Porcine deltacoronavirus infection

Porcine deltacoronavirus infection

Authors:
K JUNG - Research Scientist and Leading Expert, Food Animal Health Research Program, 1680 Madison Ave, Wooster, Ohio, OH 44691, USA
H HU - Professor, Henan, 450006, China
L J SAIF - Professor, MS, PhD, Food Animal Health Research Program, CFAES and CVM, OARDC, Ohio State University, 1680 Madison Ave, Wooster, Ohio, OH 44691, USA

Introduction

In early 2014, outbreaks of watery diarrhoea associated with porcine deltacoronavirus (PDCoV) infection occurred in sows and their suckling piglets on five farms in Ohio, United States (USA).41 Previously, PDCoV had been reported in the faeces of domestic pigs in China in 2012,44 but the role of the virus as an enteric pathogen was unclear. Porcine deltacoronavirus subsequently spread nationwide in the USA 42 causing diarrhoea and deaths in suckling pigs.3

Experimental studies verified that the PDCoV isolates from the USA are enteropathogenic in neonatal pigs, as evidenced by acute, watery diarrhoea and severe intestinal lesions.5, 17 However, the impact and disease severity of PDCoV infection is less than that of both porcine epidemic diarrhoea virus (PEDV) and transmissible gastroenteritis virus (TGEV) infections.3

Since the original outbreaks of PDCoV-associated disease in the USA, the disease also has been identified on swine farms in Canada, South Korea, China, Thailand, Vietnam, and the Lao People’s Democratic Republic, although PDCoV apparently has not spread nationwide in Canada.31

Differential diagnosis of PDCoV, PEDV, and TGEV infections is critical to in the control of coronaviral diarrhoeas in swine production systems, especially in regions where these pathogens have recently emerged or re-emerged.

Aetiology

Porcine deltacoronavirus virions are enveloped and pleomorphic with a diameter of 60 - 180 nm.28 The viral genome comprises a single-strand of positive-sense RNA of approximately 25.4 kb (excluding the poly A-tail) that encodes four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), and four nonstructural proteins.28 The genome organization and arrangement is: 5’ untranslated region (UTR), open reading frame (ORF) 1a/1b, S, E, M, nonstructural protein 6 (NS6), N, NS7, and 3’ UTR.The functions and roles of structural and nonstructural proteins of PDCoV in infection of host cells are presumed to be similar to those of TGEV and PEDV.

All global strains of PDCoV have high nucleotide (nt) identities (See Epidemiology ).45 However, a comprehensive genetic analysis of viruses of diverse geographical origin revealed that USA/Korean PDCoV viruses clustered together but Chinese viruses clustered separately and those from Thailand formed another cluster.45 Chinese PDCoV viruses had multiple mutation or deletion sites in their S, NS, or 3’ UTR genes, whereas these mutations were not found in the genomes of PDCoVs originating in the USA.43

There was lack of cross-reactivity between PDCoV and antibodies to either PEDV or TGEV.5, 28 However, a further study reported antigenic cross-reactivity between PDCoV and PEDV strains from the USA, possibly due to at least one shared epitope in the N-terminal region of their N proteins.29 Therefore, truncation of the N-terminal region of the N protein may eliminate PDCoV and PEDV shared epitope(s) and reduce possible cross-reactivity in serologic assays.11 There is no evidence that PDCoV is infectious to humans or is of public health significance.

Epidemiology

The precise origin of PDCoV is unclear. Molecular surveillance in China in 2007-2011 detected deltacoronaviruses (DCoVs) in pigs and wild birds only.44 However, DCoVs were previously detected in rectal swabs obtained from small mammals, including Asian leopard cats (Prionailurus bengalensis) and Chinese ferret badgers (Melogale moschata) at live-animal markets in China over the period 2005-2006.8 The helicase and S genes of these viruses were closely related to those of PDCoV. The data suggest the potential interspecies transmission of a DCoV between these wild small mammals and pigs, between birds and pigs, or between birds and wild small mammals. A recent study also revealed that PDCoV-inoculated gnotobiotic (Gn) calves developed acute subclinical infection without intestinal pathology, but with persisting faecal viral RNA shedding and seroconversion.18 Consequently, the potential ability of PDCoV and other DCoV isolates from birds or small mammals to infect different species needs further investigation.

In February, 2014, the Department of Agriculture in Ohio, USA reported an outbreak of diarrhoea in pigs caused by PDCoV. Among 42 faecal or intestinal samples collected from sows with diarrhoea and piglets on five Ohio farms. Of these, 39 (92.9 per cent) were positive for PDCoV by reverse transcriptase-polymerase chain reaction (RT-PCR).41 The complete genome of the PDCoV Ohio strain OH1987 had 99 per cent nt identity to the prototype strain of PDCoV, HKU15-155, first reported in Chinese pigs in 2012. During the same period, genetically similar strains, USA/IA/2014/8734 and SDCV/USA/Illinois121/2014, were identified by other diagnostic laboratories in the USA.25, 30 Since then, PDCoV has been detected in 21 USA states (www.aphis.usda.gov/animal-health/secd). The origin of PDCoV in pigs in the USA is unknown, although there is serological and virological evidence for its presence in pigs in the USA prior to its detection in February 2014.37, 40

Subsequent to the first USA report, PDCoV also has been identified in Canada (2014), South Korea (2014), mainland China (2015), Thailand (2015), and Vietnam and Laos (2015).20 In Canada diarrhoea related to PDCoV was first detected in  six Ontario farms.31 The complete genome of the Korean strain KUN14-04 had nt identities of 98.8-99.0 per cent to the Chinese HKU15-44 and HKU15-155 strains, and 99.6-99.8 per cent to US PDCoVs.24 A survey in South Korea reported that of 691 diarrhoeic faecal samples collected on 59 pig farms in 2014 to 2015, only two samples from one farm were positive for PDCoV RNA,23, indicating limited PDCoV infection in South Korean pigs during this period. The two Korean PDCoV isolates, SL2 and SL5, were also closely related to US PDCoV strains, but they differed genetically from the previous isolate KNU14-04. PDCoV has continued to evolve and spread throughout South Korea.15 Since the first identification of PDCoV in Hong Kong in 2012,44 PDCoV has been identified in diarrhoeic pigs in mainland China.10, 38 Its prevalence was high (> 30 per cent) and co-infections with PDCoV and PEDV were common (20 per cent). All Chinese PDCoV strains shared high nt identities (≥ 98.9 per cent) with each other and with the global PDCoV strains. The Thai PDCoV strains shared the highest nt identities (≥ 98.4 per cent) with the Chinese PDCoV strain CHN-AH-2004,14 but they formed a separate cluster from Chinese and US strains.45 Thai PDCoV-like strains were also found in Laos, whereas US PDCoV-like strains were detected in Vietnam.34

Transmission of PDCoV is principally by the faecal-oral route. Faeces and/or vomitus and contaminated fomites are major sources of infection. Based on experimental findings,13, 28 diarrhoea occurs in infected piglets for approximately 5-10 days, with persistent faecal shedding lasting up to 19 days. Because pigs continued to shed PDCoV RNA in the faeces after recovery from disease, another possible reservoir for PDCoV includes sub-clinically infected pigs, i.e. carriers. Introduction of PDCoV onto pig farms may occur via contaminated vehicles, feed and feed ingredients, feed bags, and, possibly, by air-borne aerosols derived from neighbouring farms or herds, as reported for PEDV.1, 7, 26, 33, 36

Pathogenesis and clinical signs

The pathogenesis of PDCoV has been studied in gnotobiotic (Gn) and “conventional” piglets inoculated orally with PDCoV isolates from the USA at 5-14 days of age.5, 13, 17, 28 Clinical signs (diarrhoea and/or vomiting) occurred at one to three days post-inoculation (dpi). Replication of PDCoV is confined to enterocytes lining the small and large intestines. PDCoV-infected enterocytes rapidly undergo necrosis,19 leading to marked villous atrophy in the small intestine. During acute infection, PDCoV antigens are detectable mainly in the villous epithelium of the atrophied mid-jejunum to ileum and to a lesser extent in the duodenum, proximal jejunum and enterocytes in the mucosa of the caecum and colon. Occasionally, some PDCoV antigen is detectable in crypt epithelial cells of the jejunum and ileum19 and macrophage-like cells in the intestinal lamina propria, Peyer’s patches, and mesenteric lymph nodes.13 Frequently, transient viraemia with low quantities of PDCoV RNA occurs in serum (RNAemia).5, 13, 28 In pigs recovered from clinical disease, significant amounts of PDCoV antigen were detected in the gut-associated lymphatic tissues by 23 dpi.13 PDCoV antigens were not detected in other organs, including the respiratory tract.20 However, by quantitative RT-PCR (qRT-PCR), PDCoV RNA was detected in low to moderate amounts in various organs, possibly resulting from concurrent viraemia.5, 28

Clinical signs of PDCoV infection in suckling and older pigs are similar, although milder than those caused by PEDV and TGEV infections. In suckling piglets, PDCoV induces acute, watery diarrhoea, frequently accompanied by mild to moderate vomiting, leading to dehydration, loss of body weight, lethargy, and death. Experimentally, the onset of diarrhoea coincided with or occurred one to two days later than the first detection of viral RNA in faeces.17, 28 Diarrhoea is a consequence of mal-digestion and malabsorption resulting from loss of absorptive enterocytes in the small intestine. That, in turn, results in decreased brush border membrane-bound digestive enzymes.16 Mild vacuolation, observed in  infected colonic epithelial cells, may interfere with the reabsorption of water and electrolytes.17 Dehydration can be exacerbated by vomiting.

Sero-negative pigs of all ages are susceptible to PDCoV infection but younger piglets are usually more severely affected than older piglets.3

Based on field observations in the USA in 2014, PDCoV infection causes up to 40 per cent mortality among suckling pigs.3 Similarly, diarrhoeal outbreaks caused by PDCoV on breeding farms in China and Thailand resulted in 64-80 per cent mortality in suckling piglets. On many farms, morbidity and mortality may be affected by co-infection with other enteric viruses such as PEDV and rotavirus.31, 38 The disease on breeding farms is self-limiting, generally ending when pregnant sows are able to provide their piglets with colostrum containing antibodies to PDCoV.

Porcine deltacoronavirus infection shares several clinical features with TGEV and PEDV infections, but the virus seems to spread more slowly among pigs. This is possibly due to its recent adaptation to pigs. Compared with PEDV infections, PDCoV-infected pigs shed less viral RNA in their faeces,17 indicating lower levels of replication of PDCoV in the intestine of affected pigs. This factor may contribute to lower mortality in suckling pigs as compared with PEDV infections. The effect of PDCoV infection on growth performance in 5-week-old pigs was lower in comparison with the significant losses in growth and production performance following PEDV infections.2, 6

Pathology and immunity

Lesions have been described in suckling piglets experimentally and naturally infected with USA, Chinese, or Thai isolates of PDCoV.5, 9, 13, 14, 17, 28, 43 The enteric lesions in PDCoV-infected pigs resemble those observed in TGEV and PEDV infections, but are less extensive. Gross lesions are limited to the gastrointestinal tract and are characterized by thin and transparent intestinal walls (proximal jejunum to colon) with accumulation of large amounts of yellowish fluid. The stomach is frequently extended with consolidated, curdled milk. The transparency and fragility of affected intestines are less apparent than in PEDV and TGEV infections.

Histological lesions are characterized by acute, multifocal to diffuse, mild to severe villous atrophy (atrophic enteritis) in the proximal jejunum to ileum, occasionally accompanied by mild vacuolation of the superficial epithelial cells in the caecum and colon.17 No villous atrophy or histological lesions were described in the duodenum, which is consistent with lower levels of PDCoV antigen-positive epithelial cells in the duodenum.5, 17 During acute infection, vacuolated enterocytes throughout the jejunum and ileum, accompanied by extensive enterocyte exfoliation were seen at the villous tips or the entire villi. Atrophic villi are frequently fused and covered with degenerated or regenerating epithelium in which the enterocytes appear flattened. There is often accompanying infiltration of inflammatory cells, such as macrophages, lymphocytes and neutrophils, in the lamina propria. Lesions in other organs have so far not been reported.

The immune responses of pigs to PDCoV infection are largely undefined, but are likely similar to those of TGEV and PEDV. Hu et al. (2016) reported the development of PDCoV antibodies in the sera of PDCoV-infected Gn pigs.13 Gnotobiotic pigs inoculated orally with the original or cell-culture propagated strain OH-FD22 of PDCoV had  IgG and IgA antibodies to PDCoV and PDCoV neutralizing (VN) antibodies in their sera by 14 dpi. These levels peaked at 24 dpi by which time the pigs had recovered from clinical disease and shedding of virus in the faeces had ceased.

By two to three weeks after the occurrence of  PDCoV infection in swine herds, neonatal piglets may be protected by maternal antibodies obtained via colostrum(lactogenic immunity) from immune dams. Infection  in sows with PDCoV activates the gut-mammary-sIgA link, as reported previously for TGEV 4, 35 and PEDV,22 leading to secretion of IgA antibodies to PDCoV in mammary secretions.

Diagnosis and differential diagnosis

Laboratory techniques are needed to differentiate PDCoV infection from PEDV, TGEV and rotavirus diarrhoea in pigs of all ages. A definitive diagnosis of PDCoV infection includes detection of PDCoV RNA or antigens in the faeces, intestinal contents or tissues from diarrhoeic pigs. This can be achieved through RT-PCR assays that target a conserved region of the PDCoV M or N genes.31, 41 Immunofluorescence (IF) or immunohistochemical staining using virus-specific monoclonal or polyclonal antibodies 5, 17, 28 and in situ hybridization17 can be employed to detect PDCoV antigens or RNA , respectively, in tissues. A real-time duplex RT-PCR assay for detection of PDCoV and/or differentiation of the virus from PEDV in intestines and faeces has been developed.46 Isolation and propagation of PDCoV can be achieved using porcine kidney (LLC-PK) and swine testicular (ST) cells supplemented with trypsin or pancreatin.12 The addition of trypsin and pancreatin to LLC-PK and ST cells inoculated with PDCoV resulted in cytopathic effects comprising enlarged and rounded cells that occurred singly or in clusters, with subsequent cell shrinkage and detachment as a result of apoptosis.19 However, the sensitivity of virus isolation from clinical samples is low.12

As was done for the original detection of PEDV in the USA,21 direct electron microscopy (EM) can be used to demonstrate PDCoV particles in faeces collected from diarrhoeic pigs, but immune EM using hyperimmune or convalescent sera is essential to differentiate PDCoV from PEDV or TGEV.17

Serological detection of PDCoV antibodies can be performed by IF, VN, and ELISA assays. Porcine deltacoronavirus antibodies in serum and milk were quantitated by ELISA using antigens consisting of cell-culture grown virus29 or individual S1, N, and M viral proteins.11, 27, 32, 39, 40

Control

Currently, there are no treatments or vaccines for control PDCoV infection. Nevertheless, prevention and control measures that have found to be effective against TGEV and PEDV infections can be applied to PDCoV.

Preventive or therapeutic antibiotic therapy is indicated in instances where concurrent infection with enteric bacterial pathogens is possible. Symptomatic treatment of suckling pigs suffering from diarrhoea includes intraperitoneal administration of bicarbonate fluids and free access to water to help alleviate acidosis and dehydration. If mortality is substantial among suckling piglets, feedback methods (intentional exposure of pregnant sows to virus using watery faeces or minced intestines from acutely-infected piglets) could stimulate lactogenic immunity and assist in reducing high mortality of suckling piglets if administered to sows at least 2 weeks before farrowing.

For farrowing to weaning herds, whole-herd feedback through a load-close-expose protocol, similar to the approach for controlling outbreaks of porcine reproductive and respiratory disease 47, could be considered to develop herd immunity to PDCoV. In general, load-close-expose approaches are to load the farm with the replacement gilts necessary for a minimum several (three to seven) month farm closure and then immunize the entire herd by intentional virus exposure. Alternatively, approaches for controlled gilt exposure to PDCoV for the acclimation period could also be considered to make gilts immune prior to their introduction into the breeding herd.

During PDCoV epidemics, neonatal pig management strategies, such as segregated early weaning or piglet depopulation, may also be used to facilitate rapid elimination of PDCoV from affected farms. Considering their practicality, however, the expected disadvantages such as decreased growth performance following early weaning also need to be considered. High-level biosecurity procedures to reduce PDCoV transmission via contaminated fomites, personnel or feed should be emphasized.

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