The Effects of Temperature and Relative Humidity on the Viability of the Coronavirus

Abstract

The main route of transmission of SARS CoV infection is presumed to be respiratory droplets. However the virus is also detectable in other body fluids and excreta. The stability of the virus at different temperatures and relative humidity on smooth surfaces were studied. The dried virus on smooth surfaces retained its viability for over 5 days at temperatures of 22–25°C and relative humidity of 40–50%, that is, typical air-conditioned environments. However, virus viability was rapidly lost (>3 log10) at higher temperatures and higher relative humidity (e.g., 38°C, and relative humidity of >95%). The better stability of SARS coronavirus at low temperature and low humidity environment may facilitate its transmission in community in subtropical area (such as Hong Kong) during the spring and in air-conditioned environments. It may also explain why some Asian countries in tropical area (such as Malaysia, Indonesia or Thailand) with high temperature and high relative humidity environment did not have major community outbreaks of SARS.

1. Introduction

Severe acute respiratory syndrome (SARS), was a new emerging disease associated with severe pneumonia and spread to involve over 30 countries in 5 continents in 2003. A novel coronavirus was identified as its cause [13]. SARS had a dramatic impact on health care services and economies of affected countries, and the overall mortality rate was estimated to be 9%, but rising to 50% in those aged 60 or above [4]. A notable feature of this disease was its predilection for transmission in the health care setting and to close family and social contacts. The disease is presumed to be spread by droplets, close direct or indirect contact, but the relative importance of these routes of transmission is presently unclear. A study showed that viral aerosol generation by a patient with SARS was possible and therefore airborne droplet transmission was a possible means of transmission [5]. However, the role of fomites and environmental contamination in transmission of infection is presently still unclear. An outbreak of disease affecting over 300 residents in high-rise apartment block (Amoy Gardens) in Hong Kong could not be explained by respiratory droplet transmission from infected patients [6]. Infectious virus is detectable in the faeces [7], and aerosolization of virus in contaminated faeces is believed to be the mode of transmission of this outbreak [8].
We and others have reported that infectivity of SARS CoV (SARS coronavirus) was lost after heating at 56°C for 15 minutes but that it was stable for at least 2 days following drying on plastic. It was completely inactivated by common fixatives used in laboratory [910]. Another study showed that it was inactivated by ultraviolet light, alkaline (), or acidic () conditions [11]. Human coronaviruses have been shown to survive in PBS or culture medium with 5–10% FCS for several days [1214] but they only survive a few hours after drying [1314]. There have been some studies reporting an association between the SARS outbreak, metrological factors, and air pollution [1517]. Thus, information on the survival of the SARS coronavirus (SCoV) in the environment at different temperature and humidity conditions is of significant interest to understanding virus transmission. A recent study using surrogate coronaviruses (transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHC)) has investigated the effect of air temperature and relative humidity on coronavirus survival on surface [18]. The survival effects of these environmental factors on SARS coronavirus remain unclear. In the present study, we report the stability of the SARS coronavirus at different temperatures and relative humidity.

2. Material and Methods

2.1. Virus Strain and Cell Line

The SARS CoV strain used in this study is HKU39849. Foetal monkey kidney cells (FRhK-4) were cultured in minimal essential medium (MEM, Gibco, USA) with 10% foetal calf serum and penicillin streptomycin (Gibco, USA) at 37°C in 5% CO2 and were used for growing stock virus and for titration of viral infectivity [12].

2.2. Preparation of Stock Virus

Stock virus was harvested when infection approximately 75% of the cell monolayer of a virus infected flask manifested cytopathic effect (CPE). Infected cells were subjected to one cycle of freeze and thaw centrifuged at 2000 rpm for 20 minutes to remove cell debris and the culture supernatant was aliquoted and stored at −80°C until use.

2.3. Determination of Tissue Culture Infectious Dose (50%) (TCID50)

96-well microtitre plates containing 100 μL of confluent FRhK-4 were infected with 100 μL of serial 10-fold of dilutions of stock virus in minimal essential medium with 1% FCS (maintenance medium) starting from 10−1 to 108. Titrations were done in quadruplicate. Infected cells were incubated for 4 days at 37°C. Appearance of CPE was recorded daily. TCID50 was determined according to Reed and the Muench method [19].

2.4. Effect of Drying, Heat, and Relative Humidity

Ten microlitre of maintenance medium containing 107 TCID50 per mL of virus was placed in individual wells of a 24-well plastic plates and allowed to dry at room temperature (22~25°C) and relative humidity of 40–50% (i.e., conditions prevailing in a typical air-conditioned room). One hundred microlitre of MM was used to resuspend the virus at 0 hr, 3 hr, 7 hr, 11 hr, 13 hr, 24 hr, and up to 4 weeks and the residual virus infectivity was titrated. Controls in closed screw cap eppendorf tube were included each time and treated similarly but without drying.
The experiment was repeated at different temperatures (38°C, 33°C, 28°C) and relative humidities (>95%, 80~89%) for 3 hr, 7 hr, 11 hr, 13 hr, and 24 hr. A nebulizer under a controlled condition was used to generate high and relative low humidity environment. All the experiments above were conducted in duplicate and the residual viral infectivity was titrated.

2.5. Infectivity Assay

The infectivity of residual virus was titrated in quadruplicate on 96-well microtitre plates containing 100 μL of confluent FRhK-4 cells. 100 μL of serial 10-fold of dilutions of virus in maintenance medium starting from 10−1 to 108 was added into FRhK-4 cells. The infected cells were incubated at 37°C for 4 days. Appearance of CPE was recorded daily. TCID50 was determined according to the Reed and Muench method [19].

3. Results

Ten microlitre of 107 TCID50 per mL of virus was placed in individual wells of a 24-well plastic plate (representing a nonporous surface) and dried. The dried virus was then incubated at different temperatures (38°C, 33°C, 28°C) at different relative humidity (>95%, 80~89%) for 3 hr, 7 hr, 11 hr, 13 hr, and 24 hr and the residual viral infectivity was titrated. A similar experiment was conducted at room temperature and relative humidity of about 40–50% (air-conditioned room) for up to 4 weeks. Virus dried on plastic retained viability for up to 5 days at 22~25°C at relative humidity of 40~50% with only  loss of titre (Figure 1). After that virus infectivity is gradually lost ever time. Loss of virus infectivity in solution was generally similar to dried virus under these environmental conditions. This indicates that SARS CoV is a stable virus that may potentially be transmitted by indirect contact or fomites, especially in air-conditioned environments.

Leave a Reply