Natural co-infection of pigs with African swine fever virus and porcine reproductive and respiratory syndrome virus in India

Abstract

Porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF) are economically important diseases of pigs throughout the world. During an outbreak, all age groups of animals except piglets < 1 month of age were affected with symptoms of high fever, cutaneous hemorrhages, vomition with blood, diarrhea, poor appetite, ataxia, and death. The outbreak was confirmed by the detection of the N gene of the porcine reproductive and respiratory syndrome virus (PRRSV) and the VP72 gene of the African swine fever virus (ASFV) by PCR in representative blood samples from affected pigs followed by Sanger sequencing. Mixed infection was also confirmed by simultaneous detection of both the viruses using multiplex PCR. Phylogenetic analysis of both the viruses revealed that the outbreak was related to ASFV and PRRSV strains from China which were also closely related to the PRRSV and ASFV strains from the recent outbreak from India. The study confirmed the involvement of genotype II of ASFV and genotype 2 of PRRSV in the present outbreak. Interestingly, PRRSV associated with the present outbreak was characterized as a highly pathogenic PRRSV. Therefore, the present study indicates the possibility of future waves or further outbreaks of these diseases (PRRS and ASF) in this region. This is the first report of ASFV and PRRSV co-infection in pigs from India.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01203-y.

Introduction

Among diseases of infectious origin, viral pathogens are a major concern for profitable pig production and are responsible for huge economic losses to the pig farmers. Two such important viral diseases which were reported from India in the last decade are African swine fever (ASF) and porcine reproductive and respiratory syndrome (PRRS). Both ASF and PRRS are highly contagious diseases that have seriously damaged the swine industry worldwide. These diseases are still prevalent in many countries and co-infections with two or more pathogens are common in some pig herds [1, 2]. As the clinical signs of both these diseases are similar, it is difficult to identify the actual causative agents based only on clinical signs [3].

PRRSV, a member of the order Nidovirales, the family Arteriviridae, and the genus Arterivirus, is the causative agent of PRRS, an economically significant swine disease with clinical manifestations characterized by a notable rise in late-term abortions, stillbirths, and the birth of weak piglets, decreased farrowing rates in sows, severe respiratory disease, and high mortality rates in suckling and weaned pigs up to 100% and 10–20% in mature pigs [4–6]. India was free from PRRS until 2012. The first PRRS outbreak in India was reported in 2013 from the State of Mizoram [7]. Since its first outbreaks in 2013 in Mizoram, subsequent years’ (in 2015, 2016, and 2018) outbreaks of the disease have been reported from India [7–9]. Interestingly, PRRSV isolated from outbreak cases in India was characterized as highly pathogenic (HP-PRRSV). Now the disease is endemic in India.

ASF is a devastating hemorrhagic disease caused by the African swine fever virus (ASFV), a large DNA virus belonging to the genus Asfivirus of the family Asfarviridae [10], which can cause mortality up to 100% [11]. Affected pigs suffer from severe depression, high fever, bloody diarrhea, and cutaneous hemorrhages and show hemorrhagic lesions in visceral organs. Since its first report in Kenya in 1921 [12], the disease has been reported from the countries in Europe, Russian federation, China, and Myanmar [13]. Presently, the disease poses a serious threat to the pig industry in India. The OIE announced the first report of an ASF outbreak in India on May 21, 2020, stating that in 11 outbreaks, a total of 3701 pigs died (morbidity 38.45% and mortality 33.89%) in the Indian states of Assam and Arunachal Pradesh [10].

Although single and multiple (including triple or more viruses) infections with common viruses like porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are reported in pigs [14], report on such co-infection with ASF is not available from India. It is also observed that although a few repots on co-infection of ASF with porcine reproductive and respiratory syndrome virus are available from some parts of the world [15, 16], such co-infection has not been reported from India to date. The present study reports the natural occurrence of mixed infection of ASF and PRRS in pigs of India including the characterization of both the viruses from filed outbreak cases. This is the first report of occurrence of mixed infection of ASFV and PRRSV in pig population of India.

Materials and methods

Sample collection, viral nucleic acid isolation, and cDNA synthesis

During June 2022, an outbreak of disease in a pig population with symptoms of inappetence, high fever, cutaneous hemorrhages, vomition with blood, nasal discharge, diarrhea, and ataxia was observed in North bank districts of Assam, India. All affected pigs are domestic pigs and managed under intensive system of farming. Pigs from two different farms in the same locality were affected. Blood samples in EDTA were collected randomly from 36 numbers of affected pigs belonging to different age groups (1 to 3 months, 12; 4 to 6 months, 10; and above 6 months, 14). The blood samples were virus-inactivated by the addition of TRIZol (Sigma-Aldrich, USA) at 1:5 (blood:TRIZol) for RNA isolation and AL buffer (QIAGEN, Germany) at 1:1 (blood:AL buffer) for DNA isolation immediately after collection and were brought to the Animal Health laboratory of ICAR-National Research Centre on Pig, Rani, Guwahati, Assam, India, for further investigation. RNA was extracted from 500 μL of each TRIZol-added sample with the RNeasy Plus Universal Mini Kit (QIAGEN, Germany). RNA was eluted in 50 μL of elution buffer. The quantity and quality of the RNA were checked by a Nanodrop spectrophotometer (Thermo Scientific). Following the manufacturer’s instructions, cDNA was synthesized using the QuantiTect Reverse Transcription Kit (QIAGEN, Germany), and 1 μg of pure RNA was used for 20 μL of total reaction volume in each sample. Simultaneously, DNA was isolated from 200 μL of each AL buffer–added sample using DNeasy Blood & Tissue Kits (QIAGEN, Germany) and DNA was eluted in 50 μL of elution buffer. The DNA concentration was then measured using a Nanodrop spectrophotometer (Thermo Scientific).

Molecular detection of viral agents

ASFV and PRRSV were detected by simplex PCR as described by Lui et al. [17] and Wernike et al. [18], respectively. The samples were also screened for the presence of other important viral pathogens such as Japanese encephalitis virus (JEV) [19], porcine circovirus 2 (PCV2) [20], classical swine fever virus (CSFV) [21], porcine parvovirus (PPV) [22], and swine influenza virus (SIV) [23]. Multiplex PCR was performed using a set of reference primers targeting the partial VP72 gene of ASFV and the partial NSP2 gene of PRRSV [24]. The primers and expected PCR product size are shown in Supplementary Table 1.

Sequencing and phylogenetic analysis

The PCR products were further purified by PCR purification kit (Thermo Scientific) and sent to a commercial company (Eurofins Genomics India, Bangalore, India) for sequencing. Sequencing was performed by using the Sanger sequencing method. The sequencing data (forward and reversed) were aligned using the Cap3 Contig Assembly tool (http://www.insilico.uni-duesseldorf.de/Cap3.html) [25]. The aligned sequences of ASFV partial VP72 gene (OP748422.1) and PRRSV partial N gene (OP748387.1) were submitted to the GenBank database. Other sequences (submitted from various parts of India as well as from neighboring countries) available in GenBank were downloaded for percent nucleotide and amino acid identity analysis (Supplementary Table 2). Nucleotide and amino acid sequences were compared by MegAlign. The phylogenetic trees based on ASFV partial VP72 gene and PRRSV partial N gene were constructed by the neighbor-joining method (1000 replications) using MEGA11 software.

Results

Molecular detection, characterization, and analysis of PRRSV and ASFV DNA sequences from the field samples

Of 36 random blood samples screened by simplex PCR using species-specific primers for the presence of important porcine viral pathogens such as ASFV, PRRSV, JEV, PCV2, PPV, CSFV, and SIV from the outbreak, all (36) samples were positive for ASFV and seven samples were positive for combined infection of ASFV and PRRSV. No single infection of PRRSV was recorded from the outbreak case. It has also been observed that all samples were negative for viruses such as JEV, PCV2, PPV, CSFV, and SIV. Using ASFV- and PRRSV-specific primers, the presence of ASF (Supplementary Fig. 1a) and PRRS (Supplementary Fig. 1b) was confirmed through simplex PCR with the amplicon sizes of 828 bp and 387 bp, respectively. The presence of mixed infection was further confirmed by multiplex PCR with reference primers. The multiplex PCR showed the specific band size of 276 bp for PRRSV NSP2 gene and 190 bp for ASFV VP72 gene (Supplementary Fig. 2). The study also confirmed that the PRRSV associated with the present outbreak was highly pathogenic PRRSV (HP-PRRSV) with a PRRSV NSP2 gene band size of 276 bp. Two representative PCR amplified products of both PRRSV N gene and ASFV VP72 gene were sequenced with Sangers sequencing. The sequencing results showed good qualities of dendrogram for each sample. BLAST analysis revealed 100% homology of the sequences of PRRSV with other PRRSV sequences available in the NCBI GenBank database and BLAST analysis also revealed 100% homology of the ASFV partial p72 gene with the ASFV sequences available in the GenBank database (Supplementary File 1).

Phylogenetic analysis

The phylogenetic analysis results are based on the partial length ASFV VP72 gene and the partial length PRRSV N gene (Supplementary Fig. 3A, 3B).

A total of 25 ASFV VP72 gene sequences were retrieved from the NCBI GenBank database and the sequences were aligned by using MEGA11. The analysis showed two different major clusters and the isolates of ASFV from the present outbreak were very closely related to the ASFV isolates from India, 2020 (OM481275, OM481276). It has also been observed that the isolates were also closely related to the ASFV isolates from China (MN393476.1, MW361944, MK645909, MN172368, MK128995), Thailand (OM461372.1, OM461371.1), the Philippines (ON963982), Hungary (MN715134), Georgia (MH910496), and Spain (MK628478) within the same sub-cluster. The nearby sub-clusters of ASFV isolates from the present outbreak were from India (MZ063771, MZ063772, MT612961, MT612963, MT612964, MT612962), Indonesia (MT851941.1), Vietnam (MW825068.1), the UK (AM999764), and South Africa (AF270706).

A total of 18 PRRSV N gene sequences were retrieved from the NCBI GenBank database and the sequences were aligned by using MEGA11. The field isolates were closely related with the PRRSV isolates from India (ON804879.1, OK283406.1, OK283404.1, MT347585.1, and KT696491.1). The current field isolate cluster was closely related to the cluster with China (MN547966.1, MN026347.1, MN119308.1), Nepal (OP037987.1), Myanmar (MT411898.1, MF991926.1), Bhutan (GU930383.1), Taiwan (AF035409, AF030306), and Denmark (AJ223079). It was observed that the PRRSV N gene accession number such as AY366525 (USA) and AJ223078 (Denmark) formed separate sub-cluster from the sequence of the current study.

Discussion

ASF and PRRS are two economically important diseases of pigs which were first detected in India from the states of Arunachal Pradesh in 2020 [10] and Mizoram in 2013, respectively [7]. Due to the absence of vaccines with protective efficacy, both ASF and PRRS represent serious threat to the Indian piggery sector and also to the piggery sector of all the pig-rearing countries of the world. The present study confirmed the presence of mix infection of ASF and PRRS in the outbreak cases from India which is the first report of co-infection of both these viruses from India. Both morbidity (95.26%, 201 out of 211) and mortality (90.99%, 192 out of 211) were higher in the outbreak. The higher mortality observed in the study might be due to involvement of ASF in the outbreaks as mortality in acute cases of ASF may reach up to 100%. It was also observed that the PRRSV associated with the present outbreak was a highly pathogenic PRRSV.

ASF was first identified in Kenya in the 1920s [12]. Based on sequence variability in the partial B646L (P72) gene sequences, a total of 24 genotypes of ASFV from different countries have been identified to date [26, 27]. Of all the 24 genotypes of ASFV found in Africa, only genotypes I and II are found to persist in countries other than Africa to date [28, 29]. Genotype I was reported from Portugal in 1957, and it has since spread to other European countries, South America, and Caribbean island countries [30]. Genotype II migrated from southern Africa to Georgia in 2007 [31], and then to European countries in 2018 [32], and then introduced into Asia, where it spread to several Chinese provinces in 2018 and reached other Asian countries such as Vietnam, Cambodia, Laos, Korea, and India [10, 29, 33]. The phylogenetic analysis of the ASFV P72 gene has revealed that ASFV from the present outbreak case was closely related to previous outbreak of ASF in India, China, Thailand, the Philippines, Hungary, Georgia, and Spain. Therefore, it suggests that the ancestors of the Indian isolates of ASFV might have originated or were derived from neighboring Asian countries or European countries and the same genotype is currently circulating in India.

On the other hand, PRRSV originated as a “mystery” disease in swine herds in both Europe and North America in the late 1980s [34] and both PRRSV genotypes were eventually defined as type 1 (European) and type 2 (North American). The novel and highly virulent variant of PRRSV (HP-PRRSV), which arose almost simultaneously in the People’s Republic of China and Vietnam in 2006, has swiftly spread in pigs in Southeast Asian nations such as Bhutan, Cambodia, Laos, Malaysia, Myanmar, the Philippines, Thailand, and Singapore [35]. The nations of Eastern and northern Asia, as well as South Korea and Russia, were also reported to be affected [35]. The epidemic affected both the commercial and backyard pig population, creating a challenge or serious problem for the global swine industry and food safety [35]. In February 2013, India has confirmed an outbreak of HP-PRRSV in pig population of Mizoram, India, and subsequently, the disease has spread to different parts of India [7]. The phylogenetic analysis of the PRRSV N gene (ORF7) revealed that the PRRSV from the present outbreak was closely related to previous outbreak of PRRS in India during 2019 and 2021. The present study also revealed that the current field isolate was closely related to the isolates from neighboring countries like China, Nepal, Myanmar, Bhutan, and Taiwan and the same genotype is circulating in India and neighboring Asian countries.

The clinical signs observed in the present outbreak of mixed infection with ASFV and PRRSV were high fever (104 °F to 105 °F), dyspnea, cutaneous hemorrhages, vomition with blood, diarrhea, lethargy, ataxia, inappetence, and oozing of frothy-blood from the nose of dead pigs. The present findings corroborated with the findings of previous studies [36–38] who also observed similar clinical signs in ASFV-infected pigs. Clinical sings like severe depression, ataxia, high fever up to 41–42 °C, respiratory distress, and huddled together have also been reported by several researchers in PRRSV-affected pigs [39–41]. A recent study has also shown that the most common virus present in co-infection in pigs is PRRSV [42]. During early infection of PRRSV, a decrease in the number of CD4+ T cells has also been described [43]. PRRSV, which targets macrophages and dendritic cells in the respiratory tract, is generally believed to be able to impair the host immune system, resulting in increased susceptibility to other pathogens [44]. The co-infections in swine with PRRSV and other pathogens such as PCV2 [45], pseudorabies virus PRV [46], SIV [47], CSFV [48], hepatitis E virus (HEV) [49], porcine group A rotavirus (PARV), and porcine epidemic diarrhea virus (PEDV) [2] were commonly observed in clinical conditions. However, this is the first report of mixed infection of ASFV and PRRSV from a natural outbreak from India. In ASF infection, it is known that the virus activates monocytes/macrophages that secrete a wide range of mediators including pro-inflammatory cytokines such as IL-6 and TNF-α, which can trigger acute phase reactions, inflammation, activation of endothelial cells, and apoptosis [50]. In addition, ASFV affects the antigen presentation and activation of lymphocytes by invading monocytes-macrophages and dendritic cells, thereby affecting the immune system of the entire body [50, 51]. However, immune response mechanism will shift owing to co-infection, which has yet to be determined during co-infection of pigs with ASFV and PRRSV. Therefore, the present study warrants further in-depth research on the host-PRRSV and ASFV interactions during co-infection, host immune response, and the effectiveness of vaccination, etc.

In conclusion, the study reports the natural occurrence of mixed infection of ASFV and PRRSV in pigs in India with the involvement of genotype II of ASFV and genotype 2 of PRRSV. Further, multiplex PCR assay was found to be very useful for the detection of mixed infection of these viral infections.

Funding

This work was supported by funding from the Department of Biotechnology, Govt. of India, in the form of project entitled “Establishment of a consortium for One Health to address Zoonotic and Transboundary diseases in India, including NE region.”

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Declarations

Conflict of interest

The authors declare no competing interests.