PHEV role in the porcine respiratory disease complex
Genetic information of circulating strains in U.S. is limited.
By Trevor T. Arunsiripate, Alyona Michael, Christopher Siepker, Eric Burrough, Pablo E. Piñeyro
One of the most economically impactful health problems in the swine industry is the development of the porcine respiratory disease complex (PRDC). This syndrome results from a combination of primary viral infection that can subsequently predispose to secondary bacterial infection, resulting in reduced growth performance, increased medication costs, and mortality. The morbidity rate of a PRDC outbreak can typically range from 30 to 70% on commercial farms, with a mortality rate of 4 to 6% or more, depending on the secondary infections.
Identifying and preventing primary pathogens, including swine influenza A virus (swIAV), porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome virus (PRRSV) has a tremendous economic benefit. With lesser clinical significance and prevalence, porcine respiratory coronavirus (PRCV) and recently porcine parainfluenza virus (PPIV-1), porcine circovirus type 3 (PCV3), porcine parvovirus 2 (PPV2), and porcine astrovirus 4 (PAstV4) have been characterized as potential primary respiratory pathogens, thus expanding the list of primary infectious agents that can contribute to the PRCD.
In 2015, porcine hemagglutinating encephalomyelitis virus (PHEV) was associated with a case of influenza-like respiratory disease of show pigs in Michigan. However, the role of PHEV in respiratory disease has not yet been confirmed experimentally.
Since then, sporadic respiratory disease submissions received at the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL), featuring necrotizing and attenuating bronchitis characteristic of epitheliotropic viral infections have notably failed to yield an etiologic diagnosis due to a lack of PCR detection of routine agents, including swIAV and PPIV-1. These findings have prompted the investigation of the role of PHEV in respiratory disease.
PHEV history, clinical presentationPHEV is the only neurotropic coronavirus affecting pigs. This virus was first isolated in Canada and later in England from piglets presenting with vomiting, anorexia and lethargy, and subsequently disease associated with PHEV infection has been known as vomiting and wasting disease (VWD) (Cartwright et al., 1969; Roe & Alexander, 1958). The disease was experimentally reproduced in pigs.
Since its first report in Canada, many countries have documented the presence of PHEV. In China, the first report of PHEV is from 1985, with a mortality rate of 80.6%. The biggest outbreak on record occurred in Taiwan in 1994, with a mortality rate of 100% (Gao et al., 2011). In August 2006, the first PHEV cases described in Argentina presented with a morbidity rate of 52.6% and mortality of 16.9%(Quiroga et al., 2008). In 2007, a new outbreak in China affecting 20-day-old pigs reported a mortality of 48%.
Serologic investigations have revealed an even wider prevalence and global dissemination of PHEV. For example, seroprevalence reported for PHEV in Canada is 31%, Ireland 46%, England 49%, Japan 52% to 82%, and United States from 11% to 99%(Mora-Díaz et al., 2020).
Infection with PHEV causes vomiting, extreme weight loss and encephalomyelitis in pigs less than 4-weeks old, specifically affecting pigs without maternal antibodies. Neonatal piglets are infected a few days after birth with up to 100% within-litter morbidity, producing vomiting, ataxia, hyperesthesia, lack of coordination, lethargy, opisthotonos, paddling and death 2 to 3 days after onset of clinical signs.
The virus ascends to the central nervous system (CNS) via peripheral nerves. Neurons of the CNS and peripheral ganglia are the target cells for viral replication. Experimentally, the virus can also replicate in porcine kidney cells. Experimental studies showed that nasopharyngeal invasion through the mucosa and tonsils is one of the infection pathways, permitting viral dissemination to local lymph nodes and cranial nerves, including the trigeminal nerve, and subsequently undergoing retrograde ascent to the CNS and sensory nucleus.
Macroscopic lesions observed experimentally are nonspecific and are also related to motor problems of the gastrointestinal tract. Affected animals exhibit cachexia, gastric dilation, and, in some cases, abdominal distension.
Histologically, there is the presence of non-suppurative encephalomyelitis associated with lymphoplasmacytic perivascular cuffs, gliosis, and neuronal degeneration. The most pronounced lesions are in the gray matter, medulla oblongata, and dorsal horns of the spinal cord. Histological changes observed in the stomach include neuronal degeneration of the gastric myenteric plexus.
Clinical diagnosis is based on a sudden increase in mortality in animals less than 4 weeks of age presenting with acute vomiting, distended stomachs, and neurologic signs. Detection of nucleic acid by PCR, viral isolation, and immunofluorescence can help to confirm the etiological diagnosis. Serological diagnosis can be made by hemagglutination-inhibition (HI), enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN).
Respiratory or neurological virus? During 2020, three diagnostic cases from pigs with reported respiratory clinical signs were received at ISU-VDL. Pulmonary lesions included bronchointerstitial pneumonia and bronchiolitis. After ruling out the presence of other respiratory and/or epitheliotropic viruses, including PRRSV, swIAV and PPIV-1, tissues were evaluated by next-generation sequencing (NGS).
Next-generation sequence analyses demonstrated the presence of PHEV in a case with bronchointerstitial pneumonia. Phylogenetic analysis revealed that PHEV associated with respiratory lesions clustered independently from neurological strains and the virus forming a unique cluster with other PHEVs previously detected in respiratory cases.
Currently the number of sequences reported is limited and the clinical information associated with those sequences is scarce. Additional studies involving a larger number of sequences to evaluate if different PHEV genotypes could lead to different phenotypes with different clinical outcomes are necessary.
The diagnosisAlthough PHEV has been described affecting swine production systems worldwide, there is limited diagnostic investigation of this pathogen in terms of its prevalence, transmission and dynamics in modern production systems. Active investigation has only been reported during acute outbreaks of neurological disease. However, one of the limitations has been the diagnostic tools available for diagnosis.
Traditionally, virus isolation has been the gold standard for PHEV diagnosis; however, this technique has its limitations, and suitable neuronal cell lines for viral isolation are not widely available. Samples most commonly used for virus isolation have also been limited. Therefore, the diagnosis of PHEV is typically made based on clinical signs, histological lesions and viral detection, by either PCR or IHC.
New technologies including next generation sequencing, metagenomics and fast and more accurate direct detection methods including in situ hybridization and small-molecule inexpensive fluorescent insitu hybridization (smiFISH) allows a visual confirmation of the viral replication within the lesions. Currently the ISU-VDL offers direct and indirect detection methods including ELISA, PCR, and in situ hybridization (ISH).
Presence of PHEVA small pilot retrospective study was performed on 18 swine respiratory disease cases received at ISU-VDL from 2019 through 2021. The inclusion criteria were based on a primary histological assessment. Animals diagnosed with bronchointerstitial pneumonia of undetermined etiology were tested for PHEV. PCR was performed on fixed tissues by sectioning a 40 µm scroll of paraffin embed blocks, followed by RNA extraction by standard procedures.
PHEV was confirmed by qPCR against the PHEV spike gene. The presence of PHEV within the bronchiolar epithelium was confirmed by ISH using Advanced Cell Diagnostics RNAscope specifically targeted against the S gene mRNA and in-house nucleotide fluorescent in situ hybridization (FISH). Specific qPCR results confirmed the presence of PHEV in 66.6% (12/18) of cases with etiologically unspecified bronchointerstitial pneumonia. The average PCR Ct value was 34.15 (2.57± SD). ISH-RNA against PHEV showed strong intracytoplasmic hybridization signal in the airway epithelium of sections with epithelial attenuation and regeneration and necrotic debris located within the lumen. Scattered PHEV-positive cells were observed in the sub-bronchiolar connective tissue and alveolar interstitium. The age, clinical signs, and state of origin of the cases are presented in Table 1.
Confirmation of causationOther betacoronaviruses, including BCoV and SARS-CoV-2, have a clear epithelial tropism, therefore it is possible that PHEV can also have similar cellular tropism. This small retrospective study provides additional information of the causative role of PHEV in respiratory lesions and its potential role in the PRDC. Other epitheliotropic viruses, including IAV and PPIV-1, are endemic in commercial pigs in the U.S.
Since all these viruses, including PHEV, can affect the respiratory epithelium, histologic evaluation may not be sufficient to confirm PHEV, since lesions are indistinguishable. In cases where no other epitheliotropic virus is detected by PCR, the next diagnostic approach could involve PHEV PCR detection. However, concurrent infections are possible, making the interpretation of PCR results not entirely diagnostic. In those cases, direct detection by ISH or IHC should be considered as a final confirmatory tool to correlate the presence of PHEV within the affected epithelium.
ConclusionPrevious serological studies have indicated a high prevalence of PHEV in the U.S., suggesting widespread circulation of the virus. However, to our knowledge, there have been no reported outbreaks or active cases of VWD, leading to the hypothesis that the high seroprevalence of PHEV could be a result of undetected or under-diagnosed respiratory PHEV infections. Currently, PHEV is considered a diagnosis of exclusion and is only explored when swIAV tests return negative results. Our pilot-retrospective study supports the current understanding of PHEV as a potential respiratory pathogen. Therefore, additional efforts to detect PHEV and confirm its causative role in flu-like clinical outbreaks affecting nursery pigs are needed.
Highlights
PHEV has been historically associated with neurologic disease (VWD).
PHEV should be considered as differential in outbreaks of flu-like clinical signs.
Numerous diagnostic techniques are available to confirm a causative role of PHEV in respiratory lesions.
Diagnosis should include clinical history, presence of microscopic lesions, and viral detection by PCR with confirmation by ISH/IHC.
Genetic information of circulating PHEV strains in the US is limited.
Clinical research is scarce and more information is necessary to understand the epidemiology of PHEV.
ReferencesCartwright, S. F., Lucas, M., Cavill, J. P., Gush, A. F., & Blandford, T. B. (1969). Vomiting and wasting disease of piglets. Vet Rec, 84(7), 175-176. https://doi.org/10.1136/vr.84.7.175
Gao, W., Zhao, K., Zhao, C., Du, C., Ren, W., Song, D., Lu, H., Chen, K., Li, Z., Lan, Y., Xie, S., He, W., & Gao, F. (2011). Vomiting and wasting disease associated with hemagglutinating encephalomyelitis viruses infection in piglets in Jilin, China. Virol J, 8, 130. https://doi.org/10.1186/1743-422x-8-130
Mora-Díaz, J. C., Magtoto, R., Houston, E., Baum, D., Carrillo-Ávila, J. A., Temeeyasen, G., Zimmerman, J., Piñeyro, P., & Giménez-Lirola, L. (2020). Detecting and Monitoring Porcine Hemagglutinating Encephalomyelitis Virus, an Underresearched Betacoronavirus. mSphere, 5(3). https://doi.org/10.1128/mSphere.00199-20
Quiroga, M. A., Cappuccio, J., Piñeyro, P., Basso, W., Moré, G., Kienast, M., Schonfeld, S., Cáncer, J. L., Arauz, S., Pintos, M. E., Nanni, M., Machuca, M., Hirano, N., & Perfumo, C. J. (2008). Hemagglutinating encephalomyelitis coronavirus infection in pigs, Argentina. Emerg Infect Dis, 14(3), 484-486. https://doi.org/10.3201/eid1403.070825
Roe, C. K., & Alexander, T. J. (1958). A Disease of Nursing Pigs Previously Unreported in Ontario. Can J Comp Med Vet Sci, 22(9), 305-307. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1614651/pdf/vetsci00346-0017.pdf
Arunsiripate is a veterinary pathology student, Michael and Siepker are clinical assistant professors, Burrough is a professor and Piñeyro is an associate professor, diagnostic pathologist all with the Iowa State University College of Veterinary Medicine.
