Development And Initial Application Of An Immunoassay For The Discrimination Between PRV-infected And Vaccinated Animals

Pseudorabies (PR), or Aujeszky’s disease (AD) is one of the most significantinfectious diseases affecting the pig rearing, causing heavy economic losses each year.The disease is characterized by acute and often fatal infections in piglets, abortions insows, and retarded growth or weight losses in growing pigs. Its causative agent,pseudorabies virus (PRV; also called Suid herpesvirus1or Aujeszky’s disease virus)has a double-stranded DNA genome of about150kb in length and infects a broadrange of wild and domestic animals. Pigs are the natural host and reservoir for PRV.The first report of PR in China was in the1950s and the disease has beenreported in many provinces. From2005to2010, PR outbreaks amongst swine herdsin Henan province were well-controlled. Since2011, however, in several parts ofChina there have been a large number of PR outbreaks which have devastated manyswine farms even though the herds had been previously immunized with gE-deletedvaccines (Bartha-K61). The emergence of these outbreak-associated PRV strainsmight indicate that Bartha-K61vaccine could not provide effective protection andposes challenges for the control and prevention of PR. Here, we performed serologicaland molecular epidemiology to investigate PR spread in Henan province, andexpressed the gE and gB protein from a typical outbreak-associated PRV strainTangyin to develop ELISAs for the discrimination between PRV-infected andvaccinated animals (DIVA) and the evaluation of antibody levels upon vaccination.Sero-epidemiology showed that PRV infection rate suddenly rose up to38.0%in2012and maintained at31.3%in2013. Infection rates in breeding pigs and fatteningpigs were36.3%and59.3%respectively. Among the347swine herds detected in2013, positive herds accounted for83.6%, with the highest infection of93.8%in thenorthern part of Henan. These data indicated that PR is widely spread in Henan andhard to control. Phylogenetic analyses based on partial gE, gB, and gC genes from thetwelve outbreak-associated strains isolated in this study displayed that those strainslocated in an independent branch and were closely related to other outbreak- associated reported in other regions of China. The genetic relationship between theseoutbreak-associated PRV strains and strain Bartha is not close, which may affect theimmune protection provided by Bartha-K61vaccination.Then we cloned the gE and gB genes from an outbreak-associated PRV strainTangyin (GenBank No. KP009871(gE) and KP009895(gB)) for protein expression.gE protein was expressed as soluble and gB protein was expressed as inclusion bodyin Escherichia coli (E. coli). Monoclonal antibodies (MAbs) againstimmunodominant epitopes on gE and gB protein were produced by immunizingBALB/c mice with Ni-NTA affinity purified or ion exchange purified protein, andscreened out using blocking enzyme-linked immunosorbent assay (blocking ELISA)and blocking immunoperoxidase monolayer assay (blocking IPMA). MAb10C3F3and10C7C10against the immunodominant epitope of gE and gB protein respectivelywere each conjugated with horseradish peroxidase (HRP). Indirect ELISA andblocking ELISA for the detection of anti-PRV antibodies were established and appliedin DIVA. The diagnostic sensitivity (DSN), diagnostic specificity (DSP), andagreement of indirect gE-ELISA with a commercial gpI-ELISA (IDEXX) were88.76%,79.15%, and94.03%respectively; the DSN, DSP, and agreement of indirectgB-ELISA with a commercial gB-ELISA (IDEXX) were93.64%,77.78%, and93.14%respectively. The DSN, DSP, and agreement of blocking gE-ELISA with acommercial gpI-ELISA were96.18%,89.90%, and94.10%respectively; the DSN,DSP, and agreement of blocking gB-ELISA with a commercial gB-ELISA were95.90%,85.37%, and94.07%respectively. The developed indirect and blockinggE-ELISA and gB-ELISA were used to evaluate PRV status (stable or active) inseveral clinical swine farms. Using pig-anti-gE hyperimmune sera and the anti-gEMAb or anti-gB MAb, a sandwich ELISA for the detection of PRV viral antigen wasdeveloped. The sandwich ELISA is able to detect PRV viral antigen treated by sonicsor NP-40with a detection limit of105.0TCID50/0.1mL. Besides, animmunochromatographic lateral flow strip test based on the sandwich format wasdeveloped and applied in detecting PRV viral antigen. The sandwich ELISA and thestrip test for gE antibody detection can react specifically with PRV viral antigen andpossess no reaction with the Bartha vaccine strain and normal BHK-21cell controls. The sandwich ELISA and the strip test for gB antibody detection react with both PRVfield strain and the Bartha vaccine strain, possessing no cross-reaction with normalBHK-21cell controls.In conclusion, we conducted serological and molecular epidemiology ofoutbreak-associated PRV strains in central China by analyzing partial gE, gB, and gCgenes and suggested that these strains might originate from earlier local isolates,established indirect and blocking gE-ELISA and gB-ELISA for DIVA and evaluatingvaccination status, and developed a sandwich ELISA and colloidal gold-based striptest for the detection of PRV viral antigen. This work is helpful for understandingPRV epidemiology, diagnostics, and vaccination, and may contribute to theelimination of PR from domestic animals in China.