Deletion of accessory genes 3a, 3b, 5a or 5b from avian coronavirus infectious bronchitis virus induces an attenuated phenotype both in vitro and in vivo.

Avian coronavirus infectious bronchitis virus (IBV) infects domestic fowl, resulting in respiratory disease and causing serious losses in unprotected birds. Its control is mainly achieved by using live attenuated vaccines. Here we explored the possibilities for rationally attenuating IBV to improve our knowledge regarding the function of IBV accessory proteins and for the development of next-generation vaccines with the recently established reverse genetic system for IBV H52 based on targeted RNA recombination and selection of recombinant viruses in embryonated eggs. To this aim, we selectively removed accessory genes 3a, 3b, 5a and 5b individually, and rescued the resulting recombinant (r) rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b. In vitro inoculation of chicken embryo kidney cells with recombinant and wild-type viruses demonstrated that the accessory protein 5b is involved in the delayed activation of the interferon response of the host after IBV infection. Embryo mortality after the inoculation of 8-day-old embryonated chicken eggs with recombinant and wild-type viruses showed that rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b had an attenuated phenotype in ovo, with reduced titres at 6 h p.i. and 12 h p.i. for all viruses, while growing to the same titre as wild-type rIBV at 48 h p.i. When administered to 1-day-old chickens, rIBV-Δ3a, rIBV-Δ3b, rIBV-Δ5a and rIBV-Δ5b showed reduced ciliostasis in comparison to the wild-type viruses. In conclusion, individual deletion of accessory genes in IBV H52 resulted in mutant viruses with an attenuated phenotype.

Recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens.

Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens, causing severe economic losses in poultry industry worldwide. Live attenuated viruses are widely used in both the broiler and layer industry because of their efficacy and ability to be mass applied. Recently, we established a novel reverse genetics system based on targeted RNA recombination to manipulate the genome of IBV strain H52. Here we explore the possibilities to attenuate IBV in a rational way in order to generate safe and effective vaccines against virulent IBV (van Beurden et al., 2017). To this end, we deleted the nonessential group-specific accessory genes 3 and/or 5 in the IBV genome by targeted RNA recombination and selected the recombinant viruses in embryonated eggs. The resulting recombinant (r) rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab could be rescued and grew to the same virus titer as recombinant and wild type IBV strain H52. Thus, genes 3ab and 5ab are not essential for replication in ovo. When administered to one-day-old chickens, rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab showed reduced ciliostasis as compared to rIBV H52 and wild type H52, indicating that the accessory genes contribute to the pathogenicity of IBV. After homologous challenge with the virulent IBV strain M41, all vaccinated chickens were protected against disease based on reduced loss of ciliary movement in the trachea compared to the non-vaccinated but challenged controls. Taken together, deletion of accessory genes 3ab and/or 5ab in IBV resulted in mutant viruses with an attenuated phenotype and the ability to induce protection in chickens. Hence, targeted RNA recombination based on virulent IBV provides opportunities for the development of a next generation of rationally designed live attenuated IBV vaccines.

A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination.

BACKGROUND: Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines. METHODS: We established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3'-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs. RESULTS: Targeted RNA recombination allowed for the modification of the 3'-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52. CONCLUSIONS: Targeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general.

Proper Timing of Foot-and-Mouth Disease Vaccination of Piglets with Maternally Derived Antibodies Will Maximize Expected Protection Levels.

We investigated to what extent maternally derived antibodies interfere with foot-and-mouth disease (FMD) vaccination in order to determine the factors that influence the correct vaccination for piglets. Groups of piglets with maternally derived antibodies were vaccinated at different time points following birth, and the antibody titers to FMD virus (FMDV) were measured using virus neutralization tests (VNT). We used 50 piglets from 5 sows that had been vaccinated 3 times intramuscularly in the neck during pregnancy with FMD vaccine containing strains of FMDV serotypes O, A, and Asia-1. Four groups of 10 piglets were vaccinated intramuscularly in the neck at 3, 5, 7, or 9 weeks of age using a monovalent Cedivac-FMD vaccine (serotype A TUR/14/98). One group of 10 piglets with maternally derived antibodies was not vaccinated, and another group of 10 piglets without maternally derived antibodies was vaccinated at 3 weeks of age and served as a control group. Sera samples were collected, and antibody titers were determined using VNT. In our study, the antibody responses of piglets with maternally derived antibodies vaccinated at 7 or 9 weeks of age were similar to the responses of piglets without maternally derived antibodies vaccinated at 3 weeks of age. The maternally derived antibody levels in piglets depended very strongly on the antibody titer in the sow, so the optimal time for vaccination of piglets will depend on the vaccination scheme and quality of vaccine used in the sows and should, therefore, be monitored and reviewed on regular basis in countries that use FMD prophylactic vaccination.

Intratypic heterologous vaccination of calves can induce an antibody response in presence of maternal antibodies against foot-and-mouth disease virus.

BACKGROUND: Maternal antibodies can interfere with foot-and-mouth disease vaccination. In this study we determined whether intratypic heterologous vaccination could help to improve herd immunity. RESULTS: In unvaccinated calves, a half-life of maternal antibodies of 21 days was determined. At two weeks of age, calves without maternal antibodies showed a good antibody response against both vaccines used in the trial, while in calves with maternal antibodies no antibody response to homologous vaccination (A Turkey 14/98) but a limited antibody response to intratypic heterologous vaccination (A22 Iraq) was observed. CONCLUSION: Two weeks old calves without maternal antibodies respond well to vaccination, but when emergency vaccination is carried out in a region that uses prophylactic vaccination, using an intratypic heterologous vaccine strain may improve the immunity in calves with maternal antibodies.

Foot-and-mouth disease vaccine potency testing: the influence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the dose-response curve in cattle.

The aim of this study was to determine a relationship between vaccine potency (amount of PD50 per dose) and fraction of clinically protected cattle following homologous challenge with infectious foot-and-mouth disease (FMD) virus, and to determine the effect of method of fractionation, serotype, type of adjuvant, valency and type of virus culture on the dose-response curve. Data from 297 potency tests of FMD vaccines, comprising 4004 vaccinated cattle, performed at the FMD vaccine production facility in the Netherlands, were used for the present study. A generalised linear mixed effect model was used to analyse the results. Our study showed that the relation between FMD vaccine potency and fraction protected was also affected by the serotype and type of adjuvant. No common level of protection could be assigned to all FMD vaccines with the same amount of PD50 per dose, this information is essential when designing a new standard FMD vaccines control.

Cedivac-FMD can be used according to a marker vaccine principle.

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine. All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD(50) vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7-14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of 'marker vaccine' applies.

Comparable sensitivity and specificity in three commercially available ELISAs to differentiate between cattle infected with or vaccinated against foot-and-mouth disease virus.

Three commercially available ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus (FMDV) were evaluated, using sera from uninfected, vaccinated, infected, inoculated, first vaccinated and subsequently infected, and first vaccinated and subsequently inoculated cattle. We compared antibody kinetics to non-structural proteins, sensitivity, and specificity. One of the ELISAs had a higher sensitivity and much lower specificity than the other two, therefore we established standardised cutoff values for the compared assays using receiver operated characteristic (ROC) curves. Using the standardised cutoff values, all three ELISAs produced comparable results with respect to sensitivity and specificity. Antibody development to non-structural proteins after infection and after vaccination/infection was not significantly different. Development of antibodies, however, both neutralising and directed to non-structural proteins, was significantly delayed after intranasal inoculation as compared to intradermolingual infection. Based on results of sera obtained after vaccination and experimental infection all three assays can be used for testing sera collected between 4 weeks and 6 months after infection. More information is needed on the prevalence of positive reactors in a situation where emergency vaccination has been used and FMD transmission was still observed.

A solid-phase blocking ELISA for detection of type O foot-and-mouth disease virus antibodies suitable for mass serology.

A simple solid-phase blocking ELISA for the detection of antibodies directed against type O foot-and-mouth disease virus (FMDV) was developed. The ELISA was validated using field sera collected from cattle, pigs and sheep originating from FMDV infected and non-infected Dutch farms, reference sera obtained from the World Reference Laboratory for foot-and-mouth disease at the Institute for Animal Health, Pirbright Laboratory, UK and sera from experimentally infected animals. Testing 2664 sera collected from non-infected cattle, pigs and sheep resulted in a specificity of 96%. A sensitivity relative to the virus neutralisation test (VNT) of >99% was achieved when testing 148 positive cattle, goat and sheep sera collected from FMDV-infected Dutch farms. All international reference sera scored consistently correct. The ELISA also correctly scored 398 of 409 positive experimentally derived sera. The sensitivity and specificity of this monoclonal antibody-based ELISA for detection of type O FMDV antibodies is sufficient for use as a screening ELISA. During the 2001 epidemic in the Netherlands, 8000 serum samples per day were regularly tested in this ELISA. The samples scoring positive were then tested by neutralisation for confirmation thus making optimum use of the neutralisation testing capacity.