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A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Published on: August 31, 2014
Two clones obtained from Urabe AM9 mumps virus vaccine differ in their replicative efficiency in neuroblastoma cells
Gerardo Santos-López1, Carlos Cruz, Nidia Pazos
1Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del IPN, Av. Instituto Politécnico Nacional 2508 Col. San Pedro Zacatenco CP 07360. México DF, Mexico.
Abstract:
A high rate of post-vaccinal aseptic meningitis for Urabe AM9 mumps virus strain is well documented. This strain is composed of two virus variants differing at the nt 1081 (A/G) region in the hemagglutinin-neuraminidase (HN) gene. An association of HN-A(1081) variant with neurovirulence has been proposed. In order to test for neurotropism we isolated the HN-A(1081) and HN-G(1081) virus variants from Urabe AM9 mumps virus vaccine. Sequential passages were performed in monkey kidney Vero cells and human neuroblastoma SH-SY5Y cells. Viral replication was determined by conventional and real-time RT-PCR. The results show that clone HN-A(1081) can replicate efficiently in both cell types. However, a defective replication of clone HN-G(1081), lacking its genetic marker, was observed after the third passage in neuroblastoma cells. Kinetics assays showed that clone HN-A(1081) replicates faster than clone HN-G(1081). Viral clones were also inoculated into the brains of newborn rats. Clone HN-A(1081) replicated 14 times, while clone HN-G(1081) merely duplicated its level over the initial inoculum. These results suggest that there is a selective replication of HN-A(1081) mumps virus variants in cells of nervous origin.
Insights
The Urabe AM9 mumps virus vaccine contains two variants. The HN-A(1081) variant shows selective replication in nervous system cells, unlike the HN-G(1081) variant.
Area of Science:
- Virology
- Neuroscience
- Vaccinology
Background:
- The Urabe AM9 mumps virus vaccine is associated with aseptic meningitis.
- This vaccine strain contains two variants: HN-A(1081) and HN-G(1081), differing in the hemagglutinin-neuraminidase (HN) gene.
- The HN-A(1081) variant has been hypothesized to be linked to neurovirulence.
Purpose of the Study:
- To investigate the neurotropism of Urabe AM9 mumps virus variants.
- To compare the replication efficiency of HN-A(1081) and HN-G(1081) variants in neural and non-neural cells.
Main Methods:
- Isolation of HN-A(1081) and HN-G(1081) variants from Urabe AM9 vaccine.
- Sequential cell culture passages in Vero and SH-SY5Y cells.
- Viral replication assessment using RT-PCR and inoculation into newborn rat brains.
Main Results:
- Clone HN-A(1081) replicated efficiently in both Vero and SH-SY5Y cells.
- Clone HN-G(1081) exhibited defective replication after the third passage in neuroblastoma cells.
- In vivo studies showed significantly higher replication of HN-A(1081) in newborn rat brains compared to HN-G(1081).
Conclusions:
- The HN-A(1081) mumps virus variant demonstrates selective replication in cells of neural origin.
- This selective replication may contribute to the neurovirulence observed with the Urabe AM9 vaccine strain.
