Isolation and molecular phylogenetic analysis of porcine circovirus 2 from Indian pigs with respiratory lesions

Introduction

Porcine circovirus type 2 (PCV2) is a small, non-enveloped virus belonging to the family Circoviridae and the genus Circovirus, characterised by a circular single-stranded DNA genome of approximately 1.7-2.0 kb (Franzo et al., 2016) [1] . The family Circoviridae comprises viruses infecting a wide range of avian and mammalian hosts, including important pathogens such as chicken anaemia virus (CAV), porcine circovirus (PCV), and psittacine beak and feather disease virus (PBFD). Among these, PCV2 is recognised as one of the most economically significant viral pathogens affecting the global swine industry (Murphy et al., 1999) [2] . PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVD), a multifactorial disease complex characterised by systemic illness, enteritis, interstitial pneumonia, and reproductive failure (Segales, 2012) [3] . In addition to overt clinical disease, PCV2 infection commonly occurs in a subclinical form, which may result in reduced growth performance and increased susceptibility to secondary infections (Segales, 2012) [3] . Owing to the substantial economic and health impact of PCVD, reliable and sensitive diagnostic methods are essential. Polymerase chain reaction (PCR)-based assays provide a practical and sensitive approach for the routine detection and epidemiological surveillance of PCV2 infection in veterinary diagnostic laboratories (Hamel et al., 2000) [4] . In this context, the present study was undertaken to investigate respiratory infections in pigs in and around the Hassan region and to confirm PCV-2 using molecular diagnostic techniques.

Materials and Methods

Sample collection

Pigs exhibiting respiratory infections were included in the study for the confirmation of porcine circovirus type 2 (PCV-2) by polymerase chain reaction (PCR) during the period from January to December 2022 in Hassan district, Karnataka, India. A total of 108 samples (Table 1) were collected, along with relevant epidemiological data, including age, sex, breed, and gross pathological lesions observed in the lungs and other organs. Tissue samples comprising lungs, tonsils of the soft palate, lymph nodes, heart, and spleen were collected aseptically and stored at -20 °C until further molecular analysis.

Polymerase chain reaction:

Using the HiPurA® Mammalian Genomic DNA Purification Kit (HiMedia Laboratories Pvt. Ltd), total DNA was recovered from pooled organ tissue samples. The sequence of primers used are as given below (Ellis et al., 1999) [5] . After 45 minutes of electrophoresis at 80 V, the amplicons were examined on a 2% agarose gel. Image Lab Software (Version 5.1, Bio-Rad Laboratories Inc.) was used to document the PCR product in the gel.

Custom sequencing:

The PCR-amplified product of the PCV-2 capsid protein gene obtained from tissue samples was subjected to custom nucleotide sequencing using a single-pass sequencing approach, employing the same primers used for PCR amplification. Sequencing was performed using an Applied Biosystems genetic analyzer (Inst Model/Name: 3730xl/XE3-IOT-2019-1406-029). Sequence data were generated using both forward and reverse primers.

Sequence data analysis and phylogenetic analysis:

The forward and reverse read sequence chromatograms were initially examined to resolve ambiguous bases and trim the sequence ends of each chromatogram. The obtained PCR sequences from different locations were then aligned using the Clustal W method (DNASTAR Lasergene programme). The programme also calculated sequence similarity and divergence. All of the putative genes included in the current investigation of Indian strains of PCV-2 capsid protein gene sequences were downloaded from the Genbank data base for comparative sequencing analysis. Sequences from both India and other countries were included. The MegAlign programme of DNASTAR Lasergene software was used to generate the maximum likelihood phylogenetic tree and the phylogenetic tree using the Porcine reproductive and respiratory syndrome virus strain with the highest homology.

Results

Out of the 108 pig samples collected from the slaughterhouses of the Hassan region, 87 animals showed respiratory-related lesions.

Molecular confirmation of PCV-2 by PCR

In the present study, tissue samples comprising lung, tonsils, bronchial lymph nodes, spleen, heart, and brain were collected and screened for PCV-2 using a nucleocapsid gene-specific PCR assay. Out of the 87 samples analyzed, 61 yielded the expected amplicon of 481 bp (Fig. 1) indicating an overall PCV-2 prevalence of 70.11%. The sequences of the five positive samples revealed similarities to other PCV-2 isolates, confirming the presence of PCV-2 in the samples. In terms of similarity, the field isolates matched the PCV-2 completely. Using NCBI blast, the nucleotide sequences obtained from the 481-base pair PCV-2 capsid protein PCR products coincided with the PCV-2 nucleotide sequences available in GenBank. In order of highest similarity, the field isolates revealed 100% resemblance to the PCV-2 previously reported ( The research areas were located in four distinct agroclimatic zones; most of the affected animals were from Hassan Taluk, which was located in the district's Southern dry zone (74.41%).

Discussion

In the present study, 61 of 87 animals exhibiting respiratory lesions tested positive for PCV-2 by PCR, resulting in a prevalence of 70.11%, which is higher than the prevalence rates of 26-60% reported in earlier studies (Deka et al., 2021) [6] . These findings indicate that PCV-2 is highly prevalent among pigs affected with porcine respiratory disease complex (PRDC) and should be considered a major respiratory pathogen, thereby supporting the observations reported previously (Deka et al., 2021; Opriessnig et al., 2006) [6,7] . The results of the present investigation are also consistent with earlier studies documenting significant respiratory disease in pigs from the Hassan district, with comparable findings reported by researchers from different regions of the country (Yaeger et al., 2019) [8] . Age-wise distribution of PCV- [11] . The polymerase chain reaction (PCR) assay is a rapid and essential tool for the detection of porcine circovirus type 2 (PCV-2), demonstrating greater sensitivity and faster turnaround than virus isolation, immunohistochemistry, and in situ hybridization. Confirmation of PCV-2 infection has been achieved through the detection of 481 bp and 225 bp amplicons corresponding to the ORF2 region in pig samples collected between 1985 and 1998 (Jacobsen et al., 2009) [12] . The detection of PCV-2 nucleic acids using PCR with specific primers designed to amplify defined genomic fragments has also been previously reported (Ellis et al., 1999) [5] . In addition, PCR assays targeting the nucleocapsid gene and yielding a 481 bp amplicon in pooled organ samples have been described, which is consistent with the findings of the present study (Keerthana et al., 2019; Sairam et al., 2019) [13,14] . Respiratory disease in pigs represents a major health concern for swine producers, with reports from the National Animal Health Monitoring System identifying it as the leading cause of mortality in nursery and growerfinisher operations (Brockmeier et al., 2002) [15] .

Conclusion

A high occurrence of porcine circovirus type 2 (PCV-2) among pigs with respiratory lesions highlights its important role in the porcine respiratory disease complex. The effective detection of PCV-2 using polymerase chain reaction underscores the value of molecular diagnostics for accurate surveillance. Future efforts should focus on largescale epidemiological studies, continuous molecular monitoring, and the evaluation of targeted prevention and management strategies to reduce the impact of PCV-2associated respiratory disease in swine production systems.

Acknowledgement

AcknowledgmentsThe authors thank Dean Veterinary College, Hassan, for laboratory facilities and DIPGS, KVAFSU, for their support.

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