Rational Design Of Avian Metapneumovirus Live Attenuated Vaccine Candidates By Inhibiting Viral Messenger RNA Cap Methyltransferase

Avian metapneumovirus (aMPV), previously known as Avian pneumovirus (APV), also called Turkey rhinotracheitis virus (TRTV) belongs to the family of Paramyxoviridae, the subfamily Pneumovirinae, and the genus Metapneumovirus. AMPV is an important pathogen that causes an acute respiratory disease in turkeys and is the etiological agent of "swollen head syndrome" in chickens. Since the first isolation of aMPV in South Africa in 1978, the virus has become prevalent worldwide. Avian metapneumovirus (aMPV) is non-segmented negative-sense RNA virus, which possess a unique mechanism for mRNA cap methylation. Messenger RNA processing is the essential issue in Human metapneumovirus (hMPV) and aMPV gene expression and replication. During viral RNA synthesis, both hMPV and aMPV produce capped, methylated, and polyadenylated mRNAs. Capping and methylation of viral mRNA are essential for mRNA stability, efficient translation, and gene expression. The large (L) polymerase protein contains all the enzymatic activities for mRNA processing, including capping, cap methylation, and polyadenylation. Sequence alignments between representative Non-segmented Negative-strand virus (NNS) RNA virus identified six regions of conservation in the L protein (CRI-CRVI) separated by regions of lower sequence homology. conserved region VI in the large (L) polymerase protein catalyzes both guanine-N-7 (G-N-7) and ribose 2’-O (R-2’-O) methylatransferases, and the two methylase activities share a binding site for the methyl donor S-adenosyl methionine(SAM).In this study, we generated a panel of recombinant aMPV (raMPV) Colorado strainscarrying mutations in SAM binding site in the CR-VI of L protein. The vitro trans methylation assay showed that these recombinantat aMPV mutants are specifically defective in ribose 2’-O (R-2-O1), but not in guanine-N-7 (G-N-7) methylation. Besides, the further two-step trans-methylation assay suggested that the G-N-7 methylation of aMPV facilitates R-2-O methylation. These MTase-defective recombinan aMPV mutants showed delayed CPE and formed smaller plaques in cell culture compared to the wildtype aMPV. Therefore, recombinan aMPV mutants were highly attenuated since the ribose 2’-O methylation was abolished.To determine whether recombinant aMPVs are attenuated in animals and if it can be used for live vaccine candidates, two-week-old SPF turkeys were were inoculated oculonasally with 5*10 5 pfu of raMPV-G1696A, raMPV-G1700A, raMPV-N1701A or raMPV-D1755A. The results showed that MTase-defective raMPVs were attenuated in viral replication in the upper and lower respiratory tracts of turkey. Importantly, turkeys immunized with raMPV-G1696A, raMPV-G1700A triggered a high level of neutralizing antibody and were protected from challenge with aMPV Colorado wild type strain or aMPV Minnesota wild type strain.Collectively, our results indicate that (ⅰ) aMPV lacking 2’-O methylation is highly attenuated in vitro and in vivo, and (ⅱ) inhibition of mRNA cap MTase can serve as a novel target to rationally design live attenuated vaccines for aMPV, and perhapsother paramyxoviruses.