Date Full Report Received

11/04/2015

Date Abstract Report Received

11/04/2015

Investigation

Institution:
Primary Investigator: ,
Porcine deltacoronavirus (PDCoV) has recently emerged in the US. The purpose of this investigation was to determine the sites of tissue localization, routes of viral shedding, duration of virus carriage, kinetics of antibody response, and potential of aerosol transmission of porcine deltacoronavirus (PDCoV) following inoculation of nursing pigs and their dams.

Experimental Animals: Approximately 100 newborn pigs and their dams were obtained from a high health commercial source. All animals were housed in five BSL2 isolation rooms at the Life Science Annex at the University of Nebraska-Lincoln.

Numbering/Grouping: The litters/animals were grouped as indicated in the table below: Group A, (estimated 8 pigs/litter) was PDCoV inoculated. The contact control Group B (2 pigs/litter) were comingled with the inoculated Group A animals. The aerosol transmission Group C (estimated 10 pigs per litter) were not inoculated, but were housed in a separate pen in the common animal room. Negative, source matched controls (Group D) were housed in a separate wing of the animal facility and will serve as proof of freedom of herd source infection since it is not known how prevalent PDCoV is in the field.

Group Treatment # of Animals
A PDCoV oronasal inoculated 47 (6 litters)
B None—Contact Control 8 (2 from 4 litters)
C None—aerosol transmission controls 20 (2 litters)
D Negative Controls 10 (1 litter)
A-Dams PDCoV oronasal inoculated 6
C-Dams None—aerosol transmission controls 2
D-Dams Negative Controls 1
Challenge: The challenge was a pool of gut derived intestinal content that was obtained from a field case and amplified in nursing age pigs to confirm infectivity and purity. The inocula was “titered” for PDCoV nucleic acid content using a real-time PCR assay. Nursing pigs to be challenged and their dams were inoculated at 2-3 days of age via intranasal and oral routes with 5 ml of inocula per route.
Sampling Requirements/Challenge Scheduling: On the days of blood collection, a serum tube was drawn. The sera were used for validation of various antibody assays (IFA, ELISA/Luminex, and SN). Sera, oral fluids, and nasal and fecal swabs were used for the determination of the presence of viral nucleic acid using RT-PCR. At regular time points, inoculated pigs were euthanized and a thorough postmortem examination was conducted. Complete sets of tissues were collected and frozen or fixed in formalin for evaluation of disease severity by histopathology and location of virus within tissues and cell types by PCR and IHC. Tissue samples collected were turbinates, tonsils, esophagus, stomach, duodenum, jejunum (two segments), ileum, cecum, spiral colon, descending colon, trachea, lungs (representative samples from cranial, middle, and caudal lobes), liver, spleen, kidney, thymus, and submandibular, tracheobronchial, mesenteric and inguinal lymph nodes.
PDCoV shedding was monitored by performing real-time PCR on fecal and nasal swab samples and oral fluids. Serum samples were collected in order to monitor viremia and antibody response. Fresh and formalized tissues were collected from randomly selected Group A pigs at days 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 14, 21, 28, 35, and 42 post-inoculation in order to monitor tissue tropism of the virus and histopathology.
Study data indicate the following:
Summary of clinical signs:
Clinical signs following infection:
• Inoculated sows developed soft feces on day 2 post-inoculation and diarrhea on day 3 post-inoculation.
• All sows were clinically normal after day 8 post-inoculation.
• Aerosol contact sows developed soft to diarrheic feces on day 5 of the study and returned to normal after day 8.
• Non-infected control sows remained clinically normal throughout the study.
• Inoculated piglets developed soft to diarrheic feces on day 2 post-inoculation, as did the contact controls.
• Morbidity was 100%.
• Mortality was variable among the litters, but was especially high in two of the six inoculated litters. All piglets from those two litters were either euthanized due to severe dehydration or had died by day 8 post-inoculation.
• Aerosol contact litters developed diarrhea on days 3 and 4. All surviving piglets had returned to normal by day 12.
• Non-infected control piglets remained clinically normal.
Virus detection via PCR demonstrated that:
• There was no significant difference between detection rates of inoculated animals versus contact control animals.
• The negative-negative control group remained free of virus throughout the duration of the study.
• Viremia was evaluated in a subset of pigs post inoculation due to IACUC restrictions for blood volume/size of the animal. Viremia was detected at one day post-inoculation in 60% of the pigs tested and remained present in all of the pigs tested until four days post-inoculation, at which time the percent positive fell to 25% at day five and all animals were negative for viremia thereafter.
• Virus detection rates in fecal and nasal samples were generally similar, with fecal samples tending to have a higher virus load than the nasal samples.
• In contrast to viremia, fecal and nasal samples were negative for virus at one day post-inoculation.
• In both sample types, virus was detected at high to moderate levels from 2 to 10 days post-inoculation. Approximately 70% of the samples were positive at day 14, 25% of the samples were positive at day 21, and no samples were positive at 28 days post-inoculation and thereafter.
• Oral fluid samples were positive at the first collection point at 14 and 21 days post-inoculation. The day 28 samples contained low levels of virus in 4 of the 6 infected litters and were negative for virus in all litters at 5 and 6 weeks post-inoculation.
• Virus is clearly detected in the lymph nodes and small intestine of all of the infected animals (sows and their nursing pigs) at 35 and 42 days post-inoculation. In contrast, fecal and nasal samples were negative during those same time points.
Virus detection via immunohistochemistry (IHC) in tissues demonstrated that:
• Rabbit anti-PDCoV nucleocapsid antibody that was kindly provided by Dr. Eric Nelson from SDSU works well as a detection antibody for IHC.
• At 1 day post-inoculation, all PDCoV-inoculated pigs displayed clear positive staining in enterocytes of the jejunum and ileum.
• All inoculated pigs sampled continued to be positive in enterocytes of the jejunum and ileum on days 2 through 6.
• The percentage of individuals with positive staining in the jejunum and ileum quickly declined through days 7 and 8, and no positive staining was observed in samples from day 10 and thereafter.
• On days 2-5 and 8, the majority (71-100%) of the mesenteric lymph nodes were positive. No mesenteric lymph nodes were positive on day 10 or thereafter.
• Other immunopositive tissues included duodenum, cecum, and spiral colon. A lower percentage of pigs had positive staining in these other intestinal segments (typically varying from 0 to 50%), and often the staining affected a low number of scattered enterocytes.
o Positive staining in the duodenum was observed days 2-6. The spiral colon was only detected positive on days 2 and 3, and cecum samples were variably positive on days 2, 3, 4, and 6.
• No positive immunoreactivity was observed in other tissues examined.
• Pigs from the contact control and aerosol control sacrificed at their selected time points were similar to the inoculated pigs.
• Consistent with the inoculated pigs, no positive immunoreactivity was observed in tissues other than the intestinal tract and mesenteric lymph node.
• The sacrificed negative control pigs remained negative by immunohistochemical analysis throughout the study.
Histologic evaluation demonstrated that:
• Mild to severe atrophic enteritis was detected in sections of jejunum and ileum from days 2-8 post-inoculation. No lesions were observed in sampled pigs on day 10 or thereafter.
• These lesions were characterized by enterocyte vacuolation and attenuation, villous blunting, and occasional villous fusion.
• These lesions correspond to the virus distribution in the jejunum and ileum detected with immunohistochemistry.
• No lesions other than atrophic enteritis were associated with virus infection.
Antibody response via the indirect fluorescent antibody (IFA) and serum neutralization (SN) assays:
• Cell culture adapted virus that was kindly provided by Dr. Sabrina Swenson from the NVSL was used to infect cells that served as the detection substrate for an indirect fluorescent antibody assay and a serum neutralization assay.
• The negative/negative control animals (sows and their pigs) were PCR negative and also negative in the IFA and SN assays throughout the entire study.
• Inoculated animals (sows and their nursing pigs), contact controls, and room aerosol controls all seroconverted and were shown to be IFA and SN positive by 14 days post-inoculation and remained positive through the conclusion of the study at 42 days post-inoculation.
Important finding that may impact transmission of the virus in the field:
• Pigs still harbored detectable virus RNA in the small intestine and mesenteric lymph nodes 42 days post-inoculation.
• Sows still harbored detectable virus RNA in the small intestine and mesenteric lymph nodes 35 days post-inoculation.
• Aerosol transmission readily occurred.
Important gaps in our knowledge of PDCoV—where there is smoke there is usually fire.
• Understand transmission.
– Biological and physical routes.
• Define age differences following PDCoV infection.
– Duration of shedding.
– Persistence.
• Develop a good understanding of virus stability
• Investigate national and regional sero prevalence
• Develop efficacious vaccines.
– Lactogenic immunity