Angela E. Pillatzki

Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences

During the 10 days commencing April 29, 2013, the Iowa State University Veterinary Diagnostic Laboratory received the first 4 of many submissions from swine farms experiencing explosive epidemics of diarrhea and vomiting affecting all ages, with 90–95% mortality in suckling pigs. Histology revealed severe atrophy of villi in all segments of the small intestines with occasional villus-epithelial syncytial cells, but testing for rotaviruses and Transmissible gastroenteritis virus (Alphacoronavirus 1) were negative. Negative-staining electron microscopy of feces revealed coronavirus-like particles and a pan-coronavirus polymerase chain reaction (PCR) designed to amplify a conserved region of the polymerase gene for all members in the family Coronaviridae produced expected 251-bp amplicons. Subsequent sequencing and analysis revealed 99.6–100% identity among the PCR amplicons from the 4 farms and 97–99% identity to the corresponding portion of the polymerase gene of Porcine epidemic diarrhea virus (PEDV) strains, with the highest identity (99%) to strains from China in 2012. Findings were corroborated at National Veterinary Services Laboratories using 2 nested S-gene and 1 nested N-gene PCR tests where the sequenced amplicons also had the highest identity with 2012 China strains. Whole genome sequence for the virus from 2 farms in 2 different states using next-generation sequencing technique was compared to PEDV sequences available in GenBank. The 2013 U.S. PEDV had 96.6–99.5% identity with all known PEDV strains and the highest identity (>99.0%) to some of the 2011–2012 Chinese strains. The nearly simultaneous outbreaks of disease, and high degree of homology (99.6–100%) between the PEDV strains from the 4 unrelated farms, suggests a common source of virus.

Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States

ABSTRACT Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 102 to 2 × 105 50% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.

Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs

Porcine epidemic diarrhea virus (PEDV) is associated with clinical diarrhea in naïve swine of all ages. This report describes timing of antibody generation and disease progression following infection with a US PEDV isolate by assessing fecal viral shedding, morphometric analysis of intestinal lesions, and magnitude of immunohistochemical staining. Sixty-three, 3-week-old pigs were randomly allocated into control (n=27) and challenged (n=36) groups. Challenged pigs were administered 1 mL of 1 × 10(3) PFU/mL of US/Iowa/18984/2013 PEDV isolate by oro-gastric gavage. Three control and four challenged pigs were necropsied on days post-inoculation (dpi) 1, 2, 3, 4, 7, and weekly thereafter, until study termination on dpi 35. Clinical disease, fecal shedding, body weight, and temperature were monitored during the study period. Diarrhea was observed in challenged pigs beginning for some on dpi 2, affecting a majority of pigs by dpi 6 and subsiding by dpi 10. Average daily gain was significantly lower (P<0.001) for one week post-infection in challenged pigs. PEDV was detected in feces by PCR on dpi 1 and continued in a subset of pigs until dpi 24. PEDV-specific antigen was detected in villous enterocytes of challenged pigs by immunohistochemistry (IHC) on dpi 1, 2, 3, 4, 7, and 14. Microscopic lesions included severe diffuse atrophic enteritis with significantly reduced (P<0.001) villous length observed on dpi 3, 4, and 7. Under the conditions of this study, fecal shedding of PEDV and IHC staining can precede and continue beyond the observation of clinical signs, thus increasing the risk of viral transmission.

Respiratory Syncytial Virus Infection in Cattle

Bovine respiratory syncytial virus (RSV) is a cause of respiratory disease in cattle worldwide. It has an integral role in enzootic pneumonia in young dairy calves and summer pneumonia in nursing beef calves. Furthermore, bovine RSV infection can predispose calves to secondary bacterial infection by organisms such as Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, resulting in bovine respiratory disease complex, the most prevalent cause of morbidity and mortality among feedlot cattle. Even in cases where animals do not succumb to bovine respiratory disease complex, there can be long-term losses in production performance. This includes reductions in feed efficiency and rate of gain in the feedlot, as well as reproductive performance, milk production, and longevity in the breeding herd. As a result, economic costs to the cattle industry from bovine respiratory disease have been estimated to approach

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model

1 billion annually due to death losses, reduced performance, and costs of vaccinations and treatment modalities. Human and bovine RSV are closely related viruses with similarities in histopathologic lesions and mechanisms of immune modulation induced following infection. Therefore, where appropriate, we provide comparisons between RSV infections in humans and cattle. This review article discusses key aspects of RSV infection of cattle, including epidemiology and strain variability, clinical signs and diagnosis, experimental infection, gross and microscopic lesions, innate and adaptive immune responses, and vaccination strategies.

Pathogenesis of Senecavirus A infection in finishing pigs

ABSTRACT Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. IMPORTANCE Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs, similarly to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV.

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

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and pathogenesis, however, remain unknown. Here the pathogenesis of SVA was investigated in finishing pigs. Animals were inoculated via the oronasal route with SVA strain SD15-26 and monitored for clinical signs and lesions associated with SVA infection. Viraemia was assessed in serum and virus shedding monitored in oral and nasal secretions and faeces by real-time reverse transcriptase quantitative PCR (RT-qPCR) and/or virus isolation. Additionally, viral load and tissue distribution were assessed during acute infection and following convalescence from disease. Clinical signs characterized by lethargy and lameness were first observed on day 4 post-inoculation (pi) and persisted for approximately 2-10 days. Vesicular lesions were first observed on day 4 pi on the snout and/or feet, affecting the coronary bands, dewclaws, interdigital space and heel/sole of SVA-infected animals. A short-term viraemia was observed between days 3 and 10 pi, whereas virus shedding was detected between days 1 and 28 pi in oral and nasal secretions and faeces. Notably, RT-qPCR and in situ hybridization (ISH) performed on tissues collected on day 38 pi revealed the presence of SVA RNA in the tonsils of all SVA-infected animals. Serological responses to SVA were characterized by early neutralizing antibody responses (day 5 pi), which coincided with decreased levels of viraemia, virus shedding and viral load in tissues. This study provides significant insights into the pathogenesis and infectious dynamics of SVA in swine.

Episodic blindness and ataxia in a horse with cholesterinic granulomas.

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.

Experimental inoculation of neonatal piglets with feed naturally contaminated with porcine epidemic diarrhea virus

An 11-year-old Oldenburg mare presented following three episodes of acute, transient blindness, ataxia, and disorientation within the preceding 7 months. Clinical improvement, including return of vision, occurred within 1 week of initiating corticosteroid therapy for each of the three episodes. However, mild right-sided miosis was a consistent finding on ophthalmic examinations. Routine clinicopathologic testing revealed no significant abnormalities, and testing of cerebral spinal fluid for selected infectious diseases was unrewarding. Computed tomography of the brain demonstrated a hyperattenuating mass with peripheral mineralization in the rostroventral aspect of each lateral ventricle. The mare was euthanized due to a guarded to poor prognosis. On histopathology, the masses consisted of clusters of cholesterol clefts admixed with leukocytes, mineral deposits, and connective tissue. Cholesterinic granulomas of the lateral ventricles and hydrocephaly were diagnosed. Cholesterinic granulomas should be considered a differential diagnosis in horses presenting for intermittent blindness.