples were isolated into SPF embryonated chicken eggs (ECEs) 10 days old in allontoic uid with daily observation. The mortalities were recorded and allonotic sac was collected and tested by rapid slide hemagglutination (HA) test (OIE 2008).

Sequencing of the viral genome
The HA gene of AI (H5, H9) and HVRs of S1 gene of IBV ampli cation were carried out by PCR using speci c primers as described by Höper Table 2.

Real time-PCR
The 48 samples/ 53 were positive for RT-PCR for different respiratory disease (twenty samples were positive for IBV, sixteen samples for HPAI (H5N8), ve samples for LPAI (H9N2), three samples for NDV (velogenic strain), two samples for HPAI (H5N8) +IBV and two samples for LPAI (H9N2) + I.BV as mentioned in Table.1. Genetic characterization of the H9N2 Virus HA gene The phylogenetic analysis of full HA sequence of three selected AI H9 isolate was named A/ CHICKEN/EGYPT/FW1/2020 from co-infected farms and A/ CHICKEN/EGYPT/FW2/2020 and A/ CHICKEN/EGYPT/FW3/2020 from single infected ocks was belonged to the A/quail/Hong Kong/G1/97like virus lineage clustered with group B (Fig.3). The viruses in our study were closely related to other Egyptian strains with identities of 94.9-98.2% (Fig.4). By mutation analysis we recorded mutation in S16N, M58K, T121I/V, I134M, T145S, N179T as speci c to all Egyptian viruses and N41G, I75V, V212I, T413N resemble to Egyptian viruses in 2018-2019 when comparing with the A/quail/Hong Kong/G1/97.
In addition, R180K/Q speci c for viruses in this study. The amino acid sequences at the cleavage site of HA contained low pathogenic RSSR/GLF motif.

Discussion
In Egypt during the last years, the outbreaks due to respiratory disease with high mortality rates has increased and effect on the poultry production in Egypt. Many avian viral pathogens were the main cause mutation speci c for Egyptian viruses. In addition, I75V, N41G and T413N mutation speci c to 2018-2019 Egyptian viruses and the 212I mutation that has important effect on increased the pathogenicity of the H9N2 in mice as previously recorded in Abozeid et al. (2017). We need further studied to study their effect on the pathogenicity of the virus. In this study, we recorded high number of infected vaccinated ocks with IBV viruses by RT-PCR but it does not distinguish between eld and vaccine strains of IBV. So partial S1 gene sequencing is important to identify the IBV strains. Furthermore, the partial IBV S1 gene was sequenced and analyzing reveal that six selected viruses of single and co-infected I.B were related to clad GI23 (Egy Var I) clad as previously recorded ( In conclusion IBV and HPAIV (H5N8) are the main causes of respiratory disease in Egypt alone or co-infected with other viruses with high mortality rate. Circulation of AI (H5N8) was detected with mutation in the antigenic site and receptor binding site, LPAI (H9N2) with speci c mutation and IBV viruses were rapidly evolved in HVRS. the IBV and HPAI H5N8 viruses was distinct from vaccine strains so it is important to surveillance of respiratory disease viruses and study the genetic evolution and its effect on pathogenicity and vaccine e cacy.

Con ict of interest
Authors declare that they have no con ict of interest.

Funding
The current work was funded by Taif University, Saudi Arabia, for nancial support through its Researchers Supporting Project (TURSP-2020-105).

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participation
The authors con rm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.    Amino acid identities and divergence of HA gene of H5N8 viruses compared to other selected strains and vaccinal strains. FN: Amino acid identity of HA gene of H5N8 viruses showed that A.A. identity percent of Egyptian H5N8 strain in this study ranged from91.9-94% and 84.6% with H5N1 and H5N2 vaccine respectively.   Phylogenetic tree of partial S1 gene of IBV. FN: The gure shows the phylogenitic analysis of HA gene of AIV H9N2 reveling that all Egyptian strains cluster were cluster to clad GI23 (Egy Var I). The IBV viruses in our study are indicated with a black dot.

Figure 6
Amino acid mutation in HVRS in S1 gene of IBV. FN: The HVR1 had 3,4,2 A.A. mutations in three, two and one samples in this study respectively. The HVRII had 7A.A. mutation in all viruses except two viruses had 6 and 4 mutations. Amino acid identities and divergence of partial S1 gene of IBV sequenced viruses compared to other selected strains and vaccinal strains. FN: Amino acid identity of partial S1 gene of IBV viruses showed