Ultrastructural Analysis of Pseudorabies Virus Infection in IB-RS-2 Cell Line and with Treatment by Persea americana Extract
Abstract
In this study, we focused on the cycle of replication of the Pseudorabies virus (PrV) in the swine IB-RS-2 cell line in absence or in presence of an infusion from Persea americana leaves. The ultrastructure of the Nova Prata strain virus presents the typical characteristics of Varicelovirus as well as lytic replication with total cell destruction between 18 and 24 hours post-infection (pi). Adsorption is immediate followed by fusion penetration of the virus membranes with those of the host. The capsid once inside the cell migrates to the nucleus where it disappears. Precursor viral particles appear from 4 hours and will form the nuclear crystalloids. The capsids with the viral DNA incorporated migrate to the nuclear membranes where they receive viral envelope after de-envelopment and re-envelopment constituted by cytoplasmic membranes. Then the virions appear grouped in vesicles that merge with the plasma membrane and finally are released out of the cell and become associated with it. Infected cells in the presence of an infusion of Persea americana leaves show few viral particles and many cells without signs of infection. In the initial stages of replication, they are shown pleomorphic with a different morphology when compared with the control. At 7 h post-infection double core with a single envelope are found both between the nuclear membranes and in the cytoplasm of the cell. These findings indicate that the extract may be interfering with virus replication as well as useful in detecting possible targets of inhibition.
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Introduction
The Aujeszky's disease virus (ADV) or pseudorabies virus (PrV), also called swine herpesvirus (SuHV-1) and belonging to the family Alfaherpesviridae, occurs in several species of mammals and attacks mainly the central nervous system. It is considered a disease primarily of swine; its control is difficult and causes many economic losses in the stock. The pigs are one of the largest reservoirs of the virus; however, in several mammalian species, such as in cattle, sheep, goats, dogs and cats the disease can also occur (Pomeranz et al., 2005 ; Nauwynck etal., 2007 ).
In this study, the strain Nova Prata isolated in Brazil in 1956 from cattle infected with PrV was investigated (Fonseca et al., 2010).
Studies in the literature describe the morphology of the virus mainly in infected cells (Granzow et al., 1997; Johnson and Baines, 2011; Goldsmith et al., 2013). The herpesviruses consist of four distinct structural components: the viral core composed of a double-stranded DNA genome and enclosed by icosahedral capsid to form the nucleocapsid, and the tegument that is a protein matrix surrounded by the viral membrane or viral envelope (Boldogköi and Nógrádi, 2003; Pomeranz et al., 2005; De Clerq and Li, 2016). The replication cycle of the virus, have been well studied in cultured cell systems and can serve as a template fordidactic and illustrative purposes, as well as for the study of targets for blockade using both synthetic and natural antiviral drugs (Goldsmith et al., 2013; Kalu et al., 2014; Paulini, 2015).
Among the natural products with antiviral properties, aqueous leaf extract (infusion) from Persea americana showed strong action on PrV with effective inhibition when the extract was added simultaneously to the virus inoculation in IB-RS2 cell line (Koseki et al., 1990; Almeida et al., 1998). In addition, Simoni et al. (1996) showed a reduction of viral infectivity of 1.5 log after 30 minutes of direct incubation with the virus.
The present study aimed to illustrate by electronic microscopy the steps of the PrV replication in IB-RS-2 infected cell line and to compare it with the replication of the same treated with Persea americana extract.
Conclusion
Persea americana leaf extract inhibited the replication of PrV, a virus of great importance in animal health. Electron microscopy of wild-type PrV infection generated results that corroborated the results found by other authors for both HSV-1 and those observed in the replication of PrV.