Development And Validation Of Antigen Capture ELISA For Detection Of Group-A Porcine Rotavirus

Rotavirus, a member of Reoviridae family, is one of the globally emerging zoonotic viruses, causing dehydrating diarrhea leading to high mortalities in children and neonates of a wide range of animals. In piglets, group- A rotavirus infection is mostly appeared in both enzootic and epizootic diarrhea form, leading to a vast range of economic losses in weaning and postweaning piglets, in terms of decreased weight gain, morbidities, and mortalities.Rotaviruses are widely reported to transfer interspecies barrier, thus pose public health concerns. The specific objective of this study was to develop a rapid, sensitive and specific antigen capture ELISA for detection of porcine rotavirus, followed by genotyping of porcine rotavirus-A (PoRVA) in China.1. Cloning, expression and purification of recombinant VP6 proteinThe genome of rotavirus consists of total 11 double-stranded (ds) RNA segments contained within triple-layered, non-enveloped, icosahedral viral particles of about 100nm diameter in size. Gene segment number 6 encodes middle capsid protienVP6, highly conserved(about 87-92%) among mammalian group-A rotaviruses (GARVs); therefore, has higher significance in the diagnosis of GARVs. Due to higher antigenic properties, the VP6 gene was cloned from a Chinese porcine rotavirus-A (PoRVA) strain TM-a, amplified by PCR. A recombinant vector pGEX-KG-VP6 was constructed, transformed into a competent prokaryotic host E.coli strain JM105, and was then culture in LB medium. IPTG was used to induce protein expression. The resulting purified recombinant VP6 protein (rVP6) of about 70kDa was then used to immunize mice and rabbits for the production of monoclonal antibodies (Mab) and polyclonal antibodies (Pab).2. Development of monoclonal antibodies based AC-ELISA2.1 Monoclonal antibodies productionFor the development of VP6 protein specific Mab, we immunized BALB/c mice with 100?g of rVP6 emulsified in Freund's Complete Adjuvant (FCA). Three booster doses were administered with the same concentration of rVP6 emulsified in Freund's Incomplete Adjuvant(FIA), at two-week intervals. The mice were injected intraperitoneally three days prior to cell fusion experiment. Mice with higher titers of antiserum were selected for cell fusion experiment. The spleens were dissected to obtain the splenocytes, which were later mixed with Sp2/0 myeloma cells (at a ratio of 5:1) to develop hybridoma. Indirect-ELISA, western-blot analysis, and indirect fluorescence assay were performed to screen for positive hybridomas against rVP6 protein and PoRVA. The hybridomas were cloned thrice by limiting dilution method. A panel of two superior hybridoma clones secreting Mab 1A7 (IgG1) and 3F5 (IgG2a)were selected on the basis of their strong reaction with both VP6 and PoRVA in indirect-ELISA.However, the antibody titer of 3F5 was two-fold higher (1:25600) than that of 1A7 (1:12800).Superior hybridoma clones were injected intraperitoneally into paraffin-primed BALB/c mice.Ten days later, ascites was harvested and IgGs were precipitated in saturated ammonium sulfate,followed by dialysis and purification on a Protein-G affinity column (GE, Sweden) using the manufacturers' instructions. The reactivities of the both Mab were further verified by western blot analysis and indirect fluorescence assay.2.2 Polyclonal antibodies productionFor the development of rabbit anti-rVP6 Pab, ten-week-old New Zealand white rabbits were injected subcutaneously at multiple sites with 1mg (1mg/ml) purified rVP6, emulsified in the same amount of FCA. Booster doses were administered at two-week intervals with 1mg of the same antigen emulsified in FIA. Ten days after the third booster dose, the serum samples were collected and checked for antibody titers by indirect ELISA, using rVP6 (1?g/well) as coating antigen. Pab were purified using commercial protein-A affinity purification kit (GE,Sweden) using the manufacturers' guidelines.2.3 Optimization of Mab-based AC-ELISAA rapid, highly specific, and sensitive Mab-based Antigen-Capture Enzyme-Linked Immunosorbent Assay (AC-ELISA) was developed for detection of PoRVA, by employing VP6 protein directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). A checkerboard titration was carried out to select the optimal concentrations of rabbit anti rVP6 Pab (capture Ab),mice anti VP6-Mab (detection Ab) and HRP-goat anti-mouse Mab,by using rVP6 as a positive control. All ninety-six wells of the polystyrene microtiter ELISA plate were coated with 250ng of rVP6 rabbit Pab in 100?l coating buffer (0.05M sodium carbonate-bicarbonate buffer, pH 9.6). Blocking was done with 200?l of blocking buffer (3% BS A diluted in phosphate buffer saline+0.05% Tween-20; PBST),to cover free sites and prevent non-specific binding. The plates were incubated with 50?l/well of a 10% (w/v) fecal suspension, 100?l mouse anti VP6 Mab (100ng/well) diluted in blocking buffer (1%BSA), HRP goat anti-mouse IgG 1: 10,000 (ABclonal, USA) 100?l/well (diluted in blocking buffer, 1%BSA). Each step received incubation at 37? for 1h, followed by washing four times with wash buffer (PBST). Finally, 100?l tetra-methyl-benzidine (TMB) was added to each well for color development. Absorbance was measured using ELx800 Absorbance Reader (BioTek, Winooski, VT, USA) at a wavelength of 630nm. The cutoff value was set at mean absorbance value of 25 porcine GARV-negative fecal samples plus 3x standard deviations (SD). The detection limit of AC-ELISA was found to be equal to that of conventional RT-PCR (about 102.5 TCIDso/ml).2.4 Validation of Mab-based AC-ELISAFor validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR, and TaqMan RT-qPCR. The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa=0.972) between these two techniques. Total detection rates with AC-ELISA, conventional RT-PCR and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without statistically significant difference. Moreover,AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudo-rabies virus,and porcine circovirus type 2. These results suggested that our developed method was rapid,highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.3. Development of Pab-based AC-ELISAA simple, swift, cost-effective, highly specific, and sensitive Pab based AC-ELISA was also developed for detection of PoRVA by employing rabbit (capture antibody) and murine anti-VP6 polyclonal antibodies (detector antibody) produced against rVP6 of PoRVA (RVA/Pig-wt/CHN/TM-a/2011/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 and PoRVA. The detection limit of AC-ELISA was found 50ng/mL of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from 295 porcine diarrhea samples, and results were compared with that of RT-qPCR and a commercial rotavirus AC-ELISA kit. The relative sensitivity and specificity of AC-ELISA, as compared to RT-qPCR were found as 94.4% and 100%, respectively. Furthermore,AC-ELISA could not detect any cross-reactivity with PEDV, TGEV, PRV and PCV type 2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples. Development of this simple Pab based AC-ELISA will particularly encourage less established diagnostic laboratories in low economic countries to manufacture their own diagnostic kits, where deficiency of funds and skills are the hurdle in producing Mab or purchase of expensive commercial diagnostic kits. However, Mab-based AC-ELISA provided balanced results in terms of sensitivity and specificity than that of Pab-based AC-ELISA, but its development is difficult and requires more funds, as compared to Pab-based AC-ELISA. These in-house developed kits will effectively serve in both timely detection and surveillance of rotavirus infection in Chinese porcine farms.4. Genotyping of PoRVA isolates in ChinaAs we know that PoRVA strains with the G5 and P[7] combination are mainly prevalent in piglets, that's why mostly focused for the vaccine production. However, recent studies have demonstrated that new RVA strains with a common G-genotype in both humans and piglets are emerging worldwide. Unfortunately, there have been no any recent studies on the proportion of different G-genotypes of PoRVA prevailing in China, therefore, in the current study, total 295 clinical samples consisting of feces of diarrheic piglets were collected from 40 pig farms in 16 provinces of Chinese mainland from January 2015 to June 2016. Out of these 40 pig farms, rotavirus was detected from 15 farms (37.7%). Rotavirus could be consistently detected throughout the year, without any significant difference among the months. In total, 17 samples were successfully sequenced. The VP7 gene of total 67 available and background-clear Chinese PoRVA strains,(including VP7 sequences of 17 strains detected in the current study and 49 downloaded from GeneBank) were subjected to phylogenetic analysis. Out of 13 G9 detections, 12 strains were closely related (99%) to Chinese PoRVA YNR strain. Results revealed that among 17 isolated strains in this study, G9 (76.5%, 13/17) was the most dominant genotype, followed by G5 (17.6%, 3/17), and G11 (5.9%, 1/17). Moreover, VP7 sequences of all of these 67 RVA strains were successfully G-typed as G2(1.5%, 1/67), G3(4.5%, 3/67),G4(13.4%, 9/67), G5(19.4%, 13/67), G9(52.2%, 35/67), G11(6%, 4/67), and G26(1.5%, 1/67).In conclusion, this study helped us to better understand the prevalence and molecular epidemiology of different PoRVA G-genotypes in China.5. ConclusionsIn conclusion, we successfully expressed and purified rVP6 protein, and applied it for the production of Mab and Pab in mice and rabbits, respectively. The resultant Mab and Pab were used for the development of swift, highly specific, and sensitive VP6 Mab and Pab-based AC-ELISA methods that efficiently detected PoRVA in fecal samples. Results obtained from the validation of theses AC-ELISA methods revealed that G9 genotype is the most dominant genotype in China.