Development of SYBR green RT-qPCR assay for titrating bivalent live infectious bronchitis vaccines.

Infectious bronchitis (IB) is a highly contagious viral disease of chickens caused by IB virus (IBV) that can cause substantial economic losses in the poultry industry. IBV variant infections have been continuously reported since the initial description in the 1930s. QX-like IBVs are the predominant circulating genotype globally. A homologous QX vaccine has superior protection efficacy compared with that of other available vaccines, and the combination of Massachusetts (Mass)-like and QX-like strains is being used to combat QX-like IBV infections. Inoculation of embryonated chicken eggs is the standard method for the titration of IBV, and the titer is expressed as 50% egg infectious dose (EID50). However, this method cannot effectively distinguish or quantify different genotypic strains in a mixture of different viruses, especially in the absence of neutralizing monoclonal antibodies. In this study, quantitative real-time PCR (RT-qPCR) was applied using specific primers for the QX- and Mass-like strains to quantitate IBV infection and for comparison with the conventional virus titration quantitative method. A strong positive correlation was observed between RT-qPCR cycle threshold values and the different EID50 concentrations. This method was further used to titrate bivalent IB vaccines, and the amount of individual genotype virus was determined based on specific primers. Thus, this RT-qPCR assay may be used as a highly specific, sensitive, and rapid alternative to the EID50 assay for titering IBVs.

Mutations in the Methyltransferase Motifs of L Protein Attenuate Newcastle Disease Virus by Regulating Viral Translation and Cell-to-Cell Spread.

The large (L) polymerase proteins of most nonsegmented, negative-stranded (NNS) RNA viruses have conserved methyltransferase motifs, (G)-G-G-D and K-D-K-E, which are important for the stabilization and translation of mRNA. However, the function of the (G)-G-G-D and K-D-K-E motifs in the NNS RNA virus Newcastle disease virus (NDV) remains unclear. We observed G-G-D and K-D-K-E motifs in all NDV genotypes. By using the infection cloning system of NDV rSG10 strain, recombinant NDVs with a single amino acid mutated to alanine in one motif (G-G-D or K-D-K-E) were rescued. The intracerebral pathogenicity index and mean death time assay results revealed that the G-G-D motif and K-D-K-E motif attenuate the virulence of NDV to various degrees. The replication, transcription, and translation levels of the K-D-K-E motif-mutant strains were significantly higher than those of wild-type virus owing to their altered regulation of the affinity between nucleocapsid protein and eukaryotic translation initiation factor 4E. When the infection dose was changed from a multiplicity of infection (MOI) of 10 to an MOI of 0.01, the cell-to-cell spread abilities of G-G-D- and K-D-K-E-mutant strains were reduced, according to plaque assay and dynamic indirect immunofluorescence assay results. Finally, we found that NDV strains with G-G-D or K-D-K-E motif mutations had less pathogenicity in 3-week-old specific-pathogen-free chickens than wild-type NDV. Therefore, these methyltransferase motifs can affect virulence by regulating the translation and cell-to-cell spread abilities of NDV. This work provides a feasible approach for generating vaccine candidates for viruses with methyltransferase motifs. IMPORTANCE Newcastle disease virus (NDV) is an important pathogen that is widespread globally. Research on its pathogenic mechanism is an important means of improving prevention and control efforts. Our study found that a deficiency in its methyltransferase motifs (G-G-D and K-D-K-E motifs) can attenuate NDV and revealed the molecular mechanism by which these motifs affect pathogenicity, which provides a new direction for the development of NDV vaccines. In addition to the (G)-G-G-D and K-D-K-E motifs of many nonsegmented, negative-stranded RNA viruses, similar motifs have been found in dengue virus, Zika virus, Japanese encephalitis virus (JEV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This suggests that such motifs may be present in more viruses. Our finding also provides a molecular basis for the discovery and functional study of (G)-G-G-D and K-D-K-E motifs of other viruses.

Identification of Novel T-Cell Epitopes on Infectious Bronchitis Virus N Protein and Development of a Multi-epitope Vaccine.

Cellular immune responses play a key role in the control of viral infection. The nucleocapsid (N) protein of infectious bronchitis virus (IBV) is a major immunogenic protein that can induce protective immunity. To screen for potential T-cell epitopes on IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. Four T-cell epitope peptides were identified by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot), intracellular cytokine staining, and carboxyfluorescein succinimidyl ester (CFSE) lymphocyte proliferation assays; among them, three peptides (N211-230, N271-290, and N381-400) were cytotoxic T lymphocyte (CTL) epitopes, and one peptide (N261-280) was a dual-specific T-cell epitope, which can be recognized by both CD8+ and CD4+ T cells. Multi-epitope gene transcription cassettes comprising four neutralizing epitope domains and four T-cell epitope peptides were synthesized and inserted into the genome of Newcastle disease virus strain La Sota between the P and M genes. Recombinant IBV multi-epitope vaccine candidate rLa Sota/SBNT was generated via reverse genetics, and its immune protection efficacy was evaluated in specific-pathogen-free chickens. Our results show that rLa Sota/SBNT induced IBV-specific neutralizing antibody and T-cell responses and provided significant protection against homologous and heterologous IBV challenge. Thus, the T-cell epitope peptides identified in this study could be good candidates for IBV vaccine development, and recombinant Newcastle disease virus-expressing IBV multi-epitope genes represent a safe and effective vaccine candidate for controlling infectious bronchitis. IMPORTANCE T-cell-mediated immune responses are critical for the elimination of IBV-infected cells. To screen conserved T-cell epitopes in the IBV N protein, 40 overlapping peptides covering the entirety of the N protein were designed and synthesized. By combining IFN-γ ELISpot, intracellular cytokine staining, and CFSE lymphocyte proliferation assays, we identified three CTL epitopes and one dual-specific T-cell epitope. The value of T-cell epitope peptides identified in the N protein was further verified by the design of an IBV multi-epitope vaccine. Results show that IBV multi-epitope vaccine candidate rLa Sota/SBNT provided cross protection against challenges with a QX-like or a TW-like IBV strain. So, T-cell-mediated immune responses play an important role in the control of viral infection, and conserved T-cell epitopes serve as promising candidates for use in multi-epitope vaccine construction. Our results provide a new perspective for the development of a safer and more effective IBV vaccine.

Experimental co-infection of variant infectious bursal disease virus and fowl adenovirus serotype 4 increases mortality and reduces immune response in chickens.

Infectious bursal disease virus (IBDV) and fowl adenovirus serotype 4 (FAdV-4) cause infectious bursal disease (IBD) and hydropericardium-hepatitis syndrome, respectively. Recently, studies have reported co-infections of poultry with IBDV and FAdV-4, which is an important problem in the poultry industry. Here, the variant IBDV strain ZD-2018-1 and FAdV-4 isolate HB1501 were used to assess the pathogenicity of co-infection in 1-day-old specific pathogen-free (SPF) chickens. Compared with chickens infected with only FAdV-4, those coinfected with IBDV and FAdV-4 showed enhanced clinical symptoms, higher mortality, more severe tissue lesions, and higher biochemical index levels. Furthermore, the expression of interleukin (IL)-6, IL-1ß, and interferon-γ mRNAs in the IBDV-FAdV-4 coinfected chickens was delayed, and the antibody response levels were significantly lower in those birds compared with the FAdV-4-infected chickens. These results indicate that co-infection with variant IBDV ZD-2018-1 and FAdV-4 HB1501 could significantly promote the pathogenicity of FAdV-4 and reduce the immune response in chickens. This study provides the foundation for further investigation of the interaction mechanism in IBDV and FAdV-4 co-infection.