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- Cohnstaedt, Lee
- Wilson, William - Bill
- Anderson, Tavis
- Mire, Chad
- Mitzel, Dana
- Noronha, Leela
- Animal Health (103)

Research Project: **Location:
                            Research Programs** Project Number: 3022-32000-018-017-S
                        
                        Project Type: Non-Assistance Cooperative Agreement Start Date: Sep 15, 2022
                        
                        End Date: Apr 30, 2027 **Objective:**
                        
                        RNA viruses have the capacity for rapid evolution due to their high mutation rates, short replication times, and large population sizes. However, in many cases rapid rates of arbovirus evolution have not been observed. This observed genetic conservation may be the result of the alternate cycles of viral replication in vertebrate hosts and arthropod vectors. This hypothesis suggests that compromises in the replication of arboviruses are made regularly by virus populations, in both the arthropod vectors and vertebrate hosts, due to the differential selection in each. Specifically, mutations advantageous to either host are purged by purifying selection if they are detrimental to replication in the alternative host; positive selection then generally results from the infrequent mutations that result in coadaptation. However, the introduction of novel serotypes into otherwise enzootic areas and the incursion of viruses into new areas suggests the need to gain a greater understanding of single strand and segmented virus evolution, fitness, and genetic diversification to better develop management and vaccination strategies to reduce the future impact of endemic and exotic virus epizootics. 
Previously we have demonstrated that cell culture infection of insect cells with the intracellular bacteria Wolbachia impacts single strand and segmented virus replication rates. Here the Cooperator proposes to use Wolbachia infected cells as a mechanism to artificially create a bottleneck wherein more virulent viral strains that bypass Wolbachia induced inhibitory effects to examine for the virus’s ability to adapt and diversify in invertebrate host cells. The Cooperator will assess the relative fitness, replicative ability, and genetic alteration of different virus serotypes during serial passage in Wolbachia infected and Wolbachia uninfected insect cell culture. The Cooperator will determine to clarify the extent to which viral variants are capable of cell-specific adaptation and the degree to which the adaption alters both the viral fitness and genetic sequences. **Approach:**
                        
                        To address the aforementioned objectives investigators will examine virus-vector host interactions to determine the success of viral infection. Deciphering these interactions will be vital to understanding viral fitness in its natural invertebrate host. This knowledge will also provide fundamental insights into the virus life cycle and rates of recombination/mutation, and will contribute to the development of novel antiviral strategies and more effective vaccines.  
1.  Determine viral fitness and replication rates of different serotypes in Wolbachia infected insect host cells.

Wolbachia has been shown to inhibit arbovirus transmission in multiple arthropods and in reduce viral replication rates in insect cell culture.  Here we propose to examine RNA viral fitness of multiple serotypes (n=3 or 4) when introduced into Wolbachia infected cell cultures. Insect cells will be cultured and passaged at a 1:4 ratio of cells: media every 5-7 days. To examine the effects of Wolbachia on viral infection, at least six replicate 25 cm2 flasks of Wolbachia infected and uninfected cells will be inoculated with different viral serotypes at MOI 0.25 in 5 mL of media. At 0, 5, and 7 days post infection (DPI), duplicate flasks will be frozen and held at -80 °C. 'Time zero' flasks will be frozen within 1 hr of the inoculum being added.

To quantify virus copy number, RNA will be isolated using RNeasy mini kits and approximately 1 μg of total RNA will be converted to cDNA using a Reverse Transcriptase. A fragment of the nonstructural NS3 gene for each virus will be amplified using cDNA and primers specific to virus serotypes. All amplifications will be performed as two technical replicates. The copy numbers of the virus serotypes will be determined using a standard curve generated from each NS3 gene by analyzing the two biological and two technical replicates. Serial passaging and subsequent examination of viral fitness will be repeated for at least 10 consecutive passages. One flask will be saved for plaque assays to determine virus titers in Wolbachia uninfected and infected cell cultures. Three biological replicates will performed for each time point and passage.

2.  Whole genome sequencing to detect the occurrence of single nucleotide variants (SNVs) over successive passages of virus in Wolbachia infected cells.

We will use Wolbachia infected cells to select for more virulent viral strains and examine for mutations after >10 consecutive serial passages.  Virus from infected insect cells from Objective 1 will be collected after passages 1, 2, 4, 6, 8, and 10 and prepared for genome sequencing as previously described (Kopanke et al. 2020). Viral population diversity will be addressed by comparing SNV for multiple viral segments for each of the passages and virus serotypes. Viral population diversity and richness will also be determined for each coding sequence SNV frequencies for each segment for each of the passages and viral serotypes.


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