Aaron Singrey

Serological evidence for the presence of influenza D virus in small ruminants

Influenza D virus (FLUDV) was isolated from diseased pigs with respiratory disease symptoms in 2011, and since then the new virus has also been spread to cattle. Little is known about the susceptibility of other agricultural animals and poultry to FLUDV. This study was designed to determine if other farm animals such as goats, sheep, chickens, and turkey are possible hosts to this newly emerging influenza virus. 648 goat and sheep serum samples and 250 chicken and turkey serum samples were collected from 141 small ruminant and 25 poultry farms from different geographical locations in the United States and Canada. Serum samples were examined using the hemagglutination inhibition (HI) assay and the sheep and goat samples were further analyzed using the serum neutralization assay. Results of this study showed FLUDV antibodies were detected in 13.5% (17/126) of the sampled sheep farms, and 5.2% (29/557) of tested sheep serum samples were positive for FLUDV antibodies. For the goat results, the FLUDV antibodies were detected in 13.3% (2/15) of the sampled farms, and 8.8% (8/91) of the tested goat serum samples were positive for FLUDV antibodies. Furthermore, all tested poultry serum samples were negative for FLUDV antibodies. Our data demonstrated that sheep and goat are susceptible to FLUDV virus and multiple states in U.S. have this virus infection already in these two species. This new finding highlights a need for future surveillance of FLUDV virus in small ruminants toward better understanding both the origin and natural reservoir of this new virus.

The S2 glycoprotein subunit of porcine epidemic diarrhea virus contains immunodominant neutralizing epitopes

Abstract The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids [aa] 744–759, 747–774 and/or 756–771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371–1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499–600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV.

Porcine epidemic diarrhea virus: An overview of current virological and serological diagnostic methods

Abstract Porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute, highly contagious, and severe enteric disease that leads to high mortality rates in suckling piglets. Therefore, accurate diagnosis of PEDV infection is critical for the implementation of control measures for the virus. Many diagnostic tests have been recently developed and are currently available for the detection of PEDV, its proteins or nucleic acid, including virus isolation, immunofluorescence (IF) or immunohistochemistry (IHC), polymerase chain reaction (PCR) and isothermal amplification assays. Additionally, several serological assays have been developed and are currently used for the detection of antibodies against PEDV. Molecular assays such as real-time reverse transcriptase-PCR (rRT-PCR) became the methods of choice for the diagnosis of PEDV infection, providing sensitive, specific and rapid detection of the virus RNA in clinical samples. Whereas serological assays have been widely used to monitor prior exposure to the virus and to evaluate the efficacy of novel vaccine candidates or vaccination strategies. Here we discuss the properties of current PEDV diagnostic assays and prospects for improving diagnostic strategies in the future.

Immunogenicity of a recombinant parapoxvirus expressing the spike protein of Porcine epidemic diarrhea virus.

The parapoxvirus Orf virus (ORFV), has long been recognized for its immunomodulatory properties in permissive and non-permissive animal species. Here, a new recombinant ORFV expressing the full-length spike (S) protein of Porcine epidemic diarrhea virus (PEDV) was generated and its immunogenicity and protective efficacy were evaluated in pigs. The PEDV S was inserted into the ORFV121 gene locus, an immunomodulatory gene that inhibits activation of the NF-κB signalling pathway and contributes to ORFV virulence in the natural host. The recombinant ORFV-PEDV-S virus efficiently and stably expressed the PEDV S protein in cell culture in vitro. Three intramuscular (IM) immunizations with the recombinant ORFV-PEDV-S in 3-week-old pigs elicited robust serum IgG, IgA and neutralizing antibody responses against PEDV. Additionally, IM immunization with the recombinant ORFV-PEDV-S virus protected pigs from clinical signs of porcine epidemic diarrhoea (PED) and reduced virus shedding in faeces upon challenge infection. These results demonstrate the suitability of ORFV121 gene locus as an insertion site for heterologous gene expression and delivery by ORFV-based viral vectors. Additionally, the results provide evidence of the potential of ORFV as a vaccine delivery vector for enteric viral diseases of swine. This study may have important implications for future development of ORFV-vectored vaccines for swine.

Serological evidence for the co‐circulation of two lineages of influenza D viruses in equine populations of the Midwest United States

Influenza D virus (IDV) is a newly described influenza type of the Orthomyxoviridae virus family that was first isolated from diseased swine in 2011 and has subsequently been detected in cattle around the world in 2014. In addition, serological evidence for IDV infection in humans has been recently established. Despite all the progress, the full range of susceptible hosts for this novel virus has yet to be determined, but includes swine, bovine, small ruminants and human. This study was designed to determine if equine is a possible host to this newly emerging influenza virus. Three hundred and sixty‐four equine serum samples were collected in 2015 from 141 farms within the Midwestern United States. Serum samples were examined using hemagglutination inhibition (HI) assay against two established IDV lineages (D/OK and D/660) and one IDV‐related human ICV lineage (C/JHB). Results of this study showed 44 (44 of 364, 12%) samples positive for antibodies against D/OK, 39 (39 of 364, 11%) samples positive for antibodies against D/660, and 41 (41 of 364, 11%) samples positive for antibodies against C/JHB. A subset of these samples was further confirmed via microtitre neutralization (MN) assay. Our data demonstrated that horses are susceptible to two lineages of IDV, and that these viruses were present in equine populations throughout multiple Midwestern states of the United States. These findings continue to support the need for further surveillance of IDV viruses in agricultural species to work towards a better understanding of the full host range and natural reservoirs of influenza D virus.

Survival of viral pathogens in animal feed ingredients under transboundary shipping models

The goal of this study was to evaluate survival of important viral pathogens of livestock in animal feed ingredients imported daily into the United States under simulated transboundary conditions. Eleven viruses were selected based on global significance and impact to the livestock industry, including Foot and Mouth Disease Virus (FMDV), Classical Swine Fever Virus (CSFV), African Swine Fever Virus (ASFV), Influenza A Virus of Swine (IAV-S), Pseudorabies virus (PRV), Nipah Virus (NiV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Swine Vesicular Disease Virus (SVDV), Vesicular Stomatitis Virus (VSV), Porcine Circovirus Type 2 (PCV2) and Vesicular Exanthema of Swine Virus (VESV). Surrogate viruses with similar genetic and physical properties were used for 6 viruses. Surrogates belonged to the same virus families as target pathogens, and included Senecavirus A (SVA) for FMDV, Bovine Viral Diarrhea Virus (BVDV) for CSFV, Bovine Herpesvirus Type 1 (BHV-1) for PRV, Canine Distemper Virus (CDV) for NiV, Porcine Sapelovirus (PSV) for SVDV and Feline Calicivirus (FCV) for VESV. For the remaining target viruses, actual pathogens were used. Virus survival was evaluated using Trans-Pacific or Trans-Atlantic transboundary models involving representative feed ingredients, transport times and environmental conditions, with samples tested by PCR, VI and/or swine bioassay. SVA (representing FMDV), FCV (representing VESV), BHV-1 (representing PRV), PRRSV, PSV (representing SVDV), ASFV and PCV2 maintained infectivity during transport, while BVDV (representing CSFV), VSV, CDV (representing NiV) and IAV-S did not. Notably, more viruses survived in conventional soybean meal, lysine hydrochloride, choline chloride, vitamin D and pork sausage casings. These results support published data on transboundary risk of PEDV in feed, demonstrate survival of certain viruses in specific feed ingredients (“high-risk combinations”) under conditions simulating transport between continents and provide further evidence that contaminated feed ingredients may represent a risk for transport of pathogens at domestic and global levels.

Passive immunity to porcine epidemic diarrhea virus following immunization of pregnant gilts with a recombinant orf virus vector expressing the spike protein

Passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (PEDV). Here, we investigated the immunogenicity of an orf virus (ORFV) vector expressing the full-length spike (S) protein of PEDV (ORFV-PEDV-S) in pregnant gilts and its ability to confer passive immunity and protection in piglets. Three doses of ORFV-PEDV-S were given to two groups of PEDV-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. One of the two groups immunized with the ORFV-PEDV-S recombinant virus was also exposed to live PEDV orally on day 31 post-immunization (pi). Antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. The protective efficacy of ORFV-PEDV-S was evaluated after challenge of the piglets with PEDV. PEDV-specific IgG, IgA and neutralizing antibody (NA) responses were detected in ORFV-PEDV-S-immunized and ORFV-PEDV-S-immunized/PEDV-exposed gilts. PEDV NA, IgG and IgA were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. Piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after PEDV challenge in comparison to control piglets. Piglets born to gilts that received ORFV-PEDV-S and were exposed to live PEDV showed stronger NA responses and lower clinical scores when compared to piglets born to gilts immunized with ORFV-PEDV-S alone. These results demonstrate the potential of ORFV as a vaccine delivery platform capable of eliciting passive immunity against PEDV.