Complete Genome of Avian coronavirus Vaccine Strains Ma5 and BR-I

ABSTRACT Avian coronavirus (AvCoV) is a ubiquitous multiple-serotype pathogen of poultry, and its control is mainly based on the use of vaccines. We report here the previously unknown full genomes of the Ma5 (27,652 nucleotides [nt]) and BR-I (27,618 nt) AvCoV vaccine strains of the GI-1 (Massachusetts) and GI-11 (Brazil) types.

A variety of vaccine strains derived from the hitherto described 6 genotypes/32 lineages have been used to control the disease (3,4). The availability of full genomes for vaccine strains allows for the search for genetic markers of attenuation, protection, and mutation, as well as for comparison with field strains. Here, we report the previously unavailable full genomes of strains Ma5 (GI-1, Massachusetts type) and BR-I (GI-11, Brazil type).
Total RNA extraction with TRIzol Reagent (Life Technologies, Inc.) and an RNeasy mini kit (Qiagen) was carried out on commercially available live vaccines with each of the two strains after the samples were filtered (0.45 m) and treated with DNase/RNase; random double-stranded cDNAs were synthesized using Superscript III and Klenow exo-DNA polymerase (Life Technologies, Inc.). Libraries were constructed using Illumina kits (Nextera XT Index and Nextera XT DNA), and paired-end reads were sequenced with a NextSeq500 instrument (Illumina) using the NextSeq500 version 2 high-output sequencing kit (300 cycles). The consensus sequences were assembled with CLC Genomics Workbench version 9.5.3 (Qiagen) with AvCoV sequences FJ888351.1 and KX258195.1 as references for Ma5 and BR-I, respectively, and with a read-trimming cutoff of 0.05. A total of 57,827,656 and 132,767,024 paired-end sequences were obtained, from which 9,382,185 and 93,704,684 were mapped for Ma5 and BR-I, respectively.
The resulting genomes for Ma5/BR-I were 27,652/27,618 nucleotides (nt) long, including the poly(A) tails, and organized as 5= UTR (nt 1 to 528/1 to 528), ORF1a (nt 529 to 12324/529 to 12324), and ORF1ab (nt 529 to 20363/529 to 20363), with a ribosomal frameshift between these two ORFs, followed by the spike gene S No recombination was found for the Ma5 and BR-I genomes using a set of AvCoV genomes in RDP4 (5). Nucleotide identities between Ma5 and BR-I were 90.5% for the full genomes and 82.6% for the spike gene. The mean Jameson-Wolf antigenic index (6) for the spike proteins of both viruses was 0.39, though at site 38, to which a role in tropism has been described (7), the index was 2.52 for Ma5 (Asp) and 0.2 for BR-I (Asn).
Because vaccination is a core tool for controlling AvCoV infection and due to the range of variables to be considered for this process (8), the integration of experimental, field, and genomic data could improve the process of developing vaccines and predicting their behavior.
Accession number(s). The complete genomes of strains BR-I and Ma5 have been deposited in GenBank under the accession numbers KY626044 and KY626045, respectively.

ACKNOWLEDGMENTS
This work was funded by CNPq (Brazilian National Board for Scientific and Technological Development) grants 400604/2016-7 and 301225/2013-3 and CAPES (Brazilian Coordination for the Improvement of Higher Education Personnel) grant 1841/2016. We are also grateful to the Swedish Research Links Program (grant 2013-6772).