PORCINE PARVOVIRUS (PPV)- AN IMPORTANT CAUSE OF SMEDI IN SWINE
Das T1 and Das NK2
Scientist, ICAR-DFMD, Bhubaneswar
BVO, Bisoi, Mayurbhanj, FARD, Government of Odisha
Corresponding author E mail id: tarenisahoo@gmail.com
Porcine parvo virus (PPV) is an important infectious agent associated with reproductive failure in naïve herds causing heavy economic losses to swine industry. The virus is associated with increased numbers of stillbirths, mummification, embryonic death, infertility in pigs, birth of weak piglets and neonatal mortality. PPV commonly replicates in lymphoid tissues. Cells with high replication index is required for productive viral infection. It causes transplacental infection after being associated with macrophages. PPV induces apoptosis of host cells. Pathological changes produced by virus depends on stage of gestation, immune status of the host and virus strain. Serology, nucleic acid based diagnostic methods, in-situ hybridization, immunohistochemistry, isolation of PPV in suitable cell culture system etc. are available diagnostic methods. As the virus is very stable, sanitary measures with higher concentration disinfectant and vaccination are important control measures to prevent virus transmission and reproductive failure.
Etiology
Porcine Parvo virus is a small non enveloped DNA virus of Genus Protoparvirus of family Parvoviridae. It is one of the most important impediments to the growth of pig industry and has been associated with reproductive failures of sows. It is the key agent of SMEDI (stillbirth, mummification, embryonic death and infertility) syndrome in pigs. It can cause overwhelming abortion storm in unvaccinated swine herd. There are seven different types of PPV (1-7) have been identified (Pallinski et al., 2016). But PPV-1 is well studied and most documented pathogen worldwide associated with reproductive failure and fetal death in sows.
The genome of PPV is around 5kb encoding ORF1 and ORF2. The ORF1 at 5’end encodes for three nonstructural protein (NS1, NS2 and NS3) which are essential for viral replication. NS1 is a highly conserved protein. The ORF2 at 3’ end encodes for three structural capsid proteins (VP1, VP2 and VP3) which are required for recognition and binding to host cell and establishment of infection. VP2 is produced by slicing from same RNA template as larger protein VP1. VP3 is a post-translational modification of VP2. VP2 is highly antigenic producing neutralizing antibodies. PPV capsid is spherical which is made up of 60 subunits (each subunit consists of 90% VP2 and 10% VP1) arranged in icosahedral symmetry (Streck AF and Truyen U, 2020; Partiban et al., 2022).
Transmission and Pathogenesis
PPV is ubiquitous in swine herd, thermostable virus and persists in the environment which is a source of infection to naïve herd. The virus is transmitted both horizontally by oronasal route, by rodents and infected fomites and vertically via placenta in naïve herd. Boars’ semen is also a source of infection. The lymphoid tissues/ tonsils are primary site of viral replication. The virus can persist in lymphoid tissue for a long period and replicates in lymphocytes proliferating under antigenic stimulation. The virus is disseminated throughout the body including central nervous system during viraemia through infected lymphocytes. PPV may be able to cross six layered epitheliochorial porcine placentas after being phagocytosed by macrophages (Paul et al., 1979). It takes usually 23-32 days for PPV to enter fetus after oral infection and somewhat less 15 days by intramuscular infection of sow (Joo et al., 1976). The fetal tissue is having high mitotic activity which supports PPV virus replication. PPV binds to sialic acid receptor of cell surface glycoprotein and enters cell by clathrin- mediated endocytosis (single particle) and micropinocytosis (aggregate of virus). The endosomal translocation to late endosomes and acidification is required for productive PPV infection which exposes phospholipase A2 of VP-1 protein and helps in release of virus from endosomes to the cytoplasm (Boisvert et al., 2010). Both microtubule and actin networks are involved in transportation of PPV to nucleus and the virus replicates in the highly mitotic host cell nucleus using host cell DNA polymerase enzyme. PPV induces host cell apoptosis via activating reactive oxygen species accumulation and mitochondrial mediated apoptosis signaling. PPV infection increased Bax expression by translocating them from cytoplasm to mitochondria and decreased bcl2 expression, enhanced release of cytochrome-c from mitochondria to cytosol. PPV infection activated only caspase-3 and 9 expressions (part of intrinsic regulated death pathway), but not caspase-8 which is a part of extrinsic regulated cell death pathway (Zhao et al., 2016). NS1 of PPV cause host tissue damage and reproductive failure by inducing apoptosis (Zhang et al., 2019). The pathogenesis of PPV depends on stage of gestation and strain of the virus. PPV infection during 1st half of pregnancy (before 56 days) results in reproductive failure in gilts/sows. Fetus infection before 70 days of gestation, before development of immune-competency resulted in fetal death and mummification (Joo et al., 1979). Low pathogenic strain like NADL-2 and MSV cannot cross placental barrier and also have limited tissue distribution ability but highly pathogenic Kresse and 27a can cross placenta easily and infect all organs of fetus (Meszaros et al., 2017; Zeeuw et al., 2007).
Clinical signs
The reproductive failure is the main clinical sign. Infection of porcine embryos before 35 days of gestation results in early embryonic death, fetal resorption, conception failure, return to estrous or small litter size. Infection after 35 days of pregnancy resulted in fetal death and mummification as fetal organs are already developed and ossification has been initiated. Infection after 70 days of gestation period may result in birth of healthy and seropositive piglets or may result in increased stillbirth, weak piglets and neonatal deaths (Leengoed et al., 1983). Infertility and long gestation period are observed in infected sow.
PPV infection in piglets may cause cutaneous lesions. Erosive and ulcerative lesions have been described in the snot, oral cavity, coronary band and interdigital space (Lager and Mengeling, 1994). Mild transient leucopenia has also been observed. In some nursing piglets, PPV was also found to be associated with diarrhea (Duhamel et al., 1991). PPV co-infection also acts as a triggering factor for porcine circovirus-2 induced post weaning multisystemic wasting syndrome in pigs by enhancing virus replication (Saikumar and Das, 2019; John et al., 2020; Das et al., 2022).
Gross lesions
Gross lesions are only observed in the fetus. Sows are clinically normal. Fetuses are infected and die at different stages of gestation due to fetus-to-fetus transmission of virus. Therefore, in a single infected litter, we usually observe fresh fetus, fetuses at different stages of autolysis and small to large mummified fetuses (Fig.1). Embryonic death and resorption are most common observations. The crown -rump length of infected fetus is usually less than 17 cm (Helke et al., 2015). The mummified fetuses are autolyzed, dehydrated and dark brown in color with shrunken eyes (Fig. 2). The discoloration of skin is due to severe bleeding. The fetus may be covered with autolyzed and partially dehydrated dark brown colored placenta (Fig. 1) with decreased extra fetal fluid amount (Joe et al., 1977). All the internal organs are autolyzed, partially dehydrated and dark brown in color. The lesions in infected stillborn fetus/ piglets are the presence of serosanguinous fluid in the body cavities like abdominal cavity, thoracic cavity and pericardial sac, congested and enlarged liver and kidney; enlarged, pale and round heart, congested lung and brain (Helke et al., 2015; Kaur et al. 2016; Leengoed et al., 1983).
| Fig.1. Different sizes of mummified fetuses covered with dark brown colored placental membrane | Fig. 2. Severely autolyzed and partially dehydrated mummified fetuses |
Histopathology
The microscopic lesions include perivascular cuffing in the myocardium and endocardium with degeneration of muscle fibers, hemorrhage and perivascular cuffing in the brain, necrosis of hepatocytes and sinusoidal congestion in liver, congestion, hemorrhage and MNCs infiltration in kidneys, necrosis of skeletal muscle etc. (Leengoed et al., 1983). So, the main lesions were vasculitis and perivascular cuffing of MNCs in liver, lung, kidney, eye, brain, heart, placenta; lymphoid depletion in spleen, gliosis in cerebellum with presence of inclusion bodies etc. (Hogg et al., 1977; Helke et al., 2015; Kaur et al., 2016). The placental mineralization and trophoblastic inclusion bodies were also observed (Woods et al., 2009).
Diagnosis
PPV is mainly associated with reproductive failure. PPV should be suspected in the swine herd when female animals recurrently return to estrous without any solid reason or there is delay in parturition with increased number of mummified fetuses of different sizes or with small litter size during first or second parity (Streck and Truyen, 2020). The best tissue samples for PPV laboratory diagnosis are mummified fetus and fetal remnants. Different methods for detection of PPV antigen and nucleic acid in tissue samples are immunofluorescence microscopy for viral antigen detection in fetal tissue (Mengeling and cutlip, 1975), Double in situ hybridization for PPV detection in tissue section (Kim and Chae, 2002), immunohistochemistry for detection of PPV in tissue samples (Kim and Chae, 2004) etc.
Many serological assays using paired serum samples are in use for diagnosis of PPV. Serological methods for detection of PPV are Gel diffusion precipitation test (Too et al., 1983), Hemagglutination inhibition test (Joo et al., 1976) against chickens, humans and guinea pigs’ erythrocytes, serum neutralization test, modified direct complement fixation test (MDCF), NS1 based ELISA (Quing et al., 2006), rVP- ELISA (Kong et al., 2014), enzyme- amplified chemiluminescence immune assay (Zhou et al., 2014), Visual protein chip and cyanide dye-3 based protein chip to detect antibodies (Wu et al., 2020).etc.
Nucleic acid-based techniques are more sensitive methods for detection of PPV DNA in clinical samples. Nano PCR assay (Cui et al., 2014), Taq Man based real time PCR (Gava et al., 2015), integrated real time and high-resolution melting curve analysis (Yu et al., 2015), multiplex PCR for simultaneous detection of PPV and other DNA and RNA virus (Hu et al., 2016), real time recombinase polymerase amplification assay (Wang et al., 2017), Loop mediated isothermal amplification (LAMP) based method for diagnosis of PPV7 (Wang et al., 2021), Cost effective SYBR green dye-based q PCR for concurrent or separate diagnosis of PPV and PCV2 (Bhattacharjee et al., 2021), One step triplex PCR assay for simultaneous detection of PPV, PCV2 and CSFV in a single tube (Rajkhowa et al., 2022) etc. were successfully applied for PPV detection with low detection limit.
For isolation of PPV, swine testis, swine testicular epithelioid, swine embryo kidney and pig kidney cell lines can be used (Mazzards et al., 2017; Streck and Truyen, 2020). PPV replication in these cell line causes cytopathic effects (CPE) like granulation, intranuclear inclusion, pyknosis and cell death (Mengeling, 1972).
Prevention and Control
General management practices like preventing susceptible animals from eating infected materials and sanitary measures should be used regularly. As the virus is very stable and heat resistant, cleaning with proper disinfectants like higher concentration of sodium hypochlorite and sodium hydroxide should be used. Maintaining long lasting herd immunity is a better method for preventing transplacental infection and associated reproductive failures. As gilts are mostly susceptible to infection, they should be vaccinated twice at two weeks interval before service and revaccination should be carried out at regular interval (Leengoed et al., 1983). Boars can also be vaccinated to prevent transmission of virus in the semen. Different vaccines are available to control PPV induced reproductive failure. Inactivated PPV vaccine was developed in 1976 (Suzuki and Fujisak, 1976). The inactivated virus vaccine is a cell cultured derived low pathogenic strain NADL-2 which is inactivated by formalin or beta propiolactone or acetyl ethyleneimine and adjuvated with oil or ammonium hydroxide (Joo and Johnson, 1977; Mengeling et al., 1979; Streck and Truyen,2020). An inactivated vaccine based on PPV 27a strain can prevent disease but cannot prevent shedding of virus after homologous challenge (Foerster et al., 2016). Another vaccine based on modified live virus (NADL-2) also prevented transplacental virus transmission but it resulted in viraemia and shedding of virus (Paul and Mengeling, 1984). A recombinant virus like particle (VLP)-PPV vaccine also found to evoke high titer antibody in water mineral oil adjuvant combination (Antonis et al., 2006). A novel VP2 based PPV1 subunit vaccine has also provided good protection to in bred pigs and their offspring’s (Gracia- Morante et al., 2019). Intrauterine vaccination of pigs against PPV is under trial (Hamonic et al., 2020). Recently, an octavalent vaccine Porcilis is developed which protects gilts and progeny against Erysipelothrix rhusiopathiae, PPV and six serogroups of Leptospira (Van Den Born et al., 2020). Due to emergence of new strains of virus, an effective vaccine needs to be developed which can provide long lasting immunity against all strains of PPV and also can prevent shedding of virus after challenge study.
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