Study On Infection HeLa Cells By Infectious Bronchitis Virus And Identification Of Viral Natural Receptor

Avian Infectious Bronchitis (IB) is a highly contacting viral infection of the domesticfowl (Gallus gallus; more commonly known as the chicken) by infectious bronchitis virus(IBV). It can cause respiratory disease, kidney inflammation with low production andpoor quality of eggs. IBV is a positive-stranded RNA virus, belonging to Coronaviridae.The previous researches indicated IBV replicated only in chicken, like most coronavirusesnaturally infecting only one animal species or, at most, a limited number of closely relatedspecies. In recent years, there is increasing evidence that IBV can infect species of birdother than the chicken. Coronaviruses have been isolated from peafowl (Pavo), guineafowl (Nurnida meleagris), partridage (Alectoris), all of which were being reared in thevicinity of domestic fowl. These viruses were closely related in genome organization toIBV, and the homology in gene sequences exceeded 99% in some strains. Moreover, somecoronaviruses have been isolated from non-gallinaceous birds, the teal (Anas) and greylaggoose. These coronaviruses are not closely related in terms of gene sequences to IBV. Andas a result, they are clearly not simply isolates of IBV, but represent new coronavirusspecies.The phenomenon of IBV is not limited to replicating in a single host species, whichwas highlighted by the spread of SARS-coronavirus from mammals, like civet cat andbat.Individual coronaviruses had often been described as infecting their hosts in aspecies-species manner. The emergence of SARS-CoV and fragments of somecoronaviruses cross-species infecting make it clear that we should not assume that a givencoronavirus species is limited to replication in a single host. It is necessary to probe intorelationship between animal coronaviruses and SARS-CoV, and the mechanism ofcoronavirus carrying out cross-species infection.Most IBV strains grow poorly in cell culture except in primary chicken kidney cells.Studies on IBV growth in cell culture would be essential to understand the interactionbetween the coronaviruses and cells, and mechanism of coronavirus cross-speciesinfection. In this thesis, the investigation was undertaken to elucidate the potential factorsthat involve in IBV infection of HeLa cells, and identify the natural receptor of IBV. Themain projects are:1: IBV propagation in HeLa cellsAllantoic fluid of IBV strains including M41, H52, H120, Gray, Holte, Connecticutand M52-19 (a Beaudette52-19 strain) containing IBV 5×10³⁰⁴ EID50 was inoculated intoconfluent HeLa monolayers or freshly digested HeLa with 0.25% trypsin. All of them could not propagate in HeLa cell monolayers. However, the strains M41, H52, H120 andGray, but not Holte, Connecticut and M52-19, could propagate in freshly digested HeLacells. Cytopathic effect was characteristic of cell rounding, congregating and detachingfrom the cellular sheets, was produced in infected cells. The infection were verified fromneutralization test, alignment sequences of S1 gene from several IBV passages,haemagglutination and haemagglutination-inhibition test, detection of viral N protein byWestern blotting and so on.2: Mutation in S1 gene contributing to the infection of IBVReverse transcription PCR (RT-PCR) was performed to amplify the spike (S1) gene(1025bp) of IBV after different passages. S1 sequences for AY561712 (GenBank), P0,and P5 remained the same. However, one nucleotide mutation was found in P21 S1leading to one amino acid replacement. In our test, the mutation in S1 gene seemed notthe major reasons responsible for IBV tropism switch.3: Relationship between HA activity and cross-species infection of IBVAfter treated with lecithinase C, the strains M41, H52, H120 and Gray, but notHolte, Connecticut and M52-19, could haemagglutinate 0.5% chicken erythrocytes.We also found the viral HA activity to chicken erythrocytes was significantlydecreased with increased passages in HeLa cells. When the HeLa adapted IBV wasinoculated back to the chicken embryo, IBV HA activity was restored. It seemed that thestrains, which have HA activity, were easier to be adapted in more species cells.4: Sialic acid was involved into IBV infecting HeLa cellsSialic acid can serve as a receptor for Group 2 coronavirus, e.g. BCoV andHCoV-OC43. Neuraminidase could remove sialic acid from cellular surface and thusblock the attachment of these viruses to host cells. Our results agreed with these findings.When the freshly dispersed HeLa cells were treated with NA, the virus TCID₅₀ wasreduced by 13 fold compared with those untreated HeLa. Furthermore, the HA activity ofIBV is determined by the capability of the virus to attach to sialic acid on the surface ofred blood cells. These results denoted that binding to sialic acid may be useful for IBV toinfect cells.5: The function of Aminopeptidase N and trypsin in IBV cross-species infectionA number of group 1 coronaviruses require the aminopeptidase N (APN) for entryinto their target cells, and trypsin was reported to be able to markedly enhance infectivityof various viruses in vitro. In our experiments, a monoclonal antibody, WM15, could notblock IBV infection of human HeLa ceil line. The trypsin also could not raise the titer ofIBV. 6: Identify natural receptor of IBVFeline APN had been reported to serve as a receptor for Ark99 strain of IBV. Wecloned full length chicken (Gallus gallus) APN (gAPN) gene, and approached thepossibility of gAPN acting as a receptor for IBV.The gAPN were cloned into prokaryotic (pET-28a) and eukaryotic (PCI-neo)expression vectors. The recombinant protein were purled from prokaryotic bacteria hadactivity of binding to IBV in ELISA and Western blotting test, which could be blocked byantiserum against gAPN.HeLa and PK-15 cell monolayers that do not permit natural infection by IBV weretransfected with gAPN-neo plasmid. The transfected and non-transfected cells wereinoculated with IBV and viral binding and replication were determinded by indirectflurescent assay, flow cytometry and semi-quantity RT-PCR. The results indicated thatthe insensitive cells were permissive to IBV after transfection with a gAPN cDNA,suggesting the gAPN plays an essential role in IBV entry.