Molecular Characterization of Hemorrhagic Enteritis Viruses (HEV) Detected in HEV-Vaccinated Commercial Turkey Flocks in Germany

Abstract

Despite the application of live hemorrhagic enteritis virus (HEV) vaccines, HEV field outbreaks are suspected to still occur in turkey flocks in Germany. Increasing secondary bacterial infections in HEV-vaccinated flocks suggest that vaccines may be losing efficacy or, possibly, that vaccine strains are causing disease. Thus, the goal of the current study was to investigate the diversity of HEV isolates from fattening turkey flocks between 2008 and 2012 by characterizing the open reading frame (ORF)1 gene at its 5' and 3' ends. Analyses of ORF1 sequences of field isolates and comparison with sequences present in databases revealed that in many cases (13 out of 16 samples), vaccine (avirulent) strains were present. In addition, data indicated the circulation of suspected virulent field isolates and these isolates (3 out of 16) cluster with an early isolate from Germany in the 1980s, but show some mutations in the predicted amino acid (aa) sequences of ORF1 compared to the early isolate. These virulent isolates clearly differ from the spleen-derived avirulent Domermuth vaccine strain used in Germany. In this study, a unique isolate was identified and showed unusual nucleotide mutations that resulted in aa exchanges at the 5' end of ORF1 between aa positions 34 and 174. This genetic drift suggests evolution of HEV including virulent and vaccine-derived strains in the field. This may lead to evasion of vaccinal immunity by drifted viruses and/or an increase in the virulence of field strains.

Molecular Characterization of Hemorrhagic Enteritis Virus (HEV) Obtained from Clinical Samples in Western Canada 2017–2018

“…Comparison between all previously published ORF1 sequences [ 40 , 41 ] and those obtained from the current work showed the presence of several unique and shared mutations. A number of mutations ( n = 39) were detected, of which 17 were synonymous and 22 were non-synonymous ( Supplement Table S1 ).…”

“…Similarly, as with previous publications, all major ORFs were located as expected [ 2 , 40 ]. As in previous research [ 6 , 40 , 41 ], no major changes in either of the genes analyzed were discovered, only single point mutations that may influence the virus ability to cause disease [ 6 , 40 , 41 ].…”

“…A number of mutations ( n = 39) were detected, of which 17 were synonymous and 22 were non-synonymous ( Supplement Table S1 ). Some mutations were detected in both vaccine sequences (ntG1485A; aaQ495R) and in German sequences obtained from vaccinated flocks suspected to have subclinical HE with increased mortality and higher incidence of Escherichia coli infections (Cases 7, 8, 12, and 17) [ 41 ] (ntG1274A; aaI425V) ( Supplement Table S1 ). The phylogenetic tree in Figure 3 shows ORF1 genes of virus 18-1234-AB-2018; 18-0943-AB-2018; and 18-0665-AB-2018 clustering with ORF1 sequences derived from vaccine strains, ORF1 sequences consist of a separate group.…”

“…NGS short reads were mapped to the splenic vaccine strain (Dindoral SPF; Merial GmbH, Hallbergmoos, Germany) (GenBank accession #AY849321.1) [ 40 , 41 ] under App Map function on CLC Genomics Workbench v 12.0.2 (Qiagen, Valencia, CA, USA) using default settings, and complemented using Geneious assembler v10.2.6 (Biomatters LTD., Auckland, New Zealand) [ 49 ]. Whole genome sequences were aligned with MAFFT v7.450 [ 50 , 51 ], and phylogenetic trees were generated using Randomized Axelerated Maximum Likelihood (RAxML) v8.2.11 by applying the nucleotide model GTR+gamma [ 52 ].…”

“…Recently, several virulence factors of HEV were identified (i.e., hexon, open reading frame 1 (ORF1), E3, and fib knob domain) [ 12 , 40 ]. HEV variants containing these factors are circulating in vaccinated flocks leading to subclinical infections [ 12 , 41 ]. Our objective was to characterize these HEV-positive samples based on whole genome sequencing and/or gene sequences (i.e., hexon, ORF1, E3, fib knob domain) for determination of HEV origin.…”