Porcine parvovirus (PPV, Protoparvovirus ungulate1), a virus of the genus Protoparvovirus in the virus family Parvoviridae, causes reproductive failure of swine characterized by embryonic and fetal infection and death, usually in the absence of outward maternal clinical signs. The disease develops mainly when seronegative dams are exposed oronasally to the virus anytime during about the first half of gestation, and conceptuses are subsequently infected transplacentally before they become immunocompetent. There is no definitive evidence that infection of swine other than during gestation is of any clinical or economic significance. The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. Diagnostic surveys have indicated that PPV is the major infectious cause of embryonic and fetal death. In addition to its direct causal role in reproductive failure, PPV can potentiate the effects of porcine circovirus type II (PCV2) infection in the clinical course of postweaning multisystemic wasting syndrome (PMWS).
Signs and symptoms Acute infection of postnatal pigs, including pregnant dams that subsequently develop reproductive failure, is usually subclinical. However, in young pigs and probably in older breeding stock as well, the virus replicates extensively and is found in many tissues and organs with a high mitotic index. Viral antigen is especially concentrated in lymphoid tissues (Fig. 3A, B). Many pigs, irrespective of age or sex, have a transient, usually mild, leukopenia sometime within 10 days after initial exposure to the virus. PPV and other structurally similar viruses have been identified in the feces of pigs with diarrhea. However, there is no experimental evidence to suggest that PPV either replicates extensively in the intestinal crypt epithelium or causes enteric disease as do parvoviruses of several other species. PPV also has been isolated from pigs with lesions described as vesiclelike. The causal role of PPV in such lesions has not been clearly defined.
The major and usually only clinical response to infection with PPV is maternal reproductive failure. Pathologic sequelae depend mainly on when exposure occurs during gestation. Dams may return to estrus, fail to farrow despite being anestrus, farrow few pigs per litter, or farrow a large proportion of mummified fetuses. All can reflect embryonic or fetal death or both. The only outward sign may be a decrease in maternal abdominal girth when fetuses die at midgestation or later and their associated fluids are resorbed. Other manifestations of maternal reproductive failure, namely, infertility, abortion, stillbirth, neonatal death, and reduced neonatal vitality, also have been ascribed to infection with PPV. These are normally only a minor component of the disease. The presence of mummified fetuses in a litter can prolong both gestation and the farrowing interval. Either may result in stillbirth of apparently normal littermates, whether or not they are infected.
There is no evidence that either fertility or libido of boars is altered by infection with PPV.
Cause
PPV is classified in the genus Parvovirus (Latin parvus = small) of the family Parvoviridae. All isolates of PPV that have been compared have been found antigenically similar if not identical. PPV is also antigenically related to several other members of the genus. However, its identity can be established by relatively stringent serologic tests such as virus neutralization (VN) and hemagglutination inhibition (HI).
Biophysical and biochemical properties The biophysical and biochemical properties of PPV have been extensively studied and are summarized as follows. A mature virion has cubic symmetry, two or three capsid proteins, a diameter of approximately 20 nm, 32 capsomeres, no envelope or essential lipids, and a weight of 5.3 million daltons. The viral genome is single-stranded deoxyribonucleic acid (DNA) with a molecular weight of 1.4 million (i.e., about 26.5% of the weight of the complete virion). Buoyant densities (g/mL in cesium chloride) of complete infectious virions, incomplete "empty" virions, and extracted virion DNA are 1.38–1.395, 1.30–1.315, and 1.724 respectively. Viral infectivity, hemagglutinating activity, and antigenicity are remarkably resistant to heat, a wide range of hydrogen ion concentrations, and enzymes.
Replication Replication of PPV in vitro is cytocidal and characterized by "rounding up," pyknosis, and lysis of cells (Fig. 1A). Many of the cell fragments often remain attached, eventually giving the affected culture a ragged appearance. Intranuclear inclusions develop but they are often sparsely distributed. Infected cultures may hemadsorb slightly (Fig. 1B). Cytopathic changes are extensive when cell culture-adapted virus is propagated under appropriate conditions. However, on initial isolation several serial passages of the virus or, better, the infected culture may be necessary before the effects are recognized. The use of immunofluorescence (IF) microscopy greatly increases the likelihood of detecting minimally infected cultures.
Primary and secondary cultures of fetal or neonatal porcine kidney cells are most often used for propagation and titration of PPV, although other kinds of cultures are also susceptible. Replication is enhanced by infection of mitotically active cultures. Many cells in such cultures are in the S phase (i.e., the DNA synthesis phase) of their cell cycle, wherein the DNA polymerases of cell origin needed for viral replication are available.
If either fetal or adult bovine serum is incorporated in the nutrient medium of cell cultures used to propagate PPV, it should be pretested for viral inhibitors. The same may apply to sera of several other species. Because replication of PPV is affected by mitotic activity, the effect of the serum on the cells is also especially important. In addition, cultures should be pretested for PPV contamination. Cultures are sometimes unknowingly prepared from infected tissues of fetal and postnatal pigs. Moreover, PPV can be accidentally introduced into cultures in several ways, including the use of contaminated trypsin. If contamination is detected before all cells are infected, the virus can be eliminated by repeatedly subculturing the cells in the presence of nutrient medium containing PPV antiserum.
Several investigators have used IF microscopy to follow the development of PPV in cell culture. In general, the sequence of events is as follows. Viral antigen is detected in the cytoplasm of cells soon after infection if the inoculum contains a high titer of virus and viral antigen. Most, if not all, of this early cytoplasmic fluorescence is the result of antigen phagocytized from the inoculum. By sequential examinations, such antigen can be demonstrated first on the external surface of the cytoplasmic membrane and later within the cytoplasm, often relatively concentrated in a juxtanuclear location. The first unequivocal evidence of viral replication is the appearance of nascent viral antigen in the nucleus (Fig. 2A). In at least some infected cells, nascent antigen next appears in the cytoplasm in sufficient quantity that both cytoplasm and nucleus are brightly fluorescent. Infected cells commonly seen in the lung of fetuses that develop a high titer of antibody for PPV probably represent this stage of replication (see Fig. 8C). Affected cells subsequently round up, become pyknotic, and disintegrate with release of virus and viral antigen (Fig. 2B). Other cells in the culture that are not at the appropriate stage to support viral replication continue to phagocytize and accumulate viral antigen in their cytoplasm (Fig. 2C). A second wave of viral replication can be induced if these cells are stimulated to enter the S phase of the cell cycle as, for example, by the addition of fresh culture medium.
Hemagglutination PPV agglutinates human, monkey, guinea pig, cat, chicken, rat, and mouse erythrocytes. Erythrocytes of other kinds of animals that have been tested are relatively or completely insensitive, or the results have been equivocal. Several parameters of the hemagglutination (HA) test—such as the temperature of incubation, the species of erythrocyte used, and in the case of chicken erythrocytes the genetic composition and age of the donor—may quantitatively affect results. The HA test is most commonly conducted at room temperature, at approximately neutral pH, and with guinea pig erythrocytes. Higher HA titers have been recorded when the diluent used in the test was veronal buffer rather than phosphate-buffered saline. Elution of virus (the hemagglutinin is part of the virion) can be induced by suspending erythrocytes in alkaline buffer, pH 9.
Infectivity assays Infectivity assays are conducted in a standard manner except that, because cytopathic changes at terminal dilutions are often vague, endpoints of infectivity are often determined either by examining cell cultures for intranuclear inclusions after appropriate staining or by examining cell culture medium for viral hemagglutinin. A titration procedure wherein infected cells are made evident by IF microscopy and a plaque assay also have been described.
Serologic Tests The HI test is frequently used for detection and quantitation of humoral antibody for PPV. Antibody sometimes can be detected as early as 5 days after swine are exposed to live virus, and it may persist for years. Sera examined by the HI test are usually pretreated by heat inactivation (56?C, 30 minutes) and by adsorption with erythrocytes (to remove naturally occurring hemagglutinins) and kaolin (to remove or reduce nonantibody inhibitors of HA). Trypsin also has been used to remove nonantibody inhibitors of HA. Parameters of the HI test have been studied in detail.
The SN test is occasionally used for detection and quantitation of humoral antibody for PPV. Neutralization of infectivity is usually confirmed by the absence or reduction either of intranuclear inclusions or fluorescent cells in cultures or of viral hemagglutinin in the culture medium. The SN test has been reported to be more sensitive than the HI test. A microtechnique for application of the SN test has been described.
Immunodiffusion, a modified direct complement-fixation test, and enzyme-linked immunosorbent assay also have been used successfully to detect antibody for PPV.
Evolution
These viruses appear to have evolved ~120 years ago with a rapid increase in their population size within the last 40–60 years. They appear to have evolved initially in wild boars and subsequently spread to domestic pigs. The rate of evolution has been estimated to be 3.86 × 10−4 – 8.23 × 10−4 substitutions per site per year. This rate is similar to other single stranded DNA viruses.
Epidemiology Porcine parvovirus is ubiquitous among swine throughout the world. In major swine-producing areas such as the midwestern United States, infection is enzootic in most herds, and with few exceptions sows are immune. In addition, a large proportion of gilts are naturally infected with PPV before they conceive, and as a result they develop an active immunity that probably persists throughout life. Collectively, the seroepidemiological data indicate that exposure to PPV is common. They also emphasize the high risk of infection and reproductive disease among gilts that have not developed immunity before conception. The most common routes of infection for postnatal and prenatal pigs are oronasal and transplacental respectively.