ENVIRONMENTAL STABILITY OF HEPATITIS C VIRUS (Hepatitis C Virus): VIABILITY OF
DRIED/STORED Hepatitis C Virus IN CHIMPANZEE INFECTIVITY STUDIES.
Author Block: Kris Krawczynski, Miriam J. Alter, Betty H. Robertson,
Ling Lu, John E. Spelbring, Karen A. McCaustland, Centers for
Disease Control and Prevention, Atlanta, GA.

Background.Epidemiologic studies of Hepatitis C Virus have indicated that
transmission among patients in health care settings is associated
with contaminated vehicles such as multi-dose medication vials and
re-used needles and syringes and among injecting drug users is
associated with contaminated drug paraphernalia such as cookers and
cotton. Determining the viability of Hepatitis C Virus and extent to which the
infectious virus can survive on environmental surfaces is critical
for developing effective recommendations for prevention and control.

Material and Methods. Aliquots of Hepatitis C Virus inoculum (CH910 second passage
chimpanzee plasma, genotype 1a) containing 105 chimpanzee infectious
doses (CID) were dried in siliconized microfuge tubes under vacuum
in the presence of Drierite® (W.A. Hammond Drierite Co.) at 250C.
After overnight drying (~16 hrs) samples were either rehydrated with
300 µL sterile water and stored at -700C or transferred to a
controlled environmental chamber (42% humidity, over saturated salt
solution) for a 4 or 7 day storage at 250C and subsequently
rehydrated with 300 µL sterile water and kept at -700C. Samples
dried/stored 7 days and dried overnight were used for reverse
transcriptase polymerase chain reaction (RT-PCR) titration. To
determine infectivity, aliquots of dried/stored plasma for 7 days, 4
days and overnight, were reconstituted in sterile water, suspended
in 1 mL of PBS and administered intravenously to a chimpanzee
(CH247). Size of the infectious dose in each inoculum was calculated
at 3.3 x 104 CID. Plasma samples from CH247 were tested for Hepatitis C Virus RNA

(Amplicor, Roche) and anti-Hepatitis C Virus (Ortho Hepatitis C Virus 3.0), and alanine
aminotransferase (ALT) twice weekly. Liver specimens were obtained
weekly or biweekly and tested for hepatitis C virus antigen (HCVAg)
and histopathology. The animal was first inoculated with Hepatitis C Virus
inoculum dried/stored for 7 days and followed during 129 days.
Subsequently, the animal was inoculated with the Hepatitis C Virus inoculum
dried/stored for 4 days and followed for 134 days, and finally
inoculated with the aliquot dried overnight and followed for 201
days. Virologic, serologic, and clinical data from three control
chimpanzees (CH1555, CH1487, CH1493) inoculated with 3 x 104 CID of
untreated Hepatitis C Virus inoculum (CH910 second passage chimpanzee plasma,
genotype 1a) were included in the study.

Results. Hepatitis C Virus RNA was detectable in plasma dried overnight and 7days,
but a ten fold decrease of detectable Hepatitis C Virus RNA was found in both of
these samples compared with the Hepatitis C Virus RNA titer of the original,
untreated Hepatitis C Virus positive plasma sample. No evidence of Hepatitis C Virus infections
was detected in CH247 after inoculation with either the 7-day or 4-
day dried/stored samples. All serum samples tested were negative for
Hepatitis C Virus RNA and anti-Hepatitis C Virus; ALT activity level remained in the normal
range. However, after inoculation with the overnight dried sample,
Hepatitis C Virus RNA was detected in serum of CH247 from day 7 post inoculation
and the viral load reached 6.0 to 7.3 logs IU/mL. HCVAg positive
hepatocytes were observed from day 11 post inoculation,
seroconversion to anti-Hepatitis C Virus was observed on day 127, and the animal
was still positive for Hepatitis C Virus RNA (4.8 logs IU/mL) at day 201 post
infection. ALT activity level was elevated over the normal range
from day 11 post inoculation and remained elevated until the end of
the observation. Virologic, serologic, and clinical evidence of Hepatitis C Virus
infection and acute hepatitis was found in all three control animals.

Conclusions. Infectivity studies in a chimpanzee suggest that Hepatitis C Virus
may survive on environmental surfaces at room temperature at least
16 hours but not longer than 4 days. The potential for Hepatitis C Virus to
survive in the environment re-emphasizes the importance of cleaning
and disinfection procedures, safe therapeutic injection practices,
and harm reduction counseling and services for injection drug users.