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The Effects of Specific Point Mutations on Infectivity and Virulence of Murine Norovirus

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Title:
The Effects of Specific Point Mutations on Infectivity and Virulence of Murine Norovirus
Series Title:
Annual UF Undergraduate Research Symposium
Creator:
Sok, Ryneth
Karst, Stephanie
Zhu, Shu
Publication Date:
Language:
English

Notes

Abstract:
My research project focuses on determining the effect of specific point mutations in the capsid protein of murine norovirus (MNV) on infectivity and virulence of the virus. The specific point mutations were selected because they arose naturally in malnourished mice chronically infected with a MNV. Together with my postdoctoral mentor, I performed site-specific PCR-based mutagenesis to introduce the appropriate mutations into a cloned version of the parental virus and determined virus yields. For those mutations that were not lethal to the virus, I generated virus stocks and performed virus growth curves to analyze replication efficiency. I performed this experiment in two known target cells of MNVs, macrophages and B cells. I have identified two mutations that reduce the viral replication efficiency in both cell types, demonstrating that these residues provide a critical function during the viral life cycle. The lab is also testing this panel of mutant viruses in antibody neutralization assays to identify mutations that alter virus-specific antibody binding; and virulence studies to identify mutations that regulate the ability of the virus to cause disease in a natural infection. Ultimately identifying residues associated with virulence and antigenicity is critical to vaccine development.
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Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Ryneth Sok.
Publication Status:
Unpublished
General Note:
Center for Undergraduate Research
Exhibitions:
Poster presented at the 2015 UF Undergraduate Research Symposium

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University of Florida Institutional Repository
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University of Florida
Rights Management:
Copyright Ryneth Sok. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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My research project focuses on determining the effect of specific point mutations in the capsid protein of murine norovirus (MNV) on its infectivity and virulence The specific point mutations were selected because they arose naturally in malnourished mice persistently infected with a MNV strain named MNV 1 Together with my postdoctoral mentor, I performed site specific PCR based mutagenesis to introduce the appropriate mutations into a cloned version of the parental virus and determined virus yields For those mutations that were not lethal to the virus, I generated virus stocks and performed virus growth curves to analyze replication efficiency I performed this experiment in two known target cells of MNVs, macrophages (RAW cells) and B cells (M 12 cells) I have identified a mutation that reduces the viral replication efficiency in both cell types, demonstrating that these residues provide a critical function during the viral life cycle The lab is also testing this panel of mutant viruses in antibody neutralization assays to identify mutations that alter virus specific antibody binding ; and virulence studies to identify mutations that regulate the ability of the virus to cause disease in a natural infection Ultimately identifying residues associated with virulence and antigenicity is critical to vaccine development The Effects of Specific Point Mutations on Infectivity and Virulence of Murine Norovirus Ryneth Sok 1 Shu Zhu 1, Stephanie Karst 1 Department of Molecular Genetics and Microbiology 1 University of Florida, Gainesville FL Introduction Results Abstract Conclusion Background and Significance Human norovirus ( HuNoVs ) are a major cause of both epidemic and sporadic gastroenteritis worldwide HuNoVs are responsible for about 12 % children less than 5 years of age hospitalized for severe gastroenteritis worldwide In healthy individuals, HuNoVs cause a mild self limited gastroenteritis ; however, in several risk groups including infants, young children, the elderly, and the immunocompromised, HuNoVs can be severe persistent and potentially life threatening As a robust culturing system for Human norovirus is not available, murine norovirus which can replicates efficiently in dendritic cells, macrophages and B cells serves as a surrogate model for studying norovirus infection The viral capsid proteins VP 1 of MNV are comprised of a conserved shell (S) domain and a more variable protruding P 1 and P 2 domains P 2 domain is the antigenic and receptor binding domains of MNV 1 Single Point Mutations Methods References Karst, S. M., Zhu, S. and Goodfellow I. G. (2015), The molecular pathology of noroviruses. J. Pathol ., 235: 206 216. doi : 10.1002/path.4463 Mutagenesis Site directed mutagenesis performed with Agilent Mutagenesis Kit DNA extraction and Purification Mini prep and identified by double digestion and sequencing Maxi prep to generate mutant virus infectious clones Generating virus stocks In vitro Transfection in 293Tcells Determine the viral yield with TCID50 Assays Propagate viruses by passaging 293T derived viruses on RAW cells Determine the titer of virus stocks with TCID50 Assays Growth Assay Infect RAW and M12 cells with Multiplicity of infection (MOI) 5 or 0.05 Harvest cell supernatants at indicated time points Determine growth kinetics of mutant viruses by TCID50 Assays Viruses Titers (293 Sup) Titers (WT ctrl) Titers (RAW) Virus stocks (mean) MNV1 G006S N/A N/A 7.52 x 10 8 7.52 x 10 8 MNV1 A097S N/A N/A 2.64 x 10 8 2.64 x 10 8 MNV1 G101D <100 (n=2) 1.95 x 10 4 MNV1 A125V <100 (n=2) 1.95 x 10 4 MNV1 H170Y <100 (n=2) 1.95 x 10 4 MNV1 P188S N/A N/A 1.95 x 10 4 4.39 x 10 8 MNV1 R204H <100 (n=2) 5.62 x 10 3 MNV1 K296E 1.00 x 10 5 7.65 x 10 4 4.83 x 10 8 4.83 x 10 8 MNV3 E296K 1.00 x 10 3 7.65 x 10 4 5.55 x 10 8 5.55 x 10 8 MNV1 G320D 6.31 x 10 4 N/A 2.57 x 10 9 2.57 x 10 9 MNV1 L359I 3.89 x 10 4 N/A 3.16 x 10 9 3.16 x 10 9 MNV1 G360D 2.15 x 10 5 NA 3.61 x 10 9 3.61 x 10 9 MNV1 Q371D <100 (n=2) N/A MNV1 Q371G 3.89 x 10 4 7.65 x 10 4 4.25 x 10 8 4.25 x 10 8 MNV1 V374L 1.31 x 10 5 7.65 x 10 5 2.52 x 10 7 2.52 x 10 7 MNV1 Q374F <100 (n=2) 1.67 x 10 4 1.95 x 10 4 MNV1 A393V <100 N/A MNV1 A445T <100 (n=2) N/A MNV1 D512G <100 N/A MNV1 V518I 4.64 x 10 4 N/A 1.95 x 10 9 1.95 x 10 9 Table 1 : single point mutations with titer of 293 supernatants < 100 are dead mutations, preventing mutant viruses from replicating Figure 1 : the RAW cells were infected with MNV 1 K 296 E, MNV 3 E 296 K and parental viruses at MOI 5 or 0 05 Figure 2 : the M 12 cells were infected with MNV 1 K 296 E, MNV 3 E 296 K and parental viruses at MOI 5 or 0 05 After incubating for 1 hour at 4 0 C, the infected cells were washed with cold PBS, harvested at 0 12 24 36 and 48 hours post infection and titrated on RAW cells using TCID 50 assays D uplicate wells were tested for both RAW and M 12 cell lines The whole experiments were repeated twice The data from the experiment for MNV 1 K 296 E, MNV 3 E 296 K with parental viruses controls was presented in figures 1 and 2 Table 1. summary of mutant viruses Position Mutation S Domain 6 G -> S 97 A -> S 101 G -> D 125 A -> V 170 H -> Y 188 P -> S 204 R -> H P2 Domain 296 K -> E (MNV1) and E -> K (MNV3) 320 G -> D 359 L -> I 360 G -> D 371 Q -> G 374 V -> F and V -> L 393 A -> V P 1 Domain 445 A -> T 512 D -> G 518 V -> I 0 2 4 6 8 10 12 0 12 24 36 48 Log (TCID50/ml) Hours post infection RAW MOI 5 MNV1 MNV3 E296K K296E 0 2 4 6 8 10 12 14 0 12 24 36 48 Log (TCID50/ml) Hours post infection RAW MOI 0.05 MNV1 MNV3 E296K K296E 0 2 4 6 8 10 12 0 12 24 36 48 Log (TCID50/ml) Hours post infection M12 MOI 5 MNV1 MNV3 E296K K296E Discussion Inconsistent results of growth assays in M 12 cells for many mutants viruses were observed in both 1 st and 2 nd experiments and the reason behind this irregularity is still unknown Growth assays should be done thrice and hours post infections should also be extended to at 72 hours to observe the full growth of viruses Figure 1. Growth kinetics of 296 amino acid mutant viruses in RAW cells Figure 2 Growth kinetics of 296 amino acid mutant viruses in RAW cells Single point mutations G101D, A125V, H170Y, R204H, Q371D, Q374F, A393V, A445T and D512G cause mutant viruses to lose replicative capacities and infectivity in vitro. In vitro, t he mutant and parental viruses have similar growth pattern at MOI 5, but K296E mutation has negative impact of viral spread in RAW cells at MOI 0.05. In vitro, K296E mutation has negative impact of viral infectivity in M12 cells at MOI 5. The attenuation growth is detected in M12 cells at low MOI 0 2 4 6 8 10 12 0 12 24 36 48 Log (TCID50/ml) Hours post infection M12 MOI 0.05 MNV1 E296K K296E


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