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Freeze-drying (lyophilization) of biologically active materials including attenuated live virus vaccine is an well accepted method for ensuring stability and preserving the vaccine potency. As freeze- drying does not ensure complete stability, studies for stability of freeze-dried vaccines after exposure at different temperatures are required. Heat stability of classical swine fever (CSF) virus vaccine is also urgently needed in tropical countries including India where the disease is still endemic. In this study, the thermostability of freeze- dried vials of live attenuated CSF vaccines were investigated at 4℃ and 25℃ for 30 days. This lyophilized vaccine containing combination of sucrose, lactose and lactalbumen hydrolysate gives good heat stability and viral infectivity was reduced by a factor of 0.2 to 1 Log10TCID50/mL at the end of freeze drying. In this experiment, we have studied storage stability of lyophilized classical swine fever vaccine produced at Division of Biological Standardization, IVRI using IVRI-CSF-BS vaccine strain which can be stored at -20℃ for more than a year without any loss. Stability at 4℃ and 25℃ may ensure easy transportation and minimize expense related to that.

Serotype-specific diagnosis of bluetongue virus (BTV) is necessary for sero-surveillance and taking effective control measures. The VP2 is the major serotype determining protein and BTV-1 is the most predominant serotype in India. In the present study, an indirect ELISA (i-ELISA) was optimized for the detection of serotype-specific antibody against BTV-1 serotype. The VP2 protein of BTV-1 was expressed in a prokaryotic expression system and used to optimize i-ELISA to detect VP2 antibodies of BTV-1 in serum samples of both small and large ruminants. Serum samples (n = 363) classified as positive and negative for antibodies to BTV-1 by serum neutralization test (SNT) and also positive and negative for BTV antibodies by c-ELSIA kit (VMRD, USA) were used to determine the cut-off value, diagnostic sensitivity (DSn), and diagnostic specificity (D-Sp) using receiver operating characteristic (ROC) analysis. The percent positivity (PP) value30.10% was accepted as the cut-off for i-ELISA at which DSn of 99.52% and D-Sp of 99.35% was observed with a 95% confidence interval. Further, there was no cross-reactivity with other available BTV serotypes in the country. The study indicated serotype-specific i-ELISA is sensitive, specific and suitable alternative to tedious SNT method for determining serotype. The assay will also help in the serotype-specific epidemiological studies and implementation of future control strategies including vaccination and selection of suitable serotype as a vaccine candidate.

Classical swine fever (CSF) is a highly contagious notifiable disease of pigs caused by CSF virus of Flaviviridae family. Previously, lapinized vaccines were used for the disease control, which has now been replaced with cell culture vaccines. Determination of virus titre is the key factor for development and quality control testing of classical swine fever (CSF) cell culture vaccines. Since CSFV is a non- cytopathic virus, an accurate method for the titration of this virus in cell culture has not yet been reported. Here we present a full proof method of titration of CSF cell culture viruses employing Fluorescent Antibody Technique (FAT) in 24 well plate cover slip culture of PK-15 cells. CSFV monoclonal antibodies (Mab) used in the test bind to the CSF virus particles in the cell cytoplasm of the infected cells and the immune-fluorescence signal is produced by subsequent binding of FITC conjugate with Mab. In this newly developed method, apple green fluorescence is observed in the cytoplasm of the infected cells as the virus multiplies only in the cytoplasm. The nucleus as well as the uninfected cells cytoplasm is stained red without any traces of green fluorescence. Thus, the test clearly differentiates a CSFV infected cell from the uninfected cells in the vicinity, if any, and also from the uninfected controls. The test can also quantify the accurate titres of CSF live viruses in the cell culture vaccines and hence it has wide application in routine virus titration applied for manufacturing of CSF cell culture vaccines, determination of accurate multiplicity of infection (m.o.i.) during infection and quality control of vaccines by the testing laboratories.

The classical swine fever (CSF) viruses are generally non-cytopathic in nature and hence titrations of these viruses are mostly done by immunological methods such as fluorescent antibody technique (FAT). Although FAT is a gold standard and virus titration can be done very accurately, it involves tedious steps and demand skilful technological interventions. Exaltation of NDV (END method) has been used in the past to detect some of the CSF viruses in cell culture wherein Newcastle Disease Virus (NDV) readily proliferates in presence of CSFV and produces cytopathic changes. In the present study, a newly developed CSF vaccine virus strain (IVRI-CSF-BS) has been characterized for NDV exaltation and the virus has been found to be END+. This feature has been exploited for titration of the vaccine virus in PK-15 cells. The virus titre obtained by the END method has been correlated with the titres obtained by FAT and a linear correlation has been established as the titre obtained by END method was always 2 log10 lesser than the FAT titre.