Stability of human metapneumovirus and human coronavirus NL63 on medical instruments and in the patient environment

Madam,

Since their discovery in 2001 and 2004, respectively, the human metapneumovirus (HMPV; a paramyxovirus) and the human coronavirus (HCoV)-NL63 have been found to be important respiratory pathogens.1, 2 Both viruses are responsible for respiratory infections in children and adults and their clinical spectrum ranges from mild to life-threatening clinical syndromes.³ Both viruses have been involved in nosocomial outbreaks, for example in a long-term care facility for elderly institutionalised persons.4, 5, 6, 7 To our knowledge, no investigations on the survival of HMPV and HCoV-NL63 have been published. Rabenau et al. have already demonstrated that the long-described virus HCoV-229E is significantly less stable than severe acute respiratory syndrome (SARS) virus, although both viruses belong to the family of coronaviruses and share many biochemical and structural characteristics.⁸ Consequently, we examined the stability of HMPV and HCoV-NL63, suspended in a medium or dried on surfaces derived from the inanimate hospital environment.

Viruses were grown under standard conditions essentially as previously described.1, 2 Supernatants were collected and viral RNA was extracted using the QIAamp MinElute Virus Spin Kit or RNeasy Protect Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For real-time reverse transcriptase–polymerase chain reaction (RT–PCR) detection and quantification of HMPV, the primers sv581s (NL-N-forward) 5′-CATATAAGCATGCTATATTAAAAGAGTCTC-3′ and sv582as (NL-N-reverse) 5′-CCTATTTCTGCAGCATATTTGTAATCAG-3′ were used. For detection and quantification of HCoV-NL63 the primers repSZ-RT (as) 5′-CCACTATAAC-3′, repSZ-1 s 5′-GTGATGCATATGCTAATTTG-3′ and repSZ-3 as 5′-CTCTTGCAGGTATAATCCTA-3′ were used. Real-time RT–PCRs were performed using the one-step real-time RT–PCR kit (Sybr green) from Qiagen. Detailed temperature profiles are available on request.

For stability analyses, virus-containing cell culture supernatants, with defined copy numbers, were seeded onto different surfaces, namely single-use latex gloves, clinical thermometer caps, stethoscopes, and the plastic surface of a bedside table. Furthermore, viral suspensions were transferred into phosphate-buffered saline (PBS) or cell culture media and stored for the time periods indicated below.

Depending on the surface, viruses were recovered by re-suspending them from the dried surface or by washing the surface. The first procedure was done by wiping the dried surface with PBS-soaked swabs and was used for stethoscopes and the table; the second procedure included washing of the gloves and the clinical thermometers in PBS. Washings and swabbing were performed immediately after seeding and once per hour for the first 8 h, and then daily for seven days. In addition, the virus suspensions transferred to PBS were stored at room temperature and harvested in parallel to the swabs and washings.

Harvesting of swabs, washings, and PBS stored virus suspensions was followed by extraction of nucleic acids (followed by real-time RT–PCR) and inoculation of LLC-MK2 cells. The latter were observed for five days for cytopathic effects before supernatants were harvested and screened for viral RNA by real-time RT–PCR.

For HMPV and HCoV-NL63, drying resulted in rapid loss of infectivity. Although viral RNA was detected up to day 7, isolation of infective particles from the washings failed. This finding was characterised by both a lack of cytopathic effects in the cell culture and no detection of increasing amounts of viral nucleic acids in the cell culture media from the inoculated cell culture dishes. This indicates that after drying no replication occurred. This observation was consistent on all inanimate surfaces tested in this investigation.

By contrast, virus suspensions diluted with PBS and stored for up to seven days remained infective at room temperature, indicating that under such conditions the stability of the viral particles is conserved.

The results support the hypothesis that direct person-to-person transmission is the major route of HMPV and HCoV-NL63 spread. Consequently, contact and droplet isolation of patients seems to be the most important intervention to contain the nosocomial spread of these pathogens. Both viruses are capable of surviving in aqueous solutions and most probably in respiratory secretions until drying is completed. Thus, environmental disinfection of hand contact surfaces and fomites in the close proximity of symptomatic patients seems to be a reasonable addendum to other hygienic precautions.

Conflict of interest statement

None declared.

Funding sources

This work was partially supported by a research grant from the Else Kröner-Fresenius-Stiftung to A. Müller (Children's Hospital, University of Bonn Medical Centre), A. Simon and O. Schildgen, a grant from the European Commission (Contract No. LSHM-CT-2006-037276), and a grant from the Deutsche Gesellschaft für Krankenhaushygiene e.V.