Rapid typing of infectious laryngotracheitis virus directly from tracheal tissues based on next-generation sequencing.

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.

Development of a rapid technique for extraction of viral DNA/RNA for whole genome sequencing directly from clinical liver tissues.

Characterisation of the entire genome of Fowl aviadenoviruses (FAdV) requires isolation and propagation of the virus in chicken embryo liver or kidney cells, a process which is not only time consuming but may occasionally fail to result in viral growth. Furthermore, in a mixed infection, isolation in cell culture may result in the loss of viral strains. In this study, we optimised a FAdV DNA extraction technique directly from affected liver tissues using kaolin hydrated aluminium silicate treatment. The whole genome of FAdV was sequenced directly from extracted DNA without any targetted PCR based enrichment. The extraction method was also tested on avian liver tissues affected with the RNA virus Avian hepatitis E virus and demonstrated to yield sequencing grade RNA. Therefore, the method described here is a simple technique which is potentially useful for the extraction of sequencing grade DNA/RNA from tissues with high fat content.

Vaccination with FAdV-8a induces protection against inclusion body hepatitis caused by homologous and heterologous strains.

Fowl aviadenoviruses (FAdV) are important avian pathogens, responsible for several poultry diseases prevalent worldwide, including inclusion body hepatitis (IBH). FAdV intraspecies cross-protection has been clearly demonstrated, but there is little evidence that any interspecies cross-protection exists. The present study aimed to assess the inter- and intraspecies protection between three FAdV field isolates (FAdV-8a, FAdV-8b, FAdV-11) identified in association with severe IBH outbreaks. Inocula prepared using inactivated plaque-purified virus with adjuvant Montanide™ ISA 71VG, were injected intramuscularly into 3-week-old SPF chickens. At 6-weeks of age, the birds were challenged with 106 TCID50 of homologous or heterologous virus intraperitoneally, and full post mortem examination performed at 4 days post-challenge. Various tissues were examined for gross and histological lesions and assessed for the presence of virus by PCR-HRM. All homologous-type vaccine/challenge groups exhibited protection against IBH lesions with no virus detected in the tissues. Unvaccinated groups challenged with virus showed evidence of FAdV-induced lesions; however, FAdV-8a demonstrated lower pathogenicity compared with FAdV-8b and FAdV-11. In the heterologous-type vaccine/challenge groups, FAdV-8a vaccine was shown to protect against challenge with both FAdV-8b and FAdV-11. FAdV-8a and 8b belong to species E and were therefore anticipated to cross-protect. However, FAdV-11 belongs to species D and therefore cross-protection by FAdV-8a was an uncharacteristic and unique finding of this study. Further research is required to disseminate the molecular basis for the interspecies cross-protection between FAdV-8a and FAdV-11. Nonetheless, the FAdV-8a isolate was shown to have substantial potential as a vaccine candidate in countries where FAdV-8a, 8b or 11 are prevalent.