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

Abstract

Hemorrhagic enteritis virus (HEV) is an immunosuppressive adenovirus that causes an acute clinical disease characterized by hemorrhagic gastroenteritis in 4-week-old turkeys and older. Recurrent incidence of secondary infections (e.g., systemic bacterial infections, cellulitis, and elevated mortality), may be associated with the presence of field-type HEV in Canadian turkey farms. We speculate that field-type HEV and vaccine/vaccine-like strains can be differentiated through analysis of the viral genomes, hexon genes, and the specific virulence factors (e.g., ORF1, E3, and fib knob domain). Nine out of sixteen spleens obtained from cases suspected of immunosuppression by HEV were analyzed. The limited data obtained showed that: (1) field-type HEV circulates in many non-vaccinated western Canadian flocks; (2) field-type HEV circulates in vaccinated flocks with increased recurrent bacterial infections; and (3) the existence of novel point mutations in hexon, ORF1, E3, and specially fib knob domains. This is the first publication showing the circulation of wild-type HEV in HEV-vaccinated flocks in Western Canada, and the usefulness of a novel procedure that allows whole genome sequencing of HEV directly from spleens, without passaging in cell culture or passaging in vivo. Further studies focusing more samples are required to confirm our observations and investigate possible vaccination failure.

Keywords: immunosuppression; meat turkeys; molecular epidemiology; turkey hemorrhagic enteritis virus; whole genome sequencing.

Figure 1

Systemic bacterial infection in 52-day-old turkeys (case 18-0374). This case was submitted due to elevated mortality in a flock without hemorrhagic enteritis virus (HEV) vaccination. The HEV sequence recovered from the spleen was found to be different from vaccine strains and to have missense mutations on the three virulence factors described by Beach et al. 2009 [40], and in the hexon protein. Airsacculitis lesions can be observed in (a,b); while pericarditis lesions can be observed in (c). Escherichia coli was isolated from pericardium and spleen.

Figure 2

Nucleotide RAxML-based phylogenetic tree of complete HEV sequences (a); and amino acid RaxML-based phylogenetic trees of hexon gene (b), respectively. The included sequences are described in Table 3, and Supplement Table S1 Sequences in bold green are the vaccines sequences derived in the present study, bold red are sequences derived from non-vaccinated flocks, and bold blue from vaccinated flocks. Sequences obtained in this study are marked with a black asterisk. GenBank accession numbers and naming structure can be found at Table 3.

Figure 3

ORF1 maximum likelihood (ML) tree. Sequences shows all ORF1 HEV sequences previously published in GenBank, and those obtained in the present project. Sequences in bold green were obtained from vaccines in the present study, bold red from non-vaccinated flocks, and bold blue from vaccinated flocks. Sequences obtained in this study are marked with a black asterisk. GenBank accession numbers and naming structure can be found at Table 3.

Figure 4

E3 (a) and fib knob domain (b) maximum likelihood (ML) trees. Sequences in bold green were obtained from vaccines in the present study, bold red from non-vaccinated flocks, and bold blue from vaccinated flocks. Sequences obtained in this study are marked with a black asterisk. GenBank accession numbers and naming structure can be found at Table 3.

Figure 5

Structure of the HEV fib knob domain. Trimeric structure can be seen from side (a) and from the top (b). Side views of a monomer observed from the outside of the molecule (c) and the inside (d) with amino-terminus (Nt), and carboxy-terminus (Ct) signaled. Mutations found in HEV Canadian sequences marked in magenta, HEV virulent sequences on red, and one mutation found in an HEV vaccine in yellow. For total list of viruses see Table 3. Figure was constructed following Singh et al. 2015 [4]. M65I and M87T are mutations of Virulent-IL-1998 when compared with an avirulent strain (splenic vaccine) resulting in a 3D change in C’C” loop [4]. R72S, and L84F mutations of sequences 17-0699-BC-2017; 18-0723-BC-2018; and 18-0665-AB-2018 target the same area (C’C”-loop).