Molecular characterisation of porcine reproductive and respiratory syndrome virus from pigs in Kerala

Abstract

Porcine reproductive and respiratory syndrome (PRRS) caused by an arterivirus is characterised by reproductive disorders in sows, and post-weaning pneumonia and growth reduction in piglets. Though the virus has been detected in Kerala, no systematic study has been carried out to ascertain its genotype and molecular epidemiology. In the present study, 7 PRRS virus (PRRSV) positive samples collected from incidences of PRRS in Kerala during 2017–2019 were subjected to ORF5, ORF7 and Nsp2 gene based reverse transcription polymerase chain reaction and the specific amplicons generated were sequenced. On BLAST analysis it was revealed that all the sequences were of genotype 2 (North American genotype). Phylogenetic analysis of ORF5 sequences, grouped them under subgenotype 4 with close clustering with other isolates from Kerala, Mizoram and Assam. Nsp2 gene sequence based phylogenetic analysis grouped the isolates under subgenotype 3 with similarities to isolates from Mizoram. Phylogenetic analysis based on ORF7, clustered the isolates under study with PRRSV isolates from Mizoram and Meghalaya. In Nsp2 sequences, a 30 amino acid discontinuous deletion was observed. On analysis of amino acid sequences of ORF5 of Kerala isolates and those from India, it was seen that the Kerala isolates showed closer similarity to PRRSV isolates from Assam than to the other Indian isolates. The study reveals that PRRSV strains prevalent in Kerala share close relationship with other PRRSV isolates in India. This may be due to spread of the virus from these regions to Kerala due to animal movement. Concerted efforts should be undertaken to check unauthorized animal movement to control spread of this economically important disease.

Electronic supplementary material

The online version of this article (10.1007/s13337-020-00634-7) contains supplementary material, which is available to authorized users.

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for causing reproductive failure and respiratory disorders along with an increased mortality rate of piglets, weanlings, growers and finishers. The reproductive failure is characterized by infertility, abortions in third trimester, stillbirths, or the birth of weak piglets that often dies soon after birth from respiratory disease and secondary infections. The respiratory syndrome is recognized by laboured breathing, fever, loss of appetite, listlessness, red discoloration of the body, and blue ears [11].

The virus has two genotypes, PRRSV 1 and 2, which are now known as Betaarterivirus suid 1 and 2 respectively and are classified under the order Nidovirales, family Arteriviridae and genus Betaarterivirus [7]. Genotype 1 is also known as the European genotype (prototype strain Lelystad virus) and genotype 2 is known as the North American genotype (prototype strain VR-2332) [1, 5]. PRRSV is an enveloped virus containing a single stranded, positive sense RNA genome which has 10 open reading frames (ORFs). Replication-related polymerase proteins are encoded by ORF1a and ORF1b, which are autoproteolytically cleaved into at least 16 non-structural proteins (Nsp) [8]. The ORFs 2, 3 and 4 encode for the minor structural glycoproteins GP2, GP3 and GP4, respectively. In addition to GP2, ORF2 encodes an alternative reading frame (ORF2b) for a small unglycosylated envelope protein (E). Further downstream, ORF5, ORF6 and ORF7 encode for the major structural protein GP5, matrix (M) and nucleocapsid (N), respectively. Besides the N-glycosylated GP5, that is involved in cell attachment, another small unglycosylated protein that is required for virus viability is translated from an alternative reading frame of ORF5, designated ORF5a [4]. Even though the two genotypes of PRRSV produce similar clinical illness, they have only 55–70% nucleotide and 50–80% amino acid similarity in their various genes [5]. PRRSV has a high degree of heterogeneity, high genetic and antigenic diversity [13] and isolates of same genotype may show remarkable differences in sequence mainly in the Nsp2 gene and ORF5 [2].

In India, PRRS was reported for the first time from Aizwal district of Mizoram in 2013 [14] and in Kerala the disease was detected in 2017 [16]. Though, the virus has been detected in Kerala, no systematic study has been carried out to ascertain the genotype and molecular epidemiology of PRRSV prevalent in pigs in Kerala. Hence, this study was undertaken to characterize PRRSV prevalent in Kerala by molecular methods.

A total of 7 PRRSV positive tissue samples (PRRSV/26MIB/PKD/2017, PRRSV/35aMIB/PKD/2017, PRRSV/74MIB/PKD/2018, PRRSV/91MIB/PKD/2018, PRRSV/165MIB/PKD/2018, PRRSV/245MIB/PKD/2018 and PRRSV/35MIB/PKD/2019) collected from incidences of PRRS in Kerala during 2017 to 2019 were taken for the study. The details of the samples are given in Supplementary Table 1. Total RNA was extracted from the tissue samples using GeneJet RNA purification kit (Thermo Scientific) and complementary DNA (cDNA) was synthesised from the RNA using RevertAid H Minus First strand cDNA Synthesis Kit (Thermo Scientific). The samples were subjected to ORF5, ORF 7 and Nsp2 gene based reverse transcription polymerase chain reaction (RT-PCR) as described previously [15]. The PCR products were electrophoresed in 1.5% agarose gels containing ethidium bromide and were visualised to identify the size of the product in a gel documentation system under UV illumination. Amplicons obtained were purified and extracted by PureLink Quick gel extraction kit (Invitrogen) and sent for sequencing (M/s AgriGenome, Kochi, Kerala).

The obtained sequences were subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm their identity. Phylogenetic analysis of 7, 6 and 2 sequences for ORF5, ORF7 and Nsp2 regions respectively along with the similar sequences obtained after BLAST and representative reference sequences (Supplementary Tables 2, 3, 4) was carried out by using MEGA X software [9]. Alignment of the sequences were done using Clustal W program of MEGAX, followed by trimming of the same to match sequence lengths obtained in this study. The evolutionary history was inferred by using Maximum Likelihood Method. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the sequences analysed. Determination of evolutionary distances were done by Kimura 2 parameter model for ORFs 5 and 7 while for Nsp2 it is done by Tamura 3 parameter model as they had the lowest Bayesian Information Criterion scores (BIC). Additionally, multiple sequence analysis of amino acid translations of ORF5 sequences of the Kerala isolates recorded previously (GenBank Accessions MG962353, MK010655, MK010654) and 32 ORF5 sequences of isolates from India (KM283189, KM283190, KM283192, KM283196–KM283198, MH220801–MH220814, KX869653–KX869658, MG561392, MG561393, MH665696, MH665697, MK764030, MK76031) was carried out with using MEGAX software.

In the present study all the seven samples yielded an amplicon of size 803 bp containing ORF5, five samples yielded amplicon of size 592 bp containing ORF7 and two samples yielded amplicon of size 558 bp containing a part of the Nsp2 region. BLAST analysis confirmed that the sequences were of genotype 2 (North American genotype) of PRRSV. The sequences have been deposited in GenBank under accession numbers MT274633–MT274645, MT277018 and MT277019.

BLAST analysis of ORF5 sequences obtained in this study revealed a high degree of homology with the other isolates from Kerala (97.97–99.89% identity). Mizoram isolates possessed 95.54–98.81% identity and isolates from Assam had a higher identity of 98.51–99.62% with Kerala isolates. The isolates had 96.35–97.51% nucleotide identity with the highly pathogenic PRRSV reference strain JXA1. ORF5 codes for a structural protein (major envelope protein) and has a high degree of genetic variation [12]. The principal neutralising epitope is located between amino acids 36–52 of this protein and is responsible for eliciting antibody production during PRRSV infection [13]. On the basis of ORF5 genotype 2 PRRSV could be further grouped under 4 subgenotypes [17]. The NADC30-like strains CHsx1401, JL580 and HENAN-HEB belonged to subgenotype (1) the low pathogenic PRRSV representative strain, CH-1a, belonged to subgenotype (2) which also included the North American type representative strain VR2332. The representative strains SHB and HB-1–3.9 belonged to subgenotype (3) All isolates from this study belonged to subgenotype (4) which also contained the highly pathogenic strains JXA1, JXwn06 and HuN4 and showed close clustering with the other isolates from Kerala and India (isolates from Mizoram and Assam) (Fig. 1).

Fig. 1.

Maximum Likelihood tree constructed from nucleotide sequences of ORF5 of PRRSV from Kerala. Legend: The symbols ‘filled circle’, ‘open circle’ and ‘open box’ depicts isolates under study, other isolates from Kerala and isolates from India, respectively. Boot strap replicates = 1000. Evolutionary distances between sequences were calculated by Kimura 2 parameter method. Branch length are given as 0.10 substitutions per position according to scale bar underneath the tree

On analysis of the amino acid sequences of the ORF5 gene, it was observed that at position 35, two isolates 74/MIB/2018 and 91/MIB/2018 had serine (S) residues whereas the other Kerala isolates and Indian isolates had asparagine (N). Similarly, at position 166, these isolates had arginine (R) instead of lysine (K). At position 38, all the Kerala isolates had arginine (R) except in isolate 165/MIB/2018 where it was histidine (H). At position 58, in isolate 35/MIB/2019, the residue was glutamic acid (E) whereas in the other Kerala isolates, it was glutamine (Q). At position 59, Kerala isolates had asparagine (N) except in 35/MIB/2019 where it was lysine (K). The most important peculiarity seen in all the Kerala isolates was at position 124, where it was Isoleucine (I). But in other Indian isolates it was valine (V) except in the case of two isolates from Assam where it was Isoleucine. Thus in the analysis of the amino acid sequences, it was seen that variations were present in Kerala isolates in positions 35, 38, 58, 59 and 166. As stated earlier, the principal neutralising epitope is located between amino acids 36 to 52 of this protein. In one of the Kerala isolates H was observed at position 38 whereas all the others had R. It was observed that most of the Indian isolates have H at this position (Table 1). Thus no unique amino acid change was observed in this region in Kerala isolates. In a study carried out in PRRSV isolates from Vietnam, it has been reported that amino acid positions 25, 33, 34, 35, 58, 59, and 104 of ORF5 were undergoing positive selection [3]. In Kerala isolates 74/MIB/2018 and 91/MIB/2018 position 35, and in isolate 35/MIB/2019 position 58, are having amino acids that have not been recorded in any Indian isolates analysed in this study (Table 1). Hence these unique changes may be due to the result of selective pressure.

Table 1.

Variations in amino acids in major envelope protein encoded by ORF5 of PRRSV from Kerala and India

Sample/accession no. (place)	Amino acid position in ORF 5
	35	38	58	59	124	166
PRRSV/26MIB/PKD/2017 (Kerala)	N	R	Q	N	I	K
PRRSV/35aMIB/PKD/2017 (Kerala)	N	R	Q	N	I	K
PRRSV/74MIB/PKD/2018 (Kerala)	S	R	Q	N	I	R
PRRSV/91MIB/PKD/2018 (Kerala)	S	R	Q	N	I	R
PRRSV/165MIB/PKD/2018 (Kerala)	N	H	Q	N	I	K
PRRSV/245MIB/PKD/2018 (Kerala)	N	R	Q	N	I	K
PRRSV/35MIB/PKD/2019 (Kerala)	N	R	E	K	I	K
MG962353 PRRSV/CDIO/MB350 (Kerala)	N	R	Q	N	I	K
MH010654 PRRSV/CDIO/MB407 (Kerala)	N	R	Q	N	I	K
MH010655 PRRSV/CDIO/MB408 (Kerala)	N	R	Q	N	I	K
MK764030 (Assam)	N	R	Q	N	I	K
MK764031 (Assam)	N	R	Q	N	I	K
Other Indian isolates	N	H	Q or K	N or K	V	K

On BLAST analysis of Nsp2 sequences, the Kerala sequences had 87.75–90.14% identity to those from Mizoram. As the Nsp2 gene region is highly variable, it is extensively used in genetic diversity studies [6]. Based on Nsp2 gene sequences, PRRSV has been categorized into 3 subgenotypes [17]. On phylogenetic analysis of Nsp2 gene sequences, the two isolates from Kerala clustered under subgenotype 3 along with isolates from Mizoram and representative strains HB-1–3.9, HB-1 sh 2002, JXA1-P80 and JX 143 (Fig. 2). Representative strains of subgenotype 1 include VR2332 and that of subgenotype 2 include CH1a and SHB. Sequence alignment also revealed discontinuous deletion of 90 nucleotides in both these Kerala isolates. These deletions have been reported in Indian isolates previously also [15]. The deletions in Nsp2 region were predicted to be immunologically important [10] but does not correlate with the virulence of the virus [19]. The presence of this deletion was previously regarded as a marker for highly pathogenic PRRSV [20]. However, it has now been detected in large number of low-pathogenicity strains also [18].

Fig. 2.

Maximum Likelihood tree constructed from partial nucleotide sequences of Nsp2 gene of PRRSV from Kerala. Legend: The symbols ‘filled circle’ and ‘open box’ depicts isolates under study and that from Mizoram, respectively Boot strap replicates = 1000. Evolutionary distances between sequences were calculated by Tamura 3 parameter method. Branch length are given as 0.5 substitutions per position according to scale bar underneath the tree

In the case of ORF7 gene sequences, BLAST analysis revealed that the Kerala isolates had 96.02–97.31% identity with Meghalaya isolates, 94.86–97.85% identity with Mizoram isolates and 95.22–97.58% identity with PRRSV reference strain JXA1. Phylogenetic analysis based on ORF7 showed close clustering between isolates from Meghalaya and Mizoram and the isolates under the study (Fig. 3).

Fig. 3.

Maximum Likelihood tree constructed from nucleotide sequences of ORF7 of PRRSV from Kerala. Legend: The symbols ‘filled circle’, ‘open circle’ and ‘open box’ depicts the isolates under study, isolates from Meghalaya and Mizoram, respectively. Boot strap replicates = 1000. Evolutionary distances between sequences were calculated by Kimura 2 parameter model method. Branch length are given as 0.5 substitutions per position according to scale bar underneath the tree

PRRS is an economically important disease of pigs characterized by reproductive failure of sows and respiratory problems of piglets and growing pigs. Results of the study indicate that PRRSV strains prevalent in Kerala are of genotype 2, are genetically diverse, and that they share close genetic relationship with strains prevalent in other parts of India. This may be due to spread of the virus from these regions to Kerala. Efforts should be undertaken to check unauthorized animal movement which will go a long way in controlling the spread of this economically important disease within the country.

Electronic supplementary material

Below is the link to the electronic supplementary material.