Extracting turkey coronaviruses from the intestinal lumen of infected turkey embryos yields full genome data with good coverage by NGS.

Currently, turkey coronaviruses (TCoV) are isolated from homogenized intestines of experimentally infected embryos to ensure a maximum recovery of viral particles from all components of the intestines. However, the process of homogenization also ensures release of a significant amount of cellular RNAs into the sample that hinders downstream viral genome sequencing. This is especially the case for next generation sequencing (NGS) which sequences molecules at random. This characteristic means that the heavily abundant cellular RNA in the sample drowns out the minority viral RNA during the sequencing process and, consequently, very little to no viral genome data are obtained. To address this problem, a method was developed, in which 10 descendent isolates of the European strain of TCoV were recovered uniquely from the intestinal lumen without homogenization of the tissue. For nine out of 10 samples, NGS produced viral RNA reads with good coverage depth over the entire TCoV genomes. This is a much-needed new, simple and cost effective method of isolating TCoV that facilitates downstream NGS of viral RNA and should be considered as an alternative method for isolating other avian enteric coronaviruses in the interest of obtaining full-length genome sequences.

Infectious Bronchitis Coronavirus: Genome Evolution in Vaccinated and Non-Vaccinated SPF Chickens.

Infectious Bronchitis virus (IBV) continues to cause significant economic losses for the chicken industry despite the use of many live IBV vaccines around the world. Several authors have suggested that vaccine-induced partial protection may contribute to the emergence of new IBV strains. In order to study this hypothesis, three passages of a challenge IBV were made in SPF chickens sham inoculated or vaccinated at day of age using a live vaccine heterologous to the challenge virus. All birds that were challenged with vaccine heterologous virus were positive for viral RNA. NGS analysis of viral RNA in the unvaccinated group showed a rapid selection of seven genetic variants, finally modifying the consensus genome of the viral population. Among them, five were non-synonymous, modifying one position in NSP 8, one in NSP 13, and three in the Spike protein. In the vaccinated group, one genetic variant was selected over the three passages. This synonymous modification was absent from the unvaccinated group. Under these conditions, the genome population of an IBV challenge virus evolved rapidly in both heterologous vaccinated and non-vaccinated birds, while the genetic changes that were selected and the locations of these were very different between the two groups.

Viral variant visualizer (VVV): A novel bioinformatic tool for rapid and simple visualization of viral genetic diversity.

Here a bioinformatic pipeline VVV has been developed to analyse viral populations in a given sample from Next Generation Sequencing (NGS) data. To date, handling large amounts of data from NGS requires the expertise of bioinformaticians, both for data processing and result analysis. Consequently, VVV was designed to help non-bioinformaticians to perform these tasks. By providing only the NGS data file, the developed pipeline generated consensus sequences and determined the composition of the viral population for an avian Metapneumovirus (AMPV) and three different animal coronaviruses (Porcine Epidemic Diarrhea Virus (PEDV), Turkey Coronavirus (TCoV) and Infectious Bronchitis Virus (IBV)). In all cases, the pipeline produced viral consensus genomes corresponding to known consensus sequence and made it possible to highlight the presence of viral genetic variants through a single graphic representation. The method was validated by comparing the viral populations of an AMPV field sample, and of a copy of this virus produced from a DNA clone. VVV demonstrated that the cloned virus population was homogeneous (as designed) at position 2934 where the wild-type virus demonstrated two variant populations at a ratio of almost 50:50. A total of 18, 10, 3 and 28, viral genetic variants were detected for AMPV, PEDV, TCoV and IBV respectively. The simplicity of this pipeline makes the study of viral genetic variants more accessible to a wide variety of biologists, which should ultimately increase the rate of understanding of the mechanisms of viral genetic evolution.

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog.

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2'-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens.