Research Progress on Detection Technologies for Porcine Circovirus

Porcine circovirus disease is a class of viral infectious diseases caused by porcine circovirus (PCV) infection and characterized by immunosuppression, with the main clinical manifestation being postweaning multisystemic wasting syndrome in piglets caused by PCV-2. This article reviews the research progress in recent years, both domestically and internationally, on detection technologies for this virus, including virus isolation, electron microscopy, polymerase chain reaction, indirect immunofluorescence assay, immunohistochemistry, enzyme-linked immunosorbent assay, and in situ nucleic acid hybridization.

Porcine circovirus disease is a chronic disease that has attracted much attention in recent years and is widely present throughout the world; it is a general term for a series of diseases. Since Tischer I et al.[1] first discovered porcine circovirus (PCV) in 1974, with the deepening of research on PCV, it can be divided, according to the pathogenicity of porcine circovirus and its genomic differences, into non-pathogenic porcine circovirus type 1 (PCV-1) and pathogenic porcine circovirus type 2 (PCV-2). PCV-1 is not pathogenic to pigs but can produce serum antibodies, and it is commonly present in pig herds; pigs inoculated at 2 days of age and 9 days of age show no clinical symptoms. PCV-2 is pathogenic to pigs and mainly causes a new disease characterized by shortness of breath or difficulty breathing, diarrhea, anemia, obvious lymphoid tissue lesions, and progressive emaciation in weaned piglets, namely postweaning multisystemic wasting syndrome (PMWS), which has caused great economic losses. Because PCV antibodies are commonly present in pig herds, a single positive antibody result cannot confirm a positive diagnosis; confirmation must rely on laboratory methods to detect PCV-2 antigen or nucleic acid. Because the virus is difficult to replicate, research on detection, prevention, and control is very difficult. At the same time, the disease is prone to mixed or secondary infection with some viral and bacterial diseases, so it is very difficult to make an accurate judgment based on symptoms, lesions, and epidemiological investigation alone. For this reason, veterinary workers in various countries have conducted a large amount of research and established a variety of highly specific virological, immunological, and molecular biological diagnostic methods. This article reviews the current research progress on PCV detection technologies at home and abroad.

1 Virus Isolation

For the isolation and culture of porcine circovirus, tissues such as lung, liver, spleen, lymph nodes, and tonsils are mostly collected from diseased pigs and made into tissue homogenate; chloroform is used to remove enveloped viruses, and the preparation is inoculated onto PK-15 cells, with short-term treatment of infected cells with glucosamine to promote virus proliferation. Lu Yanli et al.[2] isolated 3 PCV-2 strains from Beijing and Hebei, and sequenced 2 of them; sequence analysis and comparison found that they have high homology with isolates from Europe and America. Lang Hongwu et al.[3] isolated 2 circovirus strains using Dulac porcine kidney passage cells. Cui Shangjin et al. isolated 32 porcine circovirus strains from 22 provinces and cities in China, sequenced 6 of them, and submitted them to GenBank. In addition, many other researchers have isolated multiple porcine circovirus strains from various regions.

2 Electron Microscopy

Under the electron microscope, a relatively small virus can be observed, which is the spherical structure unique to porcine circovirus with a diameter of about 17 nm, as well as a large number of intracytoplasmic inclusion bodies of different forms.

3 Polymerase Chain Reaction Detection

Polymerase chain reaction (PCR), as a rapid, simple, and specific diagnostic method, is currently the most commonly used technique for virological diagnosis and molecular biology experiments; its advantages are sensitivity, specificity, speed, and accuracy. It is widely used in PCV detection, and many improved techniques have been developed.

3.1 Multiplex PCR Detection

Huang CI et al.[4] established a simple multiplex PCR method that can detect and type PCV. This method designed two sets of primers based on ORF1 and ORF2 on the PCV nucleic acid chain. Calsamiglia M et al.[5] established a multiplex PCR method that can detect and type PCV. The authors designed two sets of primers based on the ORF1 and ORF2 sequences of PCV-1 and PCV-2 isolated from Canadian PMWS pigs; both sets of primers can detect and type PCV. Lang Hongwu et al.[3] designed two pairs of PCR primers based on the species specificity of PCV and the type specificity of PCV-2 to perform multiplex PCR detection of PCV. The fragment amplified by one pair of primers has PCV species specificity; the amplified region is a relatively conserved partial nucleotide fragment of ORF1, about 900 bp in size. The fragment amplified by the other pair of primers has PCV-2 type specificity; the amplified region is a nucleotide fragment of the relatively more variable ORF2 portion, about 470 bp in size. Kim J et al.[6] established in 2003 a multiplex nested PCR method for simultaneous detection of PCV-1, PCV-2, and PPV in paraffin-embedded tissues, and tested artificially infected cases and naturally infected cases; the results were completely consistent with the in situ hybridization method.

3.2 Quantitative Competitive PCR Detection

Quantitative PCR infers the amount of the original template based on the amount of PCR product. Liu Q et al.[7] established a competitive PCR method to detect PCV DNA in serum. Two pairs of type-specific primers were designed by selecting the highly variable regions in ORF2 of PCV-1 and PCV-2, and a recombinant plasmid with a foreign fragment was introduced as a competitive template; the PCV-1 or PCV-2 fragments amplified with the above two pairs of primers can be distinguished from the amplified fragments of wild-type virus strains. When the electrophoretic band brightness of the PCR products from serially diluted plasmid DNA of known concentration and the DNA to be tested is the same, the concentration of the DNA to be tested can be calculated. Cui Shangjin et al.[8] used this method and adopted an internal standard competitive template during the experiment; the competitive template and the target template share one reaction system, which not only reduces the differences between different PCR reaction tubes for non-internal-standard templates, but also performs quality control monitoring of samples and rules out false negative results.

3.3 Composite PCR Detection