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Infectious laryngotracheitis virus (ILTV) is the causative agent of an economically important disease of chickens causing upper respiratory tract infection. Strains of ILTV are commonly identified by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and/or PCR high resolution melt (PCR-HRM) curve analysis targeting several genes. However, these techniques examine only a limited number of mutations present inside the target regions and may generate unreliable results when the sample contains more than one strain. Here, we attempted to sequence the whole genome of ILTV with known identity (class 9) directly from tracheal scrapings to circumvent in vitro culturing, which can potentially introduce variations into the genome. Despite the large number of quality reads, mapping was compromised by poor overlapping and gaps, and assembly of the complete genome sequence was not possible. In a map-to-reference alignment, the regions with low coverage were deleted, those with high coverage were concatenated and a genome sequence of 139,465 bp was obtained, which covered 91% of the ILTV genome. Sixteen single-nucleotide polymorphisms (SNPs) were found between the ILTV isolate examined and ILTV class 9 (JN804827). Despite only 91% genome coverage, using sequence analysis and comparison with previously sequenced ILTVs, we were able to classify the isolate as class 9. Therefore, this technique has the potential to replace the current PCR-HRM technique, as it provides detailed information about the ILTV isolates.

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many

Infections caused by Mycoplasma synoviae are major welfare and economic concerns in poultry industries worldwide. These infections cause chronic respiratory disease and/or synovitis in chickens and turkeys leading to reduced production and increased mortality rates. The live attenuated vaccine strain MS-H (Vaxsafe® MS), commonly used for protection against M. synoviae infection in many countries, contains 32 single nucleotide variations compared to its wildtype parent strain, 86079/7NS. Genomic analysis of vaccine strains reisolated from flocks following the administration of MS-H has identified reversions to the original 86079/7NS sequence in the obgE, oppF and gapdh genes. Here, three MS-H field reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), as well as the vaccine MS-H and the parental strain 86079/7NS were experimentally inoculated to chickens. The strains were assessed for their ability to infect and elicit immune responses in the recipient chickens, as well as in naïve in-contact chickens. Despite the loss of temperature sensitivity phenotype and colonization of the reisolates in the lower respiratory tract, there was no significant differences detected in the microscopic mucosal thickness of the middle or lower trachea of the inoculated chickens. Concurrent reversions in ObgE, OppF and GAPDH proteins were associated with higher gross air sac lesion scores and increased microscopic upper-tracheal mucosal thickness in chickens directly inoculated with the reisolates following intratracheal administration of a virulent strain of infectious bronchitis virus. The gross air sac lesions of the chickens in-contact with those inoculated with reisolates were not significantly different to those of chickens in-contact with MS-H inoculated chickens, suggesting that horizontal transmission of the reisolates in the poultry flock will not lead to higher pathogenicity or clinical signs. These results suggest a

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn’t compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos.

Avian hepatitis E virus (aHEV) is the causative agent of an important disease of broiler breeders and layers. aHEV cannot be readily propagated in cell culture and is characterised primarily by sequencing of amplicons generated through several RT-PCRs that target individual genes. This study aims to uncover the origin of current Australian aHEV isolates based on whole genome sequencing using clinical liver tissues. Complete genome sequences of the two aHEV isolates were assembled using Nanopore and Illumina reads. The two isolates possessed only four single nucleotide polymorphisms to each other. Comparison of the sequences with aHEV genome sequences available in the GenBank showed the highest nucleotide sequence identity of 88% with the prototype USA strain (AY535004), 82% with the European (AM943647) and genotype 1 Australian strains (AM943647). Recombination analysis suggested that aHEV isolates characterised in this study are progeny of a cross between a US and a Hungarian strain. Phylogenetic tree and phylogenetic networks constructed using complete genome and individual coding sequences revealed that Australian aHEV isolates formed a distinct clade closer to the USA strains and classified as genotype 2 whereas genotype 1 Australian strain clustered together with South Korean strains.

An amendment to this paper has been published and can be accessed via the original article.

Background Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo. Results Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF1 genes) could potentially restore the function of the respective protein to that of the wild-type. Conclusions These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.