The current study was conducted with CSFV positive samples representing all the North-Eastern states.
Isolation and characterization of field isolates
Overall 77 different samples were collected from domestic pigs (70), wild hogs (2), pygmy hogs (5) of North-eastern states of India including Sikkim. The presence of CSF virus in the tissue samples and lyophilized isolates were confirmed by S-ELISA and nRT-PCR. In S-ELISA, 43 samples out of 77 were positive whereas, 47 out of 77 were positive in nRT-PCR for targeting E2 gene of 271bp product size (Fig 1) from all the NE states. Total 22 samples from Assam were found S-ELISA positive and 24 in RT-PCR out of 38 samples, in Arunachal Pradesh 1 was found RT-PCR positive out of 4, in Meghalaya, all 11 samples collected were S-ELISA and RT-PCR positive, in Mizoram, out of 6, 1 positive in S-ELISA and 4 in RT-PCR, in Nagaland only 2 samples were S-ELISA positive out of 5, in Sikkim only 1 sample was RT-PCR and S-ELISA positive, out of 2 and in Tripura 6 were positive in both S-ELISA and RT-PCR out of 11. As per OIE, both S-ELISA and nRT-PCR were suitable tools for detection of CSF virus in samples
(Anonymous, 2020).
Li et al., (2018) performed ELISA for detection and confirmation of CSF virus in serum samples making it an appropriate detection assay. RT-PCR and E2 nRT-PCR can be the appropriate approach for screening of CSFV
(Rout et al., 2015; Thakuria et al., 2015).
Hence, overall positivity rate was 55.8% in S-ELISA and 61% in nRT-PCR. As RNA viruses gets degraded easily, therefore 100% samples could not be recovered emphasizing development of robust biorepository protocol.
A total 5 CSFV isolates with their known genotypes representing NE states were cultured in PK-15 cell line which showed no CPE
(Anonymous, 2020). Isolation represents a classical gold standard method for CSFV diagnosis. PK-15 has been reported to be suitable amongst other cell lines
(Basumatary, 2017;
Anonymous, 2020). Out of total 5 isolates, one isolate from Assam could not be revived in cell culture. The other four isolates representing Meghalaya, Mizoram, Tripura, Sikkim were successfully revived.
Also, molecular characterization of these selected isolates was done by successful amplification of E2 full-length gene at 1119 bp (Fig 2) as it is highly immunogenic and has neutralizing epitopes. For in-depth characterization of CSFV, E2 full-length gene was preferred to get a high-resolution analysis in order to find out genetic homology, diversity and evolution of the virus according to what the EU and OIE Reference Laboratory recommended for CSFV molecular epidemiology
(Postel et al., 2012, Beer et al., 2015). Complete E2 gene sequencing enabled precise phylogenetic analysis, distinguishing closely related CSFV isolates from outbreaks with associated epidemiology
(Postel et al., 2012). Previous findings have classified CSFV into three main genotypes- 1, 2 and 3, which have been further divided into various emerging subgenotypes. Within Genotype 1, seven subgenotypes were identified (1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7). Genotype 2, has three subgenotypes (2.1, 2.2, 2.3)
(Silva et al., 2017, Garrido
Haro et al., 2018). Moreover,
Rios et al., (2018) reported that the genotype 2 CSFV strains were reorganized into seven subgenotypes (2.1–2.7) due to suggestions to rename the formerly identified subgroups under the subgenotypes 2.1 and 2.2. Genotype 3 was also observed to encompass four subgenotypes (3.1, 3.2, 3.3, 3.4)
(Zhou, 2019).
Phylogenetic analysis on the basis of E2 full-length gene sequences formed two distinct clads. Strains CSFV_AAU_As01, CSFV_AAU_Sk01 and CSFV_AAU_Tr01 from Assam, Sikkim and Tripura, respectively were phylogene-tically close at nucleotide level to the CSFV sequences from already submitted sequences to GenBank from Assam, India (MT232843.1, Pig/India-Assam/96/2017-CSFV) and to sequence from Uttarakhand, India (KC851953.1, CSFV IND-UK-LAL-290) and clustered under Genotype 2.2. Also, CSFV_AAU_Mg01and CSFV_AAU_ Mz01 from Meghalaya and Mizoram shared close phylogenetic relationship with the standard reference sequence (KM262189.1) CSFV/IVRI/VB-131 from India and (X87939.1) Alfort/187 that belonged to genotype 1.1 (Fig 3). The current findings reveal that three isolates out of five belonged to genotype 2.2 indicating about the shift from genotype 1.1 to 2.2. The genogroup 1 (historical CSFV strain) is highly virulent causing acute cases; genogroup 2 and 3 strains are moderately virulent causing sub-acute and chronic cases
(Chander et al., 2014, Bajwa et al., 2015). This study too reveals that the isolates from Assam, Sikkim and Tripura are clustered under Genotype 2.2. Various studies indicated that genogroups other than genogroup 1.1 of CSFV strains have emerged in India too. Since 1980s, China reported emergence of genotype 2.1
(Gong et al., 2016). The shift from genotype 1 to 2 was observed globally at different time periods, as it was reported from Europe, China, Columbia and Taiwan
(Malik et al., 2020). In India too, genogroup 2.2 viruses spread recently and replaced subgroup 1.1 viruses
(Patil et al., 2012). NE India too notified genotype 2.2 outbreak from wild hog and domestic pigs in nearby sanctuary
(Barman et al., 2014). The percent nucleotide sequence similarity analysis showed that the isolates from Meghalaya and Mizoram belonging to genotype 1.1 revealed 99% nucleotide identity when compared with the standard Alfort/187 strain. Whereas nucleotide identity of isolates from Assam, Sikkim and Tripura belonging to genotype 2.2 revealed 84% nucleotide identity when compared. However, pairwise identity matrices revealed 99% nucleotide identity within the genotypes (Fig 4). The complete genome-based nucleotide for Indian 1.1 strains showed 92.075% - 96.38%, similarity and complete genome of Indian 2.2 strains showed 83.425- 84.99% similarity, which corroborates the present findings in the study
(Malik et al., 2020). The above-mentioned isolates representing each state of north-east and belonging to different genotypes were further selected for isolation in PK-15 cell line.
Analysis of conserved linear immunogenic region of E2 gene was done by comparing the amino acid sequence analysis to a standard reference virus Alfort/187, the linear immunogenic conserved motif of CSFV isolates (“TAVSPTTLR”) using CLUSTAL W. The analysis revealed that the immunogenic component remained unchanged for all the five isolates following its propagation and passage till 5 passages in PK-15 cell line (Fig 5). This result corroborated with the findings of
Kumar et al., (2015), Anindita (2018). As per reports, the linear immunogenic epitope of E2 “TAVSPTTLR” remains highly conserved
(Wang et al., 2015), although the N- terminal half of E2 gene is considered unstable
(Chen et al., 2010). But the 16% difference in pairwise identity of genotype 2.2 in comparison with genotype 1.1, in the present finding brings concern to check for the nucleotide alteration if any, which might lead to divergence of the genotypes. Further, extensive studies will be required to determine the amino acid arrangements and alterations in the immunogenic domain of E2 gene. Furthermore, the linear epitope “TAVSPTTLR” located in domain A, which has been employed in the development of epitope-based vaccines
(Reimann et al., 2010; Tarradas et al., 2011), has been extensively documented to exhibit high conservation among various CSFV strains but not in other Pestiviruses.
Neutralisation and cross-neutralisation assay based on 100 TCID50
Representing each NE states with different genogroup, total 5 isolates were propagated in PK-15 cell line and confirmed in-situ by indirect IPT test with reddish brown cytoplasm and unstained nucleus confirming replication of the non-CPE producing virus in the cytoplasm (Fig 6). For neutralization assay, hyperimmune sera was raised which showed 1:800 and 1:1600 titres in I-ELISA. Western blot analysis was done by coating with E2 protein, which reacted strongly with hyperimmune sera giving a sharp band at 40 kDa size (Fig 7). This confirmed presence of E2 specific antibodies in the hyperimmune sera raised against C-strain vaccine.
Using this hyperimmune serum, virus titration of each sample was performed by Fluorescent Antibody Virus Neutralization (FAVN)
(Anonymous, 2020). CSFV infected PK-15 cell cytoplasm exhibited bright fluorescence (Fig 8). Neutralization efficacy was evaluated by FAVN. Immunofluorescence test was preferred due to its recognized sensitivity and specificity compared to immunoperoxidase staining
(Jafari et al., 2015; Zhang et al., 2017). Although CSF virus neutralization can be done following any of the two methods namely Neutralization Peroxidase Linked Assay (NPLA) and FAVN. 100 TCID
50 was determined to perform neutralization assay. Infectivity titre at 5
th passage varied in the range of 4.49 to 5.16 among the field isolates. Highest log TCID
50 was recorded in CSFV_AAU_Mg01 of Meghalaya - 105.16 log TCID
50 per ml for CSFV (Table 2). The dilution factors for representative isolates were determined to make the final concentration of 100 TCID
50 per 100µl volume required for neutralization test.
One way cross- neutralisation titre of CSFV field isolates were compared with hyperimmune sera raised against CSFV vaccine by FAT following standard protocols
(Anonymous, 2020). The virus-antibody mixture failed to produce any infection showing unstained cell cytoplasm in PK-15 cell. The end point of 50% neutralization titre of the hyperimmune serum was found to range from 1/133 to 1/158 when assayed against the different viruses (Table 2). In between 1:160 and 1:320, 50% neutralization can be seen as 1-3 wells out of five wells showed fluorescence of the infected monolayer. Upon comparison of the field isolates with vaccine virus it was found that CSFV_AAU_Mg01, CSFV_AAU_Mz01 of genotype 1.1 reacted equally with the homologous vaccine virus. Whereas, the vaccine virus antibodies showed lower neutralizing titre for the isolates CSFV_AAU_Sk01, CSFV_AAU_Tr01 of genotype 2.2. Despite retention of protective titre, neutralization assay using an antibody specific to the C-strain vaccine, alongwith cross-neutralization assay revealed that genotype 1.1 was completely neutralized (100%), whereas 84% neutralization was observed in genotype 2.2. Genotype 2.2 depicted 16% difference in neutralization efficacy. This might also indicate an alteration in the hyper-variable region of E2 region between genogroup 1 and 2
(Liao et al., 2016). An alteration in the nucleotide sequence will reflect change in protein expression of ‘TAVSPTTLR’. Further studies are required for distinguishing the antigenic variation between the different genotypes.
Chen et al., (2010) evaluated the neutralization efficiency of monoclonal antibodies that targeted both the vaccine C-strain and heterologous subgroup 2.1 viruses which revealed reduced efficiency in binding to and neutralizing subgroup 2.1 strains. In a cross-neutralization experiment, it was observed that the anti-C-strain serum significantly diminished its capacity to neutralize RecC-HZ-E2 and QZ-14 (a 2014 subgroup 2.1d field isolate) and conversely, the efficacy of RecC-HZ-E2 and QZ-14 in neutralization was also reduced
(Liao et al., 2016). Therefore, present study findings demonstrated the emergence of genotype 2.2 that might overcome the neutralizing efficacy of the vaccine antibodies. However, an extensive study should be undertaken with large field samples from different geographical locations as well as at different time points. Cross–neutralization must be done in both ways along with challenge study for reconsideration of new candidate against CSF virus.
