Elsevier

Applied Clay Science

Volumes 124–125, May 2016, Pages 211-218
Applied Clay Science

Research paper
Montmorillonite-mediated aggregation induces deformation of influenza virus particles

https://doi.org/10.1016/j.clay.2016.02.010Get rights and content

Highlights

  • Influenza virus readily aggregates with montmorillonite particles.

  • Influenza virus particles flattened against montmorillonite faces to increase contact area.

  • Virions were predominantly adsorbed to negatively charged platelet faces.

  • Influenza virus infectivity was not compromised despite aggregation and deformation.

Abstract

The interaction between influenza virus [subtype A/Puerto Rico/8/1934 H1N1, (PR8)] and montmorillonite (Mt) is investigated by transmission electron microscopy and biochemical methods to determine how PR8 morphology and viability is affected. The majority of the PR8 particles formed aggregates with the Mt. TEM analysis showed that the virus particles retained structural integrity after aggregation but exhibited changes in morphology when compared to isolated PR8 and Mt aggregated with bromelain-treated PR8 (surface glycoproteins removed). Virus deformation shows that the virions exhibit an attraction to the Mt faces, possibly through hydrophobic interaction. The mean projection area of the aggregated PR8 was (10.4 ± 6.1) × 103 nm2 compared to (9.5 ± 3.3) × 103 nm2 for PR8 missing the surface glycoproteins; and (8.0 ± 3.9) × 103 nm2 for non-aggregated PR8 controls. The increase in projection area of the aggregated PR8 suggests that the viruses deformed to increase contact region with the Mt faces with a subsequent compression normal to the face. PR8 missing the surface protein also exhibited an increase in projection area, although to a lesser extent, indicating that both the surface glycoproteins and viral envelope are attracted to the Mt faces. Circularity calculations indicate that the aggregated PR8 (circularity: 0.69 ± 0.16) are less round, i.e. more distorted, than either control PR8 (0.78 ± 0.14) or aggregated PR8 without surface glycoproteins (0.76 ± 0.12). The pleomorphic nature of influenza virus may allow it to survive the deformation induced by the Mt platelets. High resolution TEM micrographs revealed that the otherwise-round viruses flattened when in contact with platelet faces, thus increasing contact area with the Mt.

The PR8 was found to remain infectious after aggregation although at a lower rate than PR8 controls. The apparent reduced infectivity is likely a result of each aggregate (containing ~ 102 viral particles) acting as a single infectious unit.

Introduction

Influenza is a virus whose interaction with clay minerals has not been investigated. It is a respiratory disease in humans; however, in waterfowl it is an infection of the gastrointestinal tract in which bird-to-bird transmission is fecal-oral. Infected birds secrete large numbers of virus particles into sediment-laden rivers and lakes (Dalton et al., 2009, Franklin et al., 2011, Ito et al., 1995, Nazir et al., 2011, Webster and Hulse-Poste, 2006, Webster et al., 1978). The residence time of secreted influenza viruses in aquatic environments can vary from days to months and is influenced by suspended sediment and water chemistry, thereby affecting bird-to-bird transmission rates. A number of studies (Horm et al., 2011, Horm et al., 2012a, Horm et al., 2012b, Ito et al., 1995, Keeler et al., 2013, Vong et al., 2008) have found that influenza virus persists in lake and rainwater, soil, and waterfowl fecal matter for several months. In fact, avian influenza virus has been detected in water and sediment across the Atlantic seaboard of the United States long after birds have departed indicating that environmental factors may extend virus stability. Therefore, it is of significant importance to determine how clay minerals affect the persistence of influenza virus in aquatic environments (Brown et al., 2009, Brown et al., 2007, Farnsworth et al., 2012, Negovetich and Webster, 2010, Stallknecht et al., 1990a, Stallknecht et al., 1990b).

The interaction between clay minerals and viruses can result in loss of infectivity, though this is not always the case (see Jin and Flury, 2002, Kimura et al., 2008, Theng, 2012). In near neutral pH conditions such as encountered in natural waters virus adsorption by montmorillonite (Mt) is more effective than by illite or kaolinite (Theng, 2012). Viruses such as poliovirus are found to interact with non-aggregated Mt by adsorption to positively charged edges, enhancing virus survival (Vilker et al., 1983). A similar effect on viability was observed by Shirobokov (1968) for coxsackie virus and by Lipson and Stotzky, 1983, Lipson and Stotzky, 1986 for reovirus. Furthermore, mixtures of kaolinite and reovirus have been found to be more infectious and transported more readily than virus alone (Lipson and Stotzky, 1985).

Most studies of clay mineral-virus interaction have focused on non-enveloped viruses. For the enveloped bacteriophage, ϕ6, aggregation with Mt results in disassembly, rendering the virus inactive (Block et al., 2014). Influenza is a pleomorphic, enveloped virus commonly appearing with either a quasi-ellipsoidal or filamentous morphology. The viral envelope is covered with a high density of glycoprotein surface spikes (~ 450 spikes on a typical 130 nm diameter spherical virion (Booy et al., 1985, Harris et al., 2006, Katz et al., 2014)). Approximately 80% of the surface spikes are hemagglutinin (HA) and 20% are neuraminidase (NA) (Compans et al., 1970, Nayak et al., 2009). The protein spikes protrude approximately 14 nm radially from the envelope (Booy et al., 1985, Harris et al., 2006, Katz et al., 2014). There is evidence that the pleomorphic properties of influenza virus serve to protect it from puncture and mechanical stress such as might be encountered in environmental conditions (Li et al., 2011, Schaap et al., 2012, Serebryakova et al., 2011).

Based on the literature regarding the ability for non-enveloped and enveloped viruses to aggregate with clays, we predict that influenza virus particles will aggregate with clay minerals in the water column (Chrysikopoulos and Syngouna, 2012, Syngouna and Chrysikopoulos, 2012, Syngouna and Chrysikopoulos, 2013, Block et al., 2014, Syngouna and Chrysikopoulos, 2015). In this work, transmission electron microscopy (TEM) and biochemical analysis are employed to examine heteroaggregation of Mt and influenza A (subtype virus A/Puerto Rico/8/1934 H1N1 (PR8) to (1) determine the degree to which montmorillonite interacts with the influenza virus leading to sequestration of the virus in the heteroaggregates; (2) the effect of aggregation on virus morphology; and (3) whether aggregated virus remains viable and capable of host infection.

Section snippets

Montmorillonite

The clay mineral sample was a high-purity Na-montmorillonite [commercial name: “Accofloc”; chemical formula: (Na,Ca)0.33(Al1.67Mg0.33)Si4O10(OH)2·nH2O; from American Colloid Company, Arlington Heights, IL]. Accofloc is a Na-Mt Volclay purified from Wyoming bentonite. Accofloc has a cation exchange capacity (CEC) of 79 meq/100 g (Sterte and Shabtai, 1987). Larger particles and non-clay minerals were removed by centrifugation at 3600 rpm (2700g) for 20 min. A 5% sodium hypochlorite (bleach) wash was

Formation of aggregates of influenza PR8 and Mt

Mt dispersed with PR8 and PR8δHN influenza formed aggregates within 30 min of mixing. Low speed centrifugation separated non-aggregated Mt and individual virions from PR8-Mt aggregates > 0.5 μm in size. SDS-PAGE (Fig. 2) of the non-aggregated PR8 and Mt supernatant and aggregated pellet fractions reveals that most of the PR8 aggregated with the Mt while only trace amounts of PR8 remained non-aggregated in the supernatant.

TEM of PR8 aggregated with Mt

Micrographs of aggregates of PR8-Mt and PR8δHN-Mt are shown in Fig. 3a and b,

Acknowledgments

This work was supported in part by City Seed Grant #93370-09 from The City College of New York; PSC-CUNY Grant #67709-00 45 from the City University of New York; the Research Centers in Minority Institutions (NIH/NCRR/RCMI) CCNY/Grant G12-RR03060; and a National Institute of General Medical Science Grant SC1-GM092781.

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  • Cited by (0)

    1

    Current address: Department of Earth and Environmental Sciences, University of Michigan, Ann Arbor, MI, United States.

    2

    Current address: New York Structural Biology Center, New York, NY 10031, United States.

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