Higher Concentrations of Bacterial Enveloped Virus Phi6 Can Protect the Virus from Environmental Decay

Abstract

Phage Phi6 is an enveloped virus considered as a possible non-pathogenic surrogate for SARS-CoV-2 and other viral pathogens in transmission studies. Higher input amounts of bacteriophage Phi6 are shown to delay and protect the phage from environmental decay, both when the phage are dried in plastic tubes, and when they are stored in saline solution at 4°C. By contrast, when bacteriophage Phi6 are placed in LB (Luria-Bertani) growth medium (instead of saline) prior to placement on the plastic surface, the influence of starting concentration on viral recovery is negligible. The protection is reflected in longer half-lives of the phage at higher concentrations compared to lower. Because experiments supporting the possibility of fomite transmission of SARS-CoV-2 and other viruses rely upon survival of infectious virus following inoculation of various surfaces, high initial amounts of input virus on a surface may generate artificially inflated survival times compared to realistic lower levels of virus that a subject would normally encounter. This is not only because there are extra half-lives to go through at the higher concentrations, but also because the half-lives themselves are extended at the higher virus concentrations. It is important to design surface drying experiments for pathogens with realistic levels of input virus, and to consider the role of the carrier and matrix if the results are to be clinically relevant. IMPORTANCE During the COVID-19 pandemic, a lot of attention has been paid to the environmental decay of SARS-CoV-2 due to proposed transmission of the virus via fomites. However, published experiments have commenced with very high virus titer inoculums, an experimental design not representative of real-life conditions. The study described here evaluated the impact of initial virus titer on environmental decay of an enveloped virus, using a non-pathogenic surrogate for transmission of SARS-CoV-2, enveloped bacteriophage Phi6. We establish that higher concentrations of virus can protect the virus from environmental decay, depending on conditions. This has important implications for stability studies of SARS-CoV-2 and other viruses. Our results point to a limitation in the fundamental methodology that has been used to attribute fomite transmission for almost all respiratory viruses.

Factors Impacting Persistence of Phi6 Bacteriophage, an Enveloped Virus Surrogate, on Fomite Surfaces

“…The impact of inoculum level on virus persistence on fomites has been highlighted previously ( 15 – 17 , 19 ). Lai et al ( 19 ) observed greater survival of SARS-CoV-1 at higher virus titers (4, 5, and 6 log 50% tissue culture infective dose [TCID 50 ]/mL) with survival times ranging from <5 min to 48 h following inoculation on paper, a disposable gown, and a cotton gown.…”

“…Lai et al ( 19 ) observed greater survival of SARS-CoV-1 at higher virus titers (4, 5, and 6 log 50% tissue culture infective dose [TCID 50 ]/mL) with survival times ranging from <5 min to 48 h following inoculation on paper, a disposable gown, and a cotton gown. Bangiyev et al ( 17 ) observed longer half-lives (overall range of 5 to 57 min) of Φ6 in a saline inoculum matrix when vacuum-dried on plastic tubes at approximately 4 log PFU versus when evaluated at lower concentrations (3 and 2 log PFU). However, longer half-lives at increased inoculation levels were not observed with Φ6 in a Luria-Bertani inoculum matrix on plastic (half-lives ranged from 9 to 18 h), which provides evidence that extended survival may not always result from an increased inoculum titer.…”

“…The extent of indirect contact transmission via fomites and subsequent risk of infection for SARS-CoV-2 has been controversial, as previous studies have inoculated surfaces with virus titers that do not represent real-world contamination scenarios ( 15 , 16 ). Slower inactivation kinetics of viruses have been observed at higher inoculation levels ( 17 , 18 ), although further evidence is needed, as inoculum matrix (e.g., artificial saliva, vomitus, feces, etc.) may impact this observation ( 17 ).…”

“…Slower inactivation kinetics of viruses have been observed at higher inoculation levels ( 17 , 18 ), although further evidence is needed, as inoculum matrix (e.g., artificial saliva, vomitus, feces, etc.) may impact this observation ( 17 ). Further understanding the scenarios that facilitate an increased risk of transmission due to persistence of enveloped viruses will help guide mitigation strategies in consumer-facing environments (e.g., restaurants, waiting rooms, public transportation, etc.)…”

“…To further substantiate Φ6 as a surrogate for enveloped viruses of public health importance, persistence data based on the inoculum matrix, inoculum level, and surface type are needed. Previous studies have evaluated the persistence of Φ6 on fomites ( 8 , 17 , 21 – 23 ). However, these studies have not characterized persistence on multiple unique surface types with various inoculum matrices and levels.…”