Abstract
Equine herpesvirus 1 (EHV-1), like other members of the Alphaherpesvirinae subfamily, is a neurotropic virus causing latent infections in the nervous system of the natural host. In the present study, we have investigated EHV-1 replication (wild-type Jan-E strain and Rac-H laboratory strain) during long-term infection and during the passages of the virus in cultured neurons. The studies were performed on primary murine neurons, which are an excellent in vitro model for studying neurotropism and neurovirulence of EHV-1. Using real-time cell growth analysis, we have demonstrated for the first time that primary murine neurons are able to survive long-term EHV-1 infection. Positive results of real-time PCR test indicated a high level of virus DNA in cultured neurons, and during long-term infection, these neurons were still able to transmit the virus to the other cells. We also compared the neurovirulence of Rac-H and Jan-E EHV-1 strains after multiple passages of these strains in neuron cell culture. The results showed that multiple passages of EHV-1 in neurons lead to the inhibition of viral replication as early as in the third passage. Interestingly, the inhibition of the EHV-1 replication occurred exclusively in neurons, because the equine dermal (ED) cells co-cultivated with neuroculture medium from the third passage showed the presence of large amount of viral DNA. In conclusion, our results showed that certain balance between EHV-1 and neurons has been established during in vitro infection allowing neurons to survive long-term infection.
Highlights
The natural biology of alphaherpesviruses is a primary infection with mild or no symptoms and a highly successful establishment of a long-term relationship with the host
As we mentioned in our previous paper, the use of natural host is very limited due to the difficulties associated with finding Bimmunologically naive^ test animals that have not been in contact with the virus (Cymerys et al 2010)
Results presented by the other authors provide a strong evidence that Equine herpesvirus 1 (EHV-1) causes latent infection in mice with the presence of viral DNA in mononuclear cells and in neurons of the olfactory bulbs findings were confirmed by real-time PCR results, which showed the largest copy number of viral DNA in primary murine neurons at 24 and 48 h p.i. for Jan-E strain and 48 and 96 h p.i. in the case of Rac-H strain (Fig. 6)
Summary
The natural biology of alphaherpesviruses is a primary infection with mild or no symptoms and a highly successful establishment of a long-term relationship with the host. Most of the available information about the latency establishment, maintenance and reactivation of alphaherpesviruses is derived from in vivo studies (Wilson and Mohr 2012; Sauerbrei and Wutzler 2002; Baxi 1995); it is difficult to differentiate specific effects of direct virusneuron relationship from indirect consequences mediated by immune or non-neuronal supportive cells. Presented in vitro model utilizing cultured primary murine neurons provides a simple and effective method to examine the kinetics of EHV-1 replication, to determine the differences in the influence of the virus on the neuronal cell depending on virus strain and its adaptation to the cell culture. Positive real-time PCR (quantitative PCR; qPCR) and nestedPCR (nPCR) results indicated the presence of viral DNA in neurons 24–48 h post infection (p.i.) with both
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