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Published on: November 10, 2023
Detection of MHV-RNAs in mouse intestines and in filter dust in mouse room ventilation duct by a modified RT-nested
Mitsuru Oyanagi1, Akiko Kato, Yasuko K Yamada
1Animal Resources Branch, Center for Bioresource-based Research, Brain Researches Institute, Niigata University, Niigata, Japan.
Abstract:
We applied RT-nested PCR for the detection of MHV genomic RNA in a modified manner to obtain RNA from the intestines of mice and from filter dust in the ventilation ducts of the room in which a contaminated mouse colony was kept. Since the sequences of MHV-RNA that were extracted from the intestine of a serologically MHV-positive mouse in room No. 2 (MS2) and from the filter dust in a ventilation duct in the same room (FD2) were identical, amplified product from filter dust was demonstrated to come from the MHV contaminated room. Furthermore, sequences of FD2 and of filter dust from another contaminated mouse room, No. 7 (FD7) showed 38 nucleotide exchanges among 368-bp (10.3%), suggesting that two different MHV strains were contaminating our facilities. SSCP analysis of Dra I-digested PCR product of 393 bp also showed different patterns in FD2 and FD7 samples.
Insights
This study detected Mouse Hepatitis Virus (MHV) genomic RNA in mouse intestines and ventilation systems. Identical MHV strains were found in a room
Area of Science:
- Veterinary Virology
- Environmental Monitoring
- Infectious Disease Transmission
Background:
- Mouse Hepatitis Virus (MHV) is a significant pathogen in laboratory mouse colonies.
- Contamination of animal facilities poses a risk to research integrity.
- Detecting MHV in environmental samples is crucial for effective control strategies.
Purpose of the Study:
- To develop and apply a modified RT-nested PCR method for MHV detection.
- To investigate MHV contamination in both animal hosts and the facility environment.
- To identify potential sources and diversity of MHV strains within the facility.
Main Methods:
- RNA extraction from mouse intestines and ventilation filter dust.
- Modified RT-nested PCR for MHV genomic RNA amplification.
- Sequence analysis of amplified MHV RNA.
- Single-Strand Conformation Polymorphism (SSCP) analysis.
Main Results:
- MHV genomic RNA was successfully detected in both mouse intestines and ventilation filter dust.
- Sequences from mouse intestine (MS2) and filter dust (FD2) in room 2 were identical, confirming environmental contamination.
- Significant nucleotide differences (10.3%) between FD2 and FD7 samples suggest the presence of at least two distinct MHV strains.
- SSCP analysis further differentiated the MHV strains found in different rooms.
Conclusions:
- The modified RT-nested PCR is effective for detecting MHV in environmental samples.
- Ventilation systems can harbor MHV RNA, indicating a potential route for transmission.
- The facility was contaminated with at least two different MHV strains, necessitating enhanced monitoring and control measures.
