Infectious Bronchitis Virus-Host Interaction: Viral Spike Protein Cell/Tissue Binding Specificity and Immune Responses
Type of DegreePhD Dissertation
DepartmentGeneral Veterinary Medicine
Restriction TypeAuburn University Users
MetadataShow full item record
The coronavirus avian infectious bronchitis virus (IBV) causes a highly contagious disease in chickens affecting the respiratory tract and, depending on virus strains, reproductive and urogenital tracts. Strains are distinguished and classified based on the amino acid (aa) variation in the S1 domain of spike (S) protein, the major viral attachment protein. The S1 subunit of the IBV spike protein mediates viral attachment and the S2 subunit is involved in fusion to host cells. Although S2 does not have any receptor binding domain, interplay between S1 and S2 synergistically improves host cell attachment. S is the major protein in inducing neutralizing antibodies and in determining host tropism. Adaptation of an ArkDPI IBV vaccine strain to chicken embryonic kidney (CEK) cells eliminates subpopulations that are selected in and cause problems in vaccinated chickens. The CEK-adapted virus induces protective immune responses in chicken, as demonstrated by reducing viral loads and clinical signs after virulent Ark serotype IBV challenge. Three aa changes in the S protein of CEK-adapted virus, two in S1 and one in the S2, were observed. These aa changes in the S protein of CEK-adapted vaccine virus might allow it to attach more efficiently to CEK cells compared to the ArkDPI vaccine strain, thus contributing to adaptation. Contrary to this expectation, we did not observe detectable binding of CEK-adapted S protein to CEK cells at standard protein concentration. Our results suggested that, in this case, factors other than improved attachment to CEK cells were involved in adaptation to CEK cells. We also observed severely reduced or abolished detectable binding of recombinant S protein of CEK-adapted ArkDPI IBV vaccine virus to most relevant chicken tissues in vitro, suggesting lower levels of replication in chickens for CEK-adapted ArkDPI vaccine strain than its parental strain. We compared replication in chickens of the CEK-adapted virus to a commercial ArkDPI-derived IBV vaccine, with only two aa differences in S, after ocular inoculation of 1-day-old SPF leghorn chickens with 104 or 105 EID50 CEK-adapted ArkDPI, or 104 EID50 commercial vaccine virus. Replication of the vaccine viruses in individual chickens was monitored by determining the relative levels of viral RNA in tears 3, 5 and 8-days post-vaccination (DPV) and in choanal and tracheal swabs 5 and 8 DPV. As expected, vaccine viral RNA was consistently statistically significantly lower in all three sample types in chickens inoculated with CEK-adapted vaccine virus compared to the commercial vaccine when the same dose (104 EID50) was used and in tracheal swabs even when ten times the dose was used. However, in spite of substantially lower replication of the CEK-adapted vaccine virus in some tissues in chickens, reflected in lower vaccine viral loads in tears, trachea and choana, it provided effective protection against challenge. IBV variant CalEnt with unusual enteric tropism was believed to have acquired extended tropism due to changes in its S protein. To determine whether this tropism of CalEnt was due to an increased ability of its S protein to bind to the intestinal epithelium, we compared the binding of recombinant S1 proteins derived from CalEnt and Cal99, a typical respiratory IBV variant with an S1 most closely related to CalEnt, to relevant chicken tissues. Contrary to expectations, neither the CalEnt S1 protein, S1-N-terminal domain, nor entire S-ectodomain, showed any binding at the standard protein concentration to respiratory or intestinal tissues. Thus, our results do not support better attachment to intestinal epithelia as a reason for CalEnt’s extended tropism. Bioinformatic analyses of CalEnt S protein sequences suggest that CalEnt’s S2-coding region was acquiredthrough a recombination event and encodes a unique aa sequence at the putative recognition site for the protease that activates the S protein for fusion of host membrane and viral envelope during viral entry. Thus, S2 activation by tissue-specific proteases might facilitate CalEnt entry into intestinal epithelial cells and compensate for poor binding by its S1 protein. Increasing evidence suggested that the practice common in the poultry industry of IBV vaccination early after hatching may not elicit optimal specific immunity and effectively protect chickens. We further investigated the effects of early vaccination on immune responses in chickens primed with ArkDPI-type IBV vaccine at increasing ages followed by booster vaccination. Our results confirmed that IBV vaccination on the day of hatch induces suboptimal IBV immune responses both in the systemic and mucosal compartments. However, booster vaccination seems to overcome poor initial responses. In spite of extensive vaccination against Ark-type IBV, Ark-type IBV continues to cause problems in the poultry industry. To understand how IBV field strains are able to escape IBV vaccine-induced immune responses, we compared reactivity of antibodies in IBV ArkDPI-vaccinated chickens with the vaccine strain virus and an Ark-type IBV isolated from a vaccinated flock. IBV-specific IgA antibody levels in tears and IgA and IgG antibody levels in plasma measured against the field isolate were lower compared to those against the vaccine strain, suggesting immune escape of the field strain from vaccine-induced immune responses. In order to observe whether differences in antibody levels against the vaccine strain and field isolate in vaccinated chickens included different levels of antibodies recognizing the IBV S proteins, ELISA using trimeric recombinant S-ectodomain proteins with S1 domains representing the vaccine strain and field isolate was conducted. Antibodies in both tears and plasma of vaccinated chickens both post-primary vaccination and post-boost recognized the S-ectodomain containing the field strainS1 significantly less (P<0.05) than they recognized the vaccine strain S-ectodomain. Our results were consistent with the prediction that IBV field strains escape the host humoral immune responses through aa changes within the S1 protein.
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