80 Porcine Parvovirus

80

Porcine Parvovirus Zhengyang Wang, Yu Zhu, Miaomiao Kong, and Shangjin Cui

CONTENTS 80.1 Introduction...................................................................................................................................................................... 713 80.1.1 Classification......................................................................................................................................................... 713 80.1.2 Morphology and Genome Organization............................................................................................................... 713 80.1.3 Epidemiology.........................................................................................................................................................714 80.1.4 Clinical Features and Pathogenesis.......................................................................................................................714 80.1.5 Diagnosis...............................................................................................................................................................714 80.1.5.1 Conventional Techniques........................................................................................................................714 80.1.5.2 Molecular Techniques............................................................................................................................ 715 80.2 Methods............................................................................................................................................................................ 715 80.2.1 Sample Collection and Preparation...................................................................................................................... 715 80.2.2 Detection Procedures............................................................................................................................................ 715 80.2.2.1 Nano-PCR Detection of PPV................................................................................................................. 715 80.2.2.2 Nucleic Acid Probe Detection of PPV....................................................................................................716 80.3 Conclusion and Future Perspectives..................................................................................................................................716 References...................................................................................................................................................................................716

80.1 INTRODUCTION Porcine parvovirus (PPV) is considered to be one of the major causes of reproductive failure in swine [1,2]. First described from aborted fetal swine in 1967 by Cartwright and Huck [3], PPV is linked to the reoccurrence of estrus, abortion, and the delivery of mummified or stillborn fetuses [4], the so-called SMEDI (stillbirth, mummification, embryonic death, and infertility) syndrome. The virus is endemic in most areas of the world and can be found in all pig herd categories.

80.1.1  Classification PPV is a single-stranded DNA virus classified in the family Parvoviridae. Of the two subfamilies (Parvovirinae and Densovirinae) within the family Parvoviridae, the subfamily Parvovirinae covers viruses that infect vertebrate hosts and are separated into eight genera (Amdoparvovirus, Aveparvovirus, Bocaparvovirus, Copiparvovirus, Dependoparvovirus, Erythroparvovirus, Protoparvovirus, and Tetraparvovirus), whereas the subfamily Densovirinae contains viruses that infect insect hosts and are divided into five genera (Ambidensovirus, Brevidensovirus, Hepandensovirus, Iteradensovirus, and Penstyldensovirus) [1,6]. To date, at least seven genetically divergent parvoviruses that infect pigs have been identified. These include PPV type 1 (PPV1), PPV2, PPV3 (also known as porcine PARV4, hokovirus, or partetravirus), PPV4, PPV5, and PPV6, as well as porcine bocavirus (of the genus Bocaparvovirus) [1,6–10].

Recent genetic evidence suggests that PPV2 and PPV3 demonstrate a close relationship to human parvovirus 4 (PARV4) of the genus Tetraparvovirus, whereas PPV5 and PPV6 are similar to PPV4 (of the ungulate copiparvovirus 2 group in the genus Copiparvovirus) [7–10]. On the other hand, the nonpathogenic PPV strain NADL-2 (PPV-NADL2) (which is currently used as an attenuated vaccine) and the dermatitis-associated PPV strain Kresse belong to the ungulate protoparvovirus 1 group in the genus Protoparvovirus. Interestingly, a natural attenuated PPV-N strain, isolated in 1989 from the viscera of a stillborn fetus farrowed by a gilt in Guangxi, southern China, resembles attenuated PPVNADL-2 strain [11]. Antigenically, PPV appears to be related to canine parvovirus and feline panleukopenia virus. However, it is genetically distinct from parvoviruses of all other species.

80.1.2  Morphology and Genome Organization PPV is a nonenveloped virus of about 20  nm in diameter, with a round or hexagonal appearance. The capsid of PPV is a spherical shell consisting of 60 identical copies of viral proteins arranged in an icosahedral symmetry [12]. The PPV genome consists of a single-stranded DNA molecule of about 5.2  kb with terminal palindromic sequences [1]. Two large open reading frames (ORFs) are present in the genome, one (located at the 3′ end) coding for the structural (capsid) protein (or VP) and the other (located at the 5′ end) coding for the nonstructural protein (or NSP). Sixty copies of these structural proteins assemble the icosahedral viral

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capsid. In each of these copies (capsid subunit), a spike on the threefold axis, a depression or canyon around the fivefold axis, and a dimple on the twofold axis can be observed. However, in some parvoviruses such as PPV4 and members of the genus Bocaparvovirus, an additional ORF3 (encoding accessory protein) has been found in the middle of the viral genome.

80.1.3 Epidemiology PPV infection is common in gilts, usually endemic. And occurrence of the disease seems closely related to season, from April to October each year, after farrowing and mating the disease multiples [17]. Once the disease occurs, it can continue for several years. The virus mainly infects newborn piglets, embryo, and embryo pig. When a sow becomes infected in early pregnancy, the mortality of its embryo and embryo pig can be as high as 80%–100%. In addition, PPV infection may induce viremia in the first 6 days, and from days 3 to 7 the infected swine starts to detox to the outside through feces and continue to pollute the environment by detoxing irregularly afterward. Hemagglutination inhibition antibody can be detected with infection after a week. Antibody titers can be up to 1:15,000 within 20 days and can last for several years. Sources of infection of the disease include infected sows and boar, which may sometimes be apparently healthy, asymptomatic carriers. Stillbirths, live births, low earners, and uterine secretions of infected sows contain high titers of the virus, and the environment of pens both inside and outside can be polluted. Sperm cells, spermatic cord, epididymis, and vice gonads of infected boar can contain the virus, so susceptible sows may acquire infection during mating. If pregnant sows have been infected with the virus for >55  days, the farrow may develop immune tolerance leading to the state of persistent infection. Under this circumstance, specific antibodies may not be detectable in these piglets, and these piglets will poison and detoxify in their lifetime. The virus is mainly distributed in the lymphoid tissue (such as gut lymphoid tissue, renal interstitial cells, turbinate periosteum) of the infected swine [1]. The disease can be transmitted vertically through the placenta and mating, as well as the respiratory and gastrointestinal routes. Mechanically, rodents can also play a role in the spread of the disease.

80.1.4  Clinical Features and Pathogenesis Clinical features: Pigs infected with PPV are generally in subclinical form, with reproductive failures in sows being the key clinical sign. In infected sows, the pathological sequela caused by PPV is related mainly to the gestational period in which infection occurs. In the beginning of gestation, the conceptus is protected by the zona pellucida and is therefore resistant to infection. During the embryo stage, infection with PPV results in embryonic death and resorption. From the 35th day onward, ossification starts, and the infection results

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in death and subsequent mummification. Finally, around the 70th day of gestation, the fetus is already immune competent and may resist viral infection. An infection in this stage is then usually controlled, and the piglet is born with anti-PPV antibodies [18]. Pathogenesis: The primary replication of the PPV is suggested to occur in lymphoid tissues. From there, the virus is distributed systemically via viremia. In the epitheliochorial placenta, six tissue layers completely separate the sow and the fetal blood circulation. Since these cells are closely connected, not allowing the passage of even small molecules such as antibodies, it is still not understood how PPV crosses the placental barrier and reaches the fetus [4]. Probably, as the virus was already detected in lymphoid tissue from pigs and in fetal lymphocytes and is able to remain infectious after phagocytosis by macrophages [1], PPV could use them to infect the fetus. Inside the fetus, PPV replicates due to the high mitotic activities present in the fetal tissues [5]. PPV enters cells through a series of interactions that culminate in the release of viral genetic material into a cell compartment in which replication can occur [1]. The mechanisms of PPV entry are not fully understood but may include clathrin-mediated endocytosis or macropinocytosis followed by transportation through the endosomal pathway. Endosomal trafficking and acidification are essential for PPV to enter in the nucleus [1]. Endosome acidification results in reversible modifications of the virus capsid that allow the virus to escape from the endosome [1]. This motif activity is essential for breaking the vesicular membranes, resulting in the formation of pores. After arriving in the nucleus, PPV replicates using the host cell’s own machinery, including cellular DNA polymerase for DNA replication. As virus replication takes place in the replication phase (S) of the cell, cells with a higher replication index are necessary for optimal outcome [1].

80.1.5 Diagnosis 80.1.5.1  Conventional Techniques Virus isolation represents the primary method for PPV detection. A bacterial suspension mixed with the brain, kidney, testis, lung, liver, and mesenteric lymph nodes of aborted fetuses, stillbirths, mummified and low earners of gilts or placenta, and vaginal secretions of sows is inoculated on cells and cultured. If the specimen contains the virus, the inclusion bodies can be observed in the nucleus after 16–36 h, and the characteristic cytopathic effect can be seen after 5–10 days. Initially, cells appear diffused and granular, and then they turn round, clustered, and condensed. Next, cells disintegrate and their shape is not whole. Finally, the cells detach from the bottle wall. Because the virus isolation is time-consuming and laborious, it is not conducive for routine application. Instead, a range of serological assays are commonly used. These include hemagglutinin assay (HA), hemagglutination inhibition (HI) test, ELISA, serum neutralization test, and indirect immunofluorescence. Of these, HA and HI are the most

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common methods. Sera for HI test need to be pretreated by heat ­inactivation followed by adsorption with kaolin [1]. HA and HI are simple, convenient, and fast, but their low sensitivity and low specificity limit their use for secondary diagnosis only [19–23]. 80.1.5.2  Molecular Techniques In comparison with conventional diagnostic techniques, molecular methods such as nucleic acid probes technology [24] and PCR [25] demonstrate relatively high sensitivity and specificity for the detection of PPV. Not surprisingly, multiplex PCR, real-time PCR, and nano-PCR are increasingly employed as a tool for the diagnosis of PPV infection. Multiplex PCR utilizes a plurality of pairs of primers in the same PCR system so that multiple target genes are amplified at the same time. It enables a sample to be tested simultaneously for multiple pathogens or multiple gene types so various diseases can be diagnosed in a single run, thus shortening the detection time and reducing reagent consumption. Real-time PCR combines the conventional PCR technique and fluorescence detection. By eliminating manual handling after amplification, real-time PCR is not only rapid but also reduces potential cross-contamination [26]. NanoPCR represents a further advance in PCR technology and enables amplification of DNA with complex structures [27]. Nucleic acid probe technology also has high specificity and sensitivity, but its high cost is inappropriate for use in routine diagnostic laboratories.

80.2.2 Detection Procedures 80.2.2.1  Nano-PCR Detection of PPV 1. Primers: Two pairs of primers are designed using Primer Premier 5.0 software from PPV gene sequence of PPV retrieved from GenBank (http:// www.ncbi.nlm.nih.gov/) [28] (Table 80.1). 2. Construction of a plasmid containing the NS1 gene: The complete sequence of the NS1 gene was inserted into the vector pGBKT7 as the standard plasmid, amplified in E. coli DH5a, and the recombinant plasmids were purified using the related kit. Finally, the constructs were confirmed by digestion with restriction enzymes EcoRI and SalI and sequencing. The verified products were kept at −20°C until used [28]. 3. Optimization of PPV nano-PCR assay conditions: PCR conditions such as temperature and primer volume need to be optimized. Procedure 1. Remove all aliquots of PCR reagents (2× nanobuffer, Taq DNA polymerase, ddH2O) from the freezer. Vortex and spin down all reagents before opening the tubes. 2. Prepare multiplex PCR mix as follows (volumes indicated per specimen). Component

80.2 METHODS 80.2.1 Sample Collection and Preparation Suitable samples for PPV detection include aborted fetuses, stillbirths, mummified and weak earner’s brain, kidney, testis, lung, liver, and mesenteric lymph nodes or placenta, and vaginal secretions of sows. Add 0.5% solution of hydrolyzed milk protein ­containing penicillin (1000 units/mL), streptomycin (1000 units/mL), and kanamycin (1000 units/mL) to the specimen, with the proportion of specimen and solution being 1:5–10. Grind the specimen, centrifuge it 10 min at 3000 rpm, and store the supernatant at −20°.

AQ1

2× nanobuffer F/R primers (10 µmol/L) Taq DNA polymerase (5 U/µL) ddH2O Total

Volume (µL) 6.0 1.0 0.2 3.3 11.5

3. Add 11.5 µL of PCR mix and 0.5 µL of plasmid into a 0.2mL PCR tube. 4. Turn on thermocycler and preheat the block to 94°C. 5. Spin the sample tubes in a microcentrifuge at 14,000 × g for 30 s at room temperature.

TABLE 80.1 Primers Used to Amplify the NS1 Gene (1989 bp) and the Primers Used to Amplify a Porcine Parvovirus (PPV)Specific 142-bp Portion of the NS1 Gene Fragment NS1 NS1 (nt 1694–1835)

Length (bp)

Primer Sequence (5′–3′)

Enzyme Sites

1989

F: 5′-CCGGAATTCATGGCAGCGGGAAACAC-3′ R: 5′-ACGCGTCGACGTTATTCAAGGTTTGTTG-3′ F: 5′-AGCCAAAAATGCAAACCCCAATA-3′ R: 5′-CTTTAGCCTTGGAGCCGTGGAG-3′

EcoRI SalI

142

Source: Cui, Y. et al., Lett. Appl. Microbiol., 58, 163, 2013. F, forward primer; R, reverse primer.

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AQ2

6. Place the sample tubes in thermocycler for 30 cycles at 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final elongation of 72°C for 10 min. 7. After amplification, pulse-spin the reaction tubes to pull down the condensation droplets at the inner wall of the tubes. The samples are now ready for the electrophoresis or stored frozen at –20°C. 8. Analyze the PCR products by agarose gel electrophoresis through 1% agarose gel in Tris-acetateEDTA buffer at 120 V for 30 min. 9. Stain the gel with ethidium bromide and then visualize under ultraviolet light source. 80.2.2.2  Nucleic Acid Probe Detection of PPV DNA probe: Specific gene of PPV is cloned into pUC-plasmid and then digested by endonucleases and labeled with biotin or isotope before use [22]. Procedure 1. Spotting: Take an appropriate size nylon membrane, mark the grid, add DNA samples at the centre of membrane, and air dry. 2. Denaturation: Put the nylon membrane on the double filter paper saturated with denaturing solution for 10 min. 3. Neutralization: Move the nylon membrane onto the double filter paper saturated with neutralized solution and leave it for 5 min; dry the membrane at 37°C for 30 min and then bake it in an oven at 120°C for 30 min to fix the DNA. 4. Prehybridization: Transfer the nylon membrane in hybridization tube, add prehybridization solution, and leave it in the hybridization furnace 68°C for 2 h. 5. Hybridization: Denature the probes for 10  min in boiling water, immediately quench for 5 min in ice water, remove the hybridization tube, decant prehybridization solution, and then add the denatured DNA probe, and hybridize at 68°C overnight. 6. Washing the membrane: Follow instructions of the kit insert. 7. Chromogenic reaction: Place the membrane in chromogenic solution; once spotting spots change color, add TE buffer to terminate chromogenic reaction.

80.3 CONCLUSION AND FUTURE PERSPECTIVES PPV is a small, ssDNA virus that is responsible for causing reproductive failure in swine worldwide. The significant expansion in human populations in recent decades has created a huge demand for large-scale pig farming, which in turn has contributed to a marked rise in the incidence of PPV infections.

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For the diagnosis of PPV infection, serological assays (especially hemagglutination and hemagglutination inhibition test) have been widely applied. Nonetheless, virus isolation remains valuable for precise and complete determination of PPV. Due to their high sensitivity, specificity, reliability, and rapid turnover, molecular techniques (e.g., PCR and nucleic acid probes) have been used with increasing frequency in clinical diagnostic laboratories. Further development in automation will help streamline the diagnostic operation for PPV infection. Currently, no specific treatment is available for PPV infection. The widespread and resistant characteristics of this virus make its control difficult. Regular vaccination of the breeding stock is still the most effective preventive measure for PPV infection [1,5]. Although the first vaccines were developed in the late 1970s [29,30], the constant evolution and mutation of PPV and identification of novel PPVs (e.g., PPV2, PPV3, PPV4, PPV5, and PPV6) [7–10,31–37] have reduced the effectiveness of existing vaccines. Continuing efforts to develop new vaccines, including genetic engineered vaccines, that take account of virus mutations and emergence of novel strains are vital to strengthen control and prevention campaigns against PPV infection.

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AUTHOR QUERIES [AQ1] Please provide complete web details if available throughout the chapter. [AQ2] Please check if the inserted expanded form for “TAE” is correct. [AQ3] Please provide in-text citation for Refs. [13–16]. Also references are out of sequence. Please check.

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