Studies On Molecular And Pathogenic Characteristics Of Avian Infectious Bronchitis Virus(QX-Type)

Infectious bronchitis(IB),caused by avian infectious bronchitis virus(IBV),is a prominent disease in poultry and has posed significant worldwide economic losses.Th e IBV is a prototype of Gammacoronavirus which is related to Betacoronavirus severe acute respiratory syndrome coronavirus-2(SARS Co V-2)causing COVID-19 in human.These genera belong to family of Coronaviridae and coronaviruses(Co Vs),have extensive host range include human,animals,birds and rodents.The spike glycoprotein(S)has very important biological functions and Co Vs variants have been characterized on the basis of S gene analysis.As RNA virus,IBV has the characterization of constant mutation and recombination within different serotypes.This makes the diagnosis and control of IB more and more difficult.So,the present study was carried out to assess the molecular and pathogenic characteristics of IBV isolate HH06,its evolutionary and recomb ination mechanism.In addition,we conducted phylogenetic,motif and recombinant network analysis to compare Co Vs of 4 genera including SARS Co V-2 strain of Wuhan-Hu-1 and IBV-HH06.Through current study,we may better cognize the epidemic evolution of IBV isolates,mechanism of pathobiology and tissue tropism of emerging viruses and provide a theoretical basis for the development of more effective vaccines for the betterment of poultry industry.In this study,we first isolated and identified a IBV strain(HH06).Briefly,the purification and propagation of isolate was done by the three times passage in the allantoic cavity of 9-day-old SPF embryonated chicken eggs(ECE),and distinctive IBV characteristics,likewise dwarfing,hemorrhages,curling or stunti ng of embryos were observed.Reverse transcription-polymerase chain reaction(RT-PCR)was established for amplification and sequencing of IBV variant HH06.Three sets of primers were used to amplify complete S gene,its S gene phylogenetic,recombinant net work,evolutionary mechanism was assessed.Furthermore,histopathological and immunohistochemistry(IHC)analysis were performed.Phylogenetic results showed IBV HH06 strain clustered in genotype QX(G1-19),and was closely branched with G1-18(LDT3-A and G1-22(LSC/991),whereas it was at large distance when compared to classical and vaccine strains like M41,H120 and4/13 strains.Recombination network tree indicated HH06 was closely associated with G1-18 and TW-1 type viruses,and recombination might hap pen among these strains.Selection pressure analysis demonstrated that there were 35 positively selected residual sites,and mainly attributed to the purifying selection.Keeping in mind the current pandemic SARS-Co V-2,the Co Vs has gained more attention to further study at bioinformatics on the basis of S gene,hence we selected 111 Co Vs from 4 genera(Alphacoronaviruses(Alpha-Co Vs),Betacoronaviruses(Beta-Co Vs),Gammacoronaviruses(Gamma-Co Vs)and Deltacoronaviruses(Delta-Co Vs).We also included HH06 isolate of IBV of avian origin to asses the intra species transmission among different avian species.Phylogenetically,The IBV HH06 of Avian-Co Vs was closely related to Duck-Co V and partridge S14,LDT3(Teal and chicken host).The avian Co Vs(Ph-Co V and Pi-Co V)of pheasant and pigeon origin branched closely in the same group.Beluga whale Co V(SW1)and Bottlenose dolphin-Co Vs of mammalian origin branched at distant from other animal origin viruses,however making group with Avian-Co Vs altogether into Gamma-Co Vs.SARS Co Vs-2,SARS-Co Vs clustered with Bat-Co Vs and MERS-Co V of Beta-Co Vs(C)were closely associated.The motif analysis indicated well conserved domains on S protein which were similar within the same phylogenetic class and variable at different domains of different origin.Further,chickens were infected with IBV strains of HH06 and M41 at the age of 14 days and inspected twice a day to record the clinical signs.Five chicks from each group were slaughtered on days 1,3,5,7,10 and 14 post-infection.At necropsy,macroscopically changes were observed on the killed chickens.Different specimen(trachea,lung,kidney,bursa of Fabrcius,spleen,heart and liver for haematoxylin and eosin(H&E)staining,and trachea,lung,kidney,bursa of Fabrcius,spleen for immunohistochemical assay(IHC),and trachea,lung,kidney for quantitative RT-PCR(q RT-PCR)of IBV in chickens were collected,respectively.The results revealed pathological lesion variations in trachea,lung and kidney of these 2 IBV strains.The HH06 caused early onset of clinical observation as compared to M41 group.However,both infected group express depression,ruffled feathers,minor rales from trachea and lung.The infected birds were lethargic and reluctant to move,with watery diarrhea at peak time of the disease.Two birds died at days post-infection(dpi)5 and 6respectively in group infected with HH06 strain,while M41 strain caused mortality of two birds at dpi 6 and 7 respectively.Grossly,congestion and excessive mucus was det ected in tracheal and lung,along with swollen kidney and hyperemic renal tubules.The IBV HH06 strain caused severe lesion of kidney with nephroso-nephritis as compared to M41.Histological assessment elaborated that HH06 and M41 strains caused severe histopathological changes in the different organs and extensive tissue tropism was observed.The progression of the lesions with time could be divided into degenerative,hyperplastic and recovery stages.In terms of trachea and lung,the severity of lesions w as greater in M41 as compared to HH06 infected birds,however IBV HH06 efficiently reached to renal system and replicated as compared to respiratory organs.Despite of the classical changes in trachea and lung,kidney appeared more severe lesions of renal tubular epithelia degeneration and necrosis,infiltration of inflammatory cells.Additionaly,it has caused noticeable pathological alteration in microarchitecture of bursa of Fabricius,spleen,heart and liver of infected chickens.Furthermore,IHC analys is demonstrated viral antigen intensity was higher in kidney compared to trachea and lung in HH06 infected birds,however M41 infected birds showed larger presence of antigen in respiratory organs.In HH06 infected birds,increased detection of IBV antigen was observed at dpi 5 and positive cells mainly distributed in the mucosal layer,submucous layer.Whereas M41 infected birds showed tracheal positive cells significantly increased at dpi 7 as compared to HH06.Viral antigen detection in the kidney was significantly higher in HH06 infected group as compared to M41 in the kidney,mainly renal tubular epithelial cells.IBVs copies in trachea,lung and kidney were analyzed by q RT-PCR and viral m RNA copy numbers in the tissues of chicks inoculated with M41 str ain were higher in the trachea and lung than those of infected chicks with HH06 strain,with the exception of the kidney.Regarding of T cell subset assay,in HH06 and M41 infected chicks,CD3+ and CD8+cells were present in trachea,lung and kidney at dp i 5 and 7 respectively indicating immune response.Relative expression of immune system related genes the interferons IFN-a and IFN-? in trachea and kidney of HH06 infected group were significantly higher at dpi 3,where M41 infected trachea showed increas ed expression level at dpi5.TLR 3 and MDA5 were significantly expressed at dpi 1 and 3 in trachea and kidney of HH06 infected group,whereas M41 infected chicken,tracheal and kidney TLR3 and MDA5 were expressed at dpi 5.Proinfilematoery cytokines IL-1? and IL-6 were higher in trachea and kidney at dpi 3 and 5of HH06 infected group and at dpi 5 and 7 in M41 infected group.In general,IBV HH06 strain has certain genetic characteristics,immune response,tissue tropism mainly from respiratory organs to r enal organs indicating of its nephropathogenic form.However,the classical M41 has tropism to respiratory tissues.The IBV of coronavirdae have ability of intra-host(among poultry species)transmission and replication and is constantly undergoing evoluti onary process,and should be paid more attention to be control.