Evaluation of an alternative method for determination of Protective Dose50 of Classical swine fever vaccines.

Classical Swine Fever (CSF) is still one of the most economically important viral diseases of pigs. The disease is controlled by vaccination in the endemic countries. Hence, availability or supply of efficacious and potent vaccine in the field settings is of utmost importance. Currently, as per requirement of any Pharmacopoea, a CSF vaccine must contain 100 PD50/dose which is determined by vaccinating pigs at 1/40th and 1/160th dilution of each dose followed by virulent challenge at 28 days post vaccination (dpv). Here, the control and the unprotected groups succumb to disease and need to be euthanized. Moreover, such challenge experiments are not feasible for each batch of the vaccine. In this communication, an alternate method of PD50 dose calculation of live-attenuated CSF vaccines by measuring Serum Neutralizing Titre i.e Fluorescent Antibody Virus Neutralization (FAVN) titre of the vaccinated pigs at 28 dpv was established. This alternative method do not require the vaccinated pigs to be challenged. Serum samples, generated out of QC testing of eight batches of CSF vaccines in the laboratory, were tested and found that pigs having FAVN titre ≥10 were protected against challenge. Initially this test was optimized in serum samples of 12 animals and then validated with another 56 serum samples. It was found that the alternate method is 100% correlating with the challenge experiment. Thus, based on FAVN titre of the vaccinated animal serum, it can be predicted whether the pigs would or would not come through the challenge infection. Using the predicted status (protected/succumbed), PD50 can be calculated by applying Reed and Muench formula, hence alternate method can be used as routine QC test for potency of CSF vaccines. The newly developed assay was specific since no signal was observed in controls.

Genomic characterization of Lumpy Skin Disease virus (LSDV) from India: Circulation of Kenyan-like LSDV strains with unique kelch-like proteins.

Lumpy skin disease (LSD) is an economically important poxviral disease endemic to Asia, Europe, and Africa. Recently, LSD has spread to naïve countries, including India, China, Bangladesh, Pakistan, Myanmar, Vietnam, and Thailand. Here, we describe the complete genomic characterization of LSDV from India, LSDV-WB/IND/19 isolated from an LSD affected calf in 2019 determined by Illumina next-generation sequencing (NGS). The LSDV-WB/IND/19 has a genome size of 150,969 bp encoding 156 putative ORFs. Phylogenetic analysis based on complete genome sequence suggested that LSDV-WB/IND/19 is closely related to Kenyan LSDV strains with 10-12 variants with non-synonymous changes confined to LSD_019, LSD_049, LSD_089, LSD_094, LSD_096, LSD_140, and LSD_144 genes. In contrast to complete kelch-like proteins in Kenyan LSDV strains, LSDV-WB/IND/19 LSD_019 and LSD_144 genes were found to encode truncated versions (019a, 019b, and 144a, 144b). LSD_019a and LSD_019b proteins of LSDV-WB/IND/19 resemble that of wild-type LSDV strains based on SNPs and the C-terminal part of LSD_019b except for deletion at K229, whereas the LSD_144a and LSD_144b proteins resemble that of Kenyan LSDV strains based on SNPs, however, C-terminal part of LSD_144a resembles that of vaccine-associated LSDV strains due to premature truncation. The NGS findings were confirmed by Sanger sequencing of these genes in Vero cell isolate as well as in the original skin scab along with similar findings in another Indian LSDV from scab specimen. LSD_019 and LSD_144 genes are thought to modulate virulence and host range in capripoxviruses. This study demonstrates the circulation of unique LSDV strains in India and highlights the importance of constant monitoring of the molecular evolution of LSDV and associated factors in the region in light of the emergence of recombinant LSDV strains.

Genome-wide transcriptome profiling of CSF virus challenged monocyte-derived macrophages provides distinct insights into immune response of Landrace and indigenous Ghurrah pigs.

The present study was undertaken to characterize the distinct immune response in indigenous Ghurrah and exotic Landrace pigs by challenging monocyte-derived macrophages (MDMs) with CSF virus under in-vitro conditions and assessing the variations in the transcriptome profile at 48 h post-infection (hpi). RNA-sequencing was carried out in infected and non-infected MDMs of Ghurrah (n = 3) and Landrace (n = 3) piglets prior- as well as post-stimulation. MDMs of Ghurrah showed greater immune regulation in response to CSF infection with 518 significantly differentially expressed genes (DEG) in infected versus non-infected MDMs, as compared to only 31 DEGs in Landrace MDMs. In Landrace, the principal regulators of inflammation (IL1α, IL1ß and TNF) were upregulated in infected cells while in Ghurrah, these were downregulated. Overall, macrophages from indigenous Ghurrah showed more immunological dysregulation in response to virulent CSF virus infection as compared to the exotic Landrace pigs.

Development and validation of an in vitro titrimetric method for determination of classical swine fever viruses in PK-15 cells.

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.

Stability of live attenuated classical swine fever cell culture vaccine virus in liquid form for developing an oral vaccine.

Classical swine fever (CSF) is an important viral disease of pigs and controlled by vaccination. Unorganised backyard and wild pigs are difficult to vaccinate by needle vaccination. Here we formulated liquid vaccines using an Indian CSF cell culture vaccine virus and four stabilisers and evaluated their stability at 4 °C, 25 °C and 37 °C up to 24 h for use as oral vaccine. The stabilisers were Lactalbumin hydrolysate-Trehalose, Lactalbumin hydrolysate-Trehalose-Gelatin, Lactalbumin hydrolysate-Lactose-Sucrose and Lactalbumin hydrolysate-Sucrose. The liquid vaccines, with or without stabilisers, were stable at 4 °C up to 24 h, whereas, a drop of one log10 titre was observed at 25 °C during the same period. At 37 °C, the virus titre diminished by only one log10 with the Lactalbumin hydrolysate-Trehalose (LT) stabiliser up to 24 h compared to two log10 losses in virus titre with other stabilisers and virus control. We therefore conclude that for developing a CSF oral vaccine, the vaccine virus in liquid form can be used directly during the winter, whereas for developing the oral vaccine for summer, the LT stabiliser would provide maximum stability to the virus to withstand the warm temperature while maintaining adequate therapeutic titre for inducing a protective immune response.

Comparing the efficiency of different Escherichia coli strains in producing recombinant capsid protein of porcine circovirus type 2.

The present study was aimed at comparing different E. coli strains in expressing the capsid protein of Porcine Circovirus 2 (PCV2). Full length capsid protein could be expressed only in Rosetta-gami 2 (DE3) pLysS strain using pET32b (+) vector. This confirmed that only those strains which possess tRNAs for rare codons can express the full length capsid protein. Purification of full length capsid protein could not be achieved even after several attempts using native and denaturing conditions. Subsequently, an attempt was made for expression of N-terminal truncated capsid protein using the same expression system. Truncated capsid protein was successfully expressed, purified and characterized by western blotting. The truncated capsid protein was also shown to be efficacious in testing serum samples using an optimized indirect ELISA, wherein a diagnostic sensitivity of 88.89% and specificity of 90.82% was obtained as compared to commercially available GreenSpring® porcine circovirus (PCV2) ELISA test kit. Thus, the expressed truncated capsid protein appears to be a promising diagnostic agent for PCV2. The comparative analysis suggests that cluster of arginine residues at N-terminal of capsid protein not only affects its expression in some E. coli strains but also its purification by Ni-NTA chromatography, when expressed as a histidine tagged fusion protein.

Characterization of defective interfering (DI) particles of Pestedes petitsruminants vaccine virus Sungri/96 strain-implications in vaccine upscaling.

The present investigation deals with the characterization of defective interfering (DI) particles of Peste-des-petits ruminants (PPR) vaccine Sungri/96 strain generated as a result of high MOI in Vero cells. During the serial 10 passages, infectivity titres drastically reduced from 6.5 to 2.25 log10TCID50/ml at high MOI. Further, attenuation of CPE with high MOI indicated generation of DI particles that resulted in no/slow progression of CPE during the late passages. Monoclonal antibody based cell ELISA indicated normal protein (N & H) packaging in samples with DI activity. At genomic level, inconsistency in amplicon intensity of H gene was observed in RT-PCR, indicating a possible defect of H gene. Further analysis of copy number of PPRV by RT-qPCR indicated intermittent fluctuations of viral genomic RNA copies. The significant decline of viral RNA copies with MOI 3 (314 copies) compared to low MOI (512804 copies), proved that higher DI multiplicities cause more interference with the replication process of the standard virus. Therefore, MOI is critical for manufacturing of vaccines. These investigations will help in upscaling of PPR vaccines in view of ongoing National and Global PPR control and eradication programme.

An overview of process intensification and thermo stabilization for upscaling of Peste des petits ruminants vaccines in view of global control and eradication.

Peste des petits ruminants (PPR) has been recognized as a globally distributed disease affecting the small ruminant population. The disease results in severe economic losses mainly to small land holders and low input farming systems. The control of PPR is mainly achieved through vaccination with available live attenuated vaccines. The thermo labile nature of PPR virus poses a major constraint in production of quality vaccines which often results in vaccine failures. The lack of quality vaccine production jeopardize the wide vaccination coverage especially in countries with poor infrastructure due to which PPR persists endemically. The vaccine production system may require augmentation to attain consistent and quality vaccines through efforts of process intensification integrated with suitable stabilizer formulations with appropriate freeze drying cycles for improved thermo tolerance. Manufacturing of live attenuated PPR vaccines during batch cultures might introduce defective interfering particles (DIPs) as a result of high multiplicity of infection (MOI) of inoculums, which has a huge impact on virus dynamics and yield. Accumulation of DIPs adversely affects the quality of the manufactured vaccines which can be avoided through use of appropriate MOI of virus inoculums and quality control of working seed viruses. Therefore, adherence to critical manufacturing standard operating procedures in vaccine production and ongoing efforts on development of thermo tolerant vaccine will help a long way in PPR control and eradication programme globally. The present review focuses on the way forward to achieve the objectives of quality vaccine production and easy upscaling to help the global PPR control and eradication by mass vaccination as an important tool.

Biological and molecular characterization of classical swine fever challenge virus from India.

AIM: The aim of this study was biological and molecular characterization of classical swine fever (CSF) challenge virus from India. MATERIALS AND METHODS: CSF challenge virus maintained at Division of Biological standardization was experimentally infected to two seronegative piglets. The biological characterization was done by clinical sign and symptoms along with postmortem findings. For molecular characterization 5'-nontranslated region, E2 and NS5B regions were amplified by reverse transcription polymerase chain reaction and sequenced. The sequences were compared with that of reference strains and the local field isolates to establish a phylogenetic relation. RESULTS: The virus produced symptoms of acute disease in the piglets with typical post-mortem lesions. Phylogenetic analysis of the three regions showed that the current Indian CSF Challenge virus is having maximum similarity with the BresciaX strain (USA) and Madhya Pradesh isolate (India) and is belonging to subgroup 1.2 under Group 1. CONCLUSION: Based on biological and molecular characterization of CSF challenge virus from India is described as a highly virulent virus belonging to subgroup 1.2 under Group 1 along with some field isolates from India and Brescia strain.

Recent epidemiology of peste des petits ruminants virus (PPRV).

Peste des petits ruminants (PPR) is an economically important viral disease of goats and sheep first described in west Africa in the 1940s. The virus has been circulating in parts of sub-Saharan Africa for several decades and in the Middle East and southern Asia since 1993, although the first description of the virus in India dates to 1987. To study the genetic relationship between isolates of distinct geographical origin, a selected region of the fusion (F) protein gene of the viruses was amplified using RT/PCR and the resulting DNA product sequenced for phylogenetic analysis. Viruses from 27 outbreaks in Asian and Middle Eastern countries, reported between 1993 and 2000, and two recent outbreaks from the African continent were compared with the prototype African strain. Of the four known lineages of PPR virus, lineage 1 and 2 viruses have been found exclusively in west Africa. Virus from an outbreak in Burkina Faso in 1999 fell into the lineage 1 group. Viruses of lineage 3 have been found in east Africa, where an outbreak in Ethiopia in 1996 was of this type, and also in Arabia and in southern India. However, there have been no further isolations of lineage 3 virus from India since the one reported in 1992 from Tamil Nadu. A virus of this lineage was found circulating in Yemen in 2001. In the past 8 years virus exclusively of the fourth lineage has spread across the Middle East and the Asian sub-continent, reaching east as far as Nepal and Bangladesh. This virus lineage was also reported from Kuwait in 1999. The geographical source of the new lineage 4 virus is unknown although it is most closely related to African lineage 1. The possibility that its earlier presence in northern India was masked by the circulation of Rinderpest virus, a related virus of cattle, is considered unlikely.