Replication cycle of PRRSV

Once the virus enters in susceptible cells and starts the replication process, viral antigen can be detected in the cytoplasm from 6 h post-infection and completely assembled virions can be detected in infected cells at 9 h post-infection (Pol et al., 1997). Viral release takes place by lysis of infected cells and seems to be also involved in the induction of apoptosis in neighbour uninfected cells (Kim et al., 2002; Lee and

Kleiboeker, 2007). Cellular necrosis and apoptosis, as well as the secretion of pro-inflammatory cytokines from PRRSV infected macrophages (i.e. IL-10 and TNF-α), have important implication on the pathogenesis of the disease (Labarque et al., 2003).

Pigs can be infected by several routes of exposure, including intranasal, intramuscular, oral, sexual and transplacental. Following exposure, replication occurs primarily in local permissive macrophages of lymphoid tissues and then the virus rapidly spreads throughout the body by the lympho-hematic way. Viremia starts as early as 12 h post-infection (Rossow et al., 1995) and viral load peaks in serum around 7-10 days post-infection (dpi). The duration of viremia varies depending on the PRRSV strain and on the age of the animal (Cho et al., 2006a; Klinge et al., 2009; Van Der Linden et al., 2003a). Younger pigs generally replicate virus to higher titers and for longer time than older pigs (Klinge et al., 2009; Van Der Linden et al., 2003a). Several studies indicated that the period of viremia may range from few weeks (generally less than four) in older animals to up to three months in young pigs (Allende et al., 2000; Díaz et al., 2012; Horter et al., 2002; Van Der Linden et al., 2003a; Wills et al., 2003). The lung and the lymphoid organs, such as tonsil, Peyer’s patches, thymus and spleen (Duan et al., 1997b; Lamontagne et al., 2003; Lawson et al., 1997; Sur et al., 1996) are the tissues with the higher viral loads. The virus in lung is usually detected from 1 day post-exposure until 28 dpi (Halbur et al., 1996; Sur et al., 1996) although it has been described until 72 days post-exposure in young pigs (Bierk et al., 2001b).

The viremic phase of the infection is followed by a period of confinement of the virus in secondary lymphoid tissues and low viral replication. PRRSV antigen could be detected by RT-PCR in serum and tonsils until 251 dpi (Wills et al., 2003) and infectious virus could be isolated from oropharyngeal scrapings until 157 dpi (Wills et al., 1997c). Moreover, viral genome can be present in serum and tonsils until 132 days after birth in piglets surviving congenital infection (Benfield et al., 2000b).

Transmission of PRRSV from congenitally infected piglets to sentinel animals was shown until 112 days after birth (Rowland et al., 2003).

In a study by Horter et al. (2002), 51/59 pigs were confirmed to carry the virus in oropharyngeal scrapings or tonsil between 63 and 105 dpi. Moreover, 10/11 pigs

euthanized at 105 dpi, harboured infectious virus as demonstrated by viral isolation and/or swine bioassay. Infectious PRRSV was also detected by swine bioassay in 2/5 pigs at 150 dpi (Allende et al., 2000). Nevertheless, the proportion of animals harbouring the virus dropped substantially from 84 dpi. This fact indicated that most of pigs clear the virus between 3 and 4 months after infection and that the presence of PRRSV in tissues for long time after the end of viremia cannot be considered as a true state of viral persistence (Allende et al., 2000; Wills et al., 2003).

Obviously, the mere detection of PRRSV genome in tissues of chronically infected pigs cannot be considered as an evidence of shedding of the virus or as an evidence of contagiousness. The only evidence of contagiousness of an infected pig is its ability to infect a susceptible one after a period of contact. Therefore, Bierk et al. (2001a) demonstrated that non-viremic sows were able to transmit the infection by direct contact to PRRSV-naïve sows at 49, 56 and 84 dpi. Likewise, non viremic grower pigs (6-7 months of age) transmitted the virus to naïve sentinels until 62 dpi (Wills et al., 2002). Conversely, sows with a detectable amount of viral genome in sera and nasal secretions until 77 and 48 dpi, respectively, were infectious by contact to naïve sows only until 42 dpi (Charpin et al., 2012).

Regarding the ability of chronically infected pigs to transmit the virus to susceptible animals, it is worth to note that circumstances causing stress such as farrowing, regrouping etc., might induce a reactivation of viral replication and shedding. For example, Albina et al. (1994) demonstrated reactivation of PRRSV shedding after corticosteroid treatment at 15 weeks after the initial seroconversion of the animal.

Mechanisms of viral persistence in the host have not been clearly identified yet.

Rowland et al. (1999) suggested that persistence could be associated with the selection of viral subpopulations/quasispecies. It was thought that the immune system can play a role in this selection. For instance, changes in the sequence of GP5 (Allende et al., 2000; Rowland et al., 1999), GP4 and GP3 (Allende et al., 2000) were observed in viruses isolated from chronically infected pigs. However, the effect of positive natural selection for immune evasiveness in maintaining quasispecies variation did not was demonstrated (Chang et al., 2002; Goldberg et al., 2003).

A possible explanation for the maintenance of PRRSV in lymphoid tissues was offered by Díaz et al. (2005). The authors suggested that early in the infection, the virus can induce the release of immunomodulatory cytokines, i.e. interleukin 10 (IL-10), which can inhibit the cell-mediate immune response (CMI) against the virus. As the infection progresses, the number of permissive macrophages available in the lung decreases and the CMI achieves the confinement of the virus in the lymphoid tissues. Here, the viral replication continues but gradually declines as the number of permissive macrophages decreases. Thus, the immune response is finally able to clear the infection, and this moment would correspond with the increase of interferon-γ-secreting cells (IFN-γ-SC) in blood and the development of neutralizing antibodies (NA).