Dataset Information

Magnetic bead purification of enveloped alphavirus and flavivirus.

ABSTRACT: Virus production is an essential part of virology. The quality of virus preparation, in terms of purification and yield, can affect the outcome of experiments. Purified virus is critical for assays, as contamination from cell culture supernatants may affect results. Purified virus is also needed for vaccine preparations. The traditional process of gradient purification of virus is time consuming, with multiple steps spanning over days, and produces significant amounts of biohazard waste. Developing a more efficient alternative that provides a purified virus product is highly desirable. In this study, we performed magnetic bead purification of Zika virus (ZIKV) and Mayaro virus (MAYV) with the Mag4C LV kit from OZ Biosciences. Currently, this kit is only indicated for use with lentiviruses and retroviruses. We found that we were able to successfully concentrate and purify both ZIKV and MAYV using the kit. Therefore, we concluded that the Mag4C LV kit provides a quick and simple alternative to traditional virus purification methods.•Our protocol is customized by using an alphavirus (MAYV) and flavivirus (ZIKV). This method has not been previously used for these viruses.

SUBMITTER: Galasso B

PROVIDER: S-EPMC8720801 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Magnetic bead purification of enveloped alphavirus and flavivirus.

Galasso Bianca B Sharma Shreya S Knollmann-Ritschel Barbara B Sharma Anuj A

MethodsX 20211014

Virus production is an essential part of virology. The quality of virus preparation, in terms of purification and yield, can affect the outcome of experiments. Purified virus is critical for assays, as contamination from cell culture supernatants may affect results. Purified virus is also needed for vaccine preparations. The traditional process of gradient purification of virus is time consuming, with multiple steps spanning over days, and produces significant amounts of biohazard waste. Develop ...[more]

PMID: 35004196

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Project description:The impact of the host microbiota on arbovirus infections is currently not well understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that preincubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivity in vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of the Alphavirus genus and Zika virus of the Flavivirus genus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after preincubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses. IMPORTANCE During the past decades, the world has experienced a vast increase in epidemics of alphavirus and flavivirus infections. These viruses can cause severe diseases, such as hemorrhagic fever, encephalitis, and arthritis. Several alpha- and flaviviruses, such as chikungunya virus, Zika virus, and dengue virus, are significant global health threats because of their high disease burden, their widespread (re-)emergence, and the lack of (good) anti-arboviral strategies. Despite the clear health burden, alphavirus and flavivirus infection and disease are not fully understood. A knowledge gap in the interplay between the host and the arbovirus is the potential interaction with host skin bacteria. Therefore, we studied the effect of (skin) bacteria and bacterial cell wall components on alphavirus and flavivirus infectivity in cell culture. Our results show that certain bacterial cell wall components markedly reduced viral infectivity by interacting directly with the virus particle.

Project description:Alphaviruses and flaviviruses have class II fusion glycoproteins that are essential for virion assembly and infectivity. Importantly, the tip of domain II is structurally conserved between the alphavirus and flavivirus fusion proteins, yet whether these structural similarities between virus families translate to functional similarities is unclear. Using in vivo evolution of Zika virus (ZIKV), we identified several novel emerging variants, including an envelope glycoprotein variant in β-strand c (V114M) of domain II. We have previously shown that the analogous β-strand c and the ij loop, located in the tip of domain II of the alphavirus E1 glycoprotein, are important for infectivity. This led us to hypothesize that flavivirus E β-strand c also contributes to flavivirus infection. We generated this ZIKV glycoprotein variant and found that while it had little impact on infection in mosquitoes, it reduced replication in human cells and mice and increased virus sensitivity to ammonium chloride, as seen for alphaviruses. In light of these results and given our alphavirus ij loop studies, we mutated a conserved alanine at the tip of the flavivirus ij loop to valine to test its effect on ZIKV infectivity. Interestingly, this mutation inhibited infectious virion production of ZIKV and yellow fever virus, but not West Nile virus. Together, these studies show that shared domains of the alphavirus and flavivirus class II fusion glycoproteins harbor structurally analogous residues that are functionally important and contribute to virus infection in vivo. IMPORTANCE Arboviruses are a significant global public health threat, yet there are no antivirals targeting these viruses. This problem is in part due to our lack of knowledge of the molecular mechanisms involved in the arbovirus life cycle. In particular, virus entry and assembly are essential processes in the virus life cycle and steps that can be targeted for the development of antiviral therapies. Therefore, understanding common, fundamental mechanisms used by different arboviruses for entry and assembly is essential. In this study, we show that flavivirus and alphavirus residues located in structurally conserved and analogous regions of the class II fusion proteins contribute to common mechanisms of entry, dissemination, and infectious-virion production. These studies highlight how class II fusion proteins function and provide novel targets for development of antivirals.

Dataset Information

Magnetic bead purification of enveloped alphavirus and flavivirus.

Publications

Magnetic bead purification of enveloped alphavirus and flavivirus.

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