Acanthamoeba polyphaga mimivirus stability in environmental and clinical substrates: implications for virus detection and isolation

Abstract

Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

Figure 1. APMV recovery from samples after 1 hour.

10⁶ TCID₅₀ of purified APMV were added to salt water (10 ml), fresh water (10 ml), topsoil (1 g) and three different brands of VD substrates, and after one hour, virus recovery was evaluated by titration in A. castellanii. The fresh water and soil samples were collected from three different Brazilian biomes: Amazon and Mata Atlântica, two highly biodiverse rainforests, and Cerrado, a savanna-like biome. The salt water samples were collected from 3 points on the coast of South and Southeast Brazil, for a total of five samples per substrate per biome. The results are the means+SD of an experiment performed in quintuplicate.

Figure 2. APMV stability in different substrates.

To evaluate the long-term stability of APMV in different substrates for one year, 10⁶ TCID₅₀ of purified APMV were added to salt water, soil, fresh water and VD substrates, which were maintained in 15 ml tubes at room temperature. At monthly intervals, the samples were titrated in amoebae. (A) Salt water; (B) Fresh water; (C) Soil; (D) VD. The results are the means+SD of an experiment performed in quintuplicate. I, II and III represent independent experiments performed with samples collected from distinct locations.

Figure 3. APMV isolation in different substrates after enrichment.

As above, substrates were inoculated with APMV (10⁶ viral particles) and then enriched. At one-month intervals, the samples were titrated in amoebae. (A) Salt water; (B) Fresh water; (C) Soil; (D) VD. The results are the means+SD of an experiment performed in quintuplicate. I, II and III represent independent experiments performed with samples collected from distinct locations.

Figure 4. APMV stability in VD enriched after BAL addition.

Purified APMV (10⁶ viral particles) were added to VD samples, and subsequently, BAL samples were added. The BAL-VD samples were or were not enriched and maintained at room temperature. The samples were titrated in amoebae monthly. (A) Samples without enrichment; (B) Enriched samples. The results are the means+SD of an experiment performed in quintuplicate.

Figure 5. APMV one-step growth curves with or without enrichment.

Purified APMV (10⁶ viral particles) was added to salt water, soil, fresh water and VD substrates, which were then enriched. After viral isolation from each substrate, A. castellanii were infected at an MOI of 10. The infectivity was measured by TCID₅₀ after 25 hours (0 to 25 hours) by observing CPE in amoeba. The results are the means of experiments performed in duplicate.