ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2010, p. 452–459
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 1
Inhibition of B Virus (Macacine herpesvirus 1) by Conventional and
Experimental Antiviral Compounds?
P. W. Krug,1† R. F. Schinazi,2and J. K. Hilliard1*
Viral Immunology Center, Georgia State University,1and Laboratory of Biochemical Pharmacology, Department of Pediatrics,
Emory University School of Medicine and Veterans Affairs Medical Center,2Atlanta, Georgia
Received 25 October 2008/Returned for modification 8 December 2008/Accepted 19 October 2009
B virus infection of humans results in high morbidity and mortality in as many as 80% of identified cases.
The main objective of this study was to conduct a comparative analysis of conventional and experimental
antiviral drug susceptibilities of B virus isolates from multiple macaque species and zoonotically infected
humans. We used a plaque reduction assay to establish the effective inhibitory doses of acyclovir, ganciclovir,
and vidarabine, as well as those of a group of experimental nucleoside analogs with known anti-herpes simplex
virus activity. Four of the experimental drugs tested were 10- to 100-fold more potent inhibitors of B virus
replication than conventional antiviral agents. Drug efficacies were similar for multiple B virus isolates tested,
with variations within 2-fold of the median effective concentration (EC50) for each drug, and each EC50was
considerably lower than those for B virus thymidine kinase (TK) mutants. We observed no differences in the
viral TK amino acid sequence between B virus isolates from rhesus monkeys and those from human zoonoses.
Differences in the TK protein sequence between cynomolgus and pigtail macaque B virus isolates did not affect
drug sensitivity except in the case of one compound. Taken together, these data suggest that future B virus
zoonoses will respond consistently to conventional antiviral treatment. Further, the considerably higher
potency of FEAU (2?-fluoro-5-ethyl-Ara-U) than of conventional antiviral drugs argues for its compassionate
use in advanced human B virus infections.
In its natural host, macaque monkeys, B virus (Macacine
herpesvirus 1; Simplexvirus, Herpesviridae) causes lesions on
epithelial surfaces (2, 33) and establishes reactivatable latent
infection in sensory neurons (30, 37), like herpes simplex virus
(HSV) in humans. B virus often results in severe pathogenesis,
including paralysis, encephalitis, and in many cases, rapid
death, following infection of humans (reviewed in reference
22). Nearly all reported cases of B virus zoonosis have been
associated with individuals handling macaques during the
course of research or technology development (33). Five fatal-
ities, along with at least 23 cases in which the patient survived,
have occurred in the past 20 years, underscoring that zoonotic
infections remain a problem in the laboratory animal environ-
The CDC’s B Virus Working Group currently recommends
treatment of confirmed zoonotic infections with herpesvirus-
specific antiviral drugs, including acyclovir (ACV) and ganci-
clovir (GCV) (10). Both agents in this class of compounds are
phosphorylated to active form by virus-encoded thymidine ki-
nase (TK). The resulting nucleoside triphosphate analog in-
hibits viral DNA replication by termination of chain elongation
and by direct inhibition of herpesvirus DNA polymerase (24).
While ACV is effective against B virus both in cell culture and
in animal models (6), the dose required for 50% plaque re-
duction is more than 10-fold higher for B virus than for HSV
type 1 (HSV-1) (38). GCV is twice as potent as ACV in B virus
plaque reduction, yet the median effective concentration
(EC50) of GCV for B virus is almost 10 times higher than that
for HSV-1 (38). In some, but not all, cases of zoonotic B virus
infection, acyclovir and ganciclovir have proven to be effective
at curtailing disease progression (7, 8). Zoonotic infections
that have progressed to extensive central nervous system
(CNS) involvement, including respiratory arrest, appear to be
refractory to conventional antiviral intervention (12, 15; J.
Hilliard, unpublished data).
Vidarabine (9-?-D-arabinofuranosyladenine [ara-A, or VDB]),
an antiviral agent effective against HSV, may be useful for the
treatment of early stages of zoonotic B virus infections, but it
has not been used alone in previous cases. Prior to the use of
ACV for treatment of HSV, intravenous VDB was used for
treatment of encephalitic infections (34). In cell culture, it has
been shown to be equally potent against B virus and HSV-1
(5). VDB does not require selective phosphorylation by the
viral TK for activity (4); however, since VDB can be converted
to its active form by host enzymes, it has potential toxicity in
Experimental drugs effective in cell culture against HSV
include the ?-D-2?-fluoro-5-substituted arabinosyl pyrimidines
FMAU (2?-fluoro-5-methyl-Ara-U) and FEAU (2?-fluoro-5-
ethyl-Ara-U), both of which require phosphorylation by viral
thymidine kinase for activation (19). FMAU and FEAU have
potencies similar to that of ACV for inhibition of HSV-1 in cell
culture, but FMAU has 10-fold greater potency than ACV
against HSV-2 (19). Unfortunately, FMAU has been reported
to be toxic to humans at elevated doses used for cancer che-
motherapy (1, 13); FMAU can be incorporated into uninfected
cell DNA by host DNA polymerase, suggesting the basis of its
* Corresponding author. Mailing address: Viral Immunology Center,
Georgia State University, P.O. Box 4118, Atlanta, GA 30302. Phone:
(404) 413-6560. Fax: (404) 413-6569. E-mail: firstname.lastname@example.org.
† Present address: Plum Island Animal Disease Center, Agricultural
Research Service, United States Department of Agriculture, Orient
?Published ahead of print on 26 October 2009.
toxicity in vivo (11). FEAU has been shown to be a selective
inhibitor of HSV DNA synthesis in cell culture (7), but its
toxicity in humans has not been investigated. Other ?-D-2?-
fluoro-5-substituted arabinosyl pyrimidines, such as FMAC
(2?-fluoro-5-methyl-Ara-C) and FBrAC (2?-fluoro-5-bro-
myl-Ara-C), have been synthesized and tested against HSV
(17, 32; R. F. Schinazi, unpublished results). No information
on FBrAC toxicity is known.
The goal of this study was to determine the general variabil-
ity of drug susceptibility and the efficacy of a class of experi-
mental antiviral agents by using a panel of B virus isolates from
multiple macaque species and humans. The results presented
here suggest that B virus isolates in the wild are susceptible to
antiviral agents that require the viral TK for activity. Further,
our findings that specific experimental nucleoside analogs are
appreciably more effective than conventionally used antiviral
agents at blocking B virus replication suggest that these drugs
may benefit the treatment of high-morbidity human cases.
MATERIALS AND METHODS
Cells and viruses. African green monkey kidney (Vero) cells (ATCC CCL-81)
were obtained from the American Type Culture Collection (Manassas, VA) and
propagated in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech) sup-
plemented with 7% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). Infec-
tions were done using DMEM supplemented with 1% FBS. All B virus plaque
reduction assays were performed under biosafety level 3 conditions acceptable at
the time of these experiments performed in compliance with the 4th edition of
the Biosafety in Microbiological and Biomedical Laboratories. B virus infections
for viral DNA purification and virus stock production were done under biosafety
level 4 conditions, as mandated by the 5th edition of Biosafety in Microbiological
and Biomedical Laboratories (28a) for propagating and handling B virus.
The E2490 reference strain of rhesus macaque B virus was originally obtained
from R. N. Hull, Eli Lilly Research Laboratories (Indianapolis, IN), and passage
72 was used. All other wild-type viruses used in this study were diagnostic isolates
obtained from the NIH’s NCRR-supported National B Virus Resource Center,
Georgia State University, Atlanta, GA. Restriction endonuclease digestions of
these isolates’ viral genomes are shown in Fig. 1.
The TK mutants were generated by cotransfection of NaI gradient-purified
E2490 viral DNA with plasmids containing the TK gene with either a deletion
(pBV?TK) or a premature stop codon (pBVTKstop) into rabbit skin cells
(ATCC CCL-68) (Fig. 2A) (technique described in reference 27). For pBVTK-
stop, a SpeI linker containing stop codons in all frames (catalog no. 1061; New
England Biolabs) was inserted into the BglII site of pBVTK. Dilutions of the
subsequent transfection stocks were plated on Vero cells, and individual plaques
were screened by PCR and Southern hybridization. After three (BV?TK) or four
(BVTKstop) rounds of plaque purification, one plaque with no evidence of
wild-type virus was chosen as the representative mutant of each virus for prep-
aration of stocks.
The DNA sequence of both viral mutants was confirmed by sequencing.
Confirmation of the deletion and stop codon insertion using PCR is shown in Fig.
2B. The parent virus PCR amplimer is 1,143 bp (Fig. 2B, lane 1). The deletion
from ApaI to BspEI removes a 759-bp fragment, resulting in a 384-bp amplimer
(lane 2). The insertion of the stop codon linker into the BglII site results in a
1,157-bp amplimer (lane 4) that, when cut with SpeI, yields 856-bp and 301-bp
fragments (lane 3). Manipulation of these drug-resistant TKnullviruses is re-
stricted to a biosafety level 4 laboratory in accordance with the Georgia State
University Institutional Biosafety Committee and the Office of Biotechnology
Activities at the National Institutes of Health.
Antiviral compounds. Acyclovir (ACV), ganciclovir (GCV), and vidarabine
(VDB) were purchased from Sigma (St. Louis, MO). The compounds
1-(2-fluoro-5-methyl-beta-D-arabinofuranosyl)uracil (FMAU), 1-(2-deoxy-2-
fluoro-beta-D-arabinofuranosyl)-5-ethyluracil (FEAU), 1-(2-deoxy-2-fluoro-
beta-D-arabinofuranosyl)-5-bromylcytosine(FBrAC), and 1-(2-deoxy-2-
FIG. 1. Confirmation of B virus isolate genotype by RFLP. Viral DNA was digested with the SalI restriction enzyme and was separated on a
0.8% agarose gel. MR, rhesus macaque isolate; E2490, prototype laboratory strain; A, human isolate; MJ, Japanese macaque isolate; MC,
cynomolgous macaque isolate; MP, pigtail macaque isolate. Size markers are indicated in kilobase pairs and reflect the positions of BstEII-digested
lambda DNA fragments.
VOL. 54, 2010B VIRUS ANTIVIRALS 453
fluoro-beta-D-arabinofuranosyl)-5-methylcytosine (FMAC) were synthesized
in one of our laboratories (R.F.S.).
Plaque reduction assay. Confluent monolayers of Vero cells in 12-well micro-
plates were infected with approximately 200 PFU of virus and incubated at 37°C
with twofold serial dilutions of antiviral drug. After 1 h, the inoculum was
removed and replaced with medium containing 1% methylcellulose and antiviral
drug. Control wells did not contain antiviral drugs. The cells were fixed in 100%
methanol 40 h after infection and were stained with Giemsa stain. Plaques were
visually inspected and counted using a dissecting microscope. The number of
plaques at each drug concentration was plotted versus the log of the drug
concentration, and the slope of the regression line in the linear range was
determined. The amount of drug required to reduce the number of plaques by
50% from those in the control wells (EC50) was calculated from the equation of
the regression line. Each drug was tested against each isolate at least twice in
replicate wells, and the EC50was calculated as an average value. Cytotoxicity in
Vero cells for all the compounds was determined previously using a 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. None
of the compounds were cytotoxic when evaluated up to 10 ?M (data not shown).
Viral DNA analysis. Viral DNA was isolated from infected cells by the method
of Poffenberger and Roizman (23). Restriction digests were separated on 0.8%
agarose gels. Each PCR amplification of the TK gene from rhesus, Japanese
macaque, cynomolgus, and human B virus isolates was performed with primers
5?-CCAACGCTCCGTAAAACCG-3? and 5?-ACCATCTTTATTGCGCCAG
G-3?. Amplimers were purified after extraction from 1% agarose gels, and both
strands were sequenced using an automated sequencer, model 377 (Applied
Biosystems), with the primers given above and three internal primers, 5?-AAG
ATCGTGTCCTCGATCTC-3?, 5?-GAGATCGAGGACACGATCTT-3?, and
5?-CACCTGAGCGCTGGCCATTG-3?. Because PCR amplification of the pig-
tail macaque B virus TK gene was not possible with the rhesus macaque-based
PCR primers, restriction fragments containing the pigtail macaque B virus TK
gene were cloned. These plasmids were subcloned, and the pigtail B virus TK
gene was then sequenced using vector-specific primers and two internal gene-
specific primers, 5?-AAGATCGTGTCCTCGATCTC-3? and 5?-GAGATCGAG
GACACGATCTT-3?. Sequence alignment was performed using MegAlign, ver-
sion 4.03 (DNAStar, Madison, WI), with the Jotun Hein algorithm.
Characterization of the virus panel. One of the goals of this
work was to examine the variability of drug potency and sus-
ceptibility for multiple B virus isolates from macaques and
humans. We chose a large panel of B virus isolates from var-
ious species of macaques, as well as human isolates; many of
the viruses were isolated recently and have been maintained at
3 or fewer passages. To verify the genotype and observe the
genetic variability of the B virus isolates, we performed restric-
tion fragment length polymorphism (RFLP) analysis with SalI
restriction endonuclease (Fig. 1). Numerous polymorphisms
were observed, similar to those noted in a previous study of
multiple isolates from cynomolgus macaques (31). It is also
noted that the seven B virus isolates from human zoonoses
were genotyped as rhesus B virus. B virus isolates from cyno-
molgus monkeys and pigtail macaques had restriction profiles
distinct from those of isolates from rhesus macaques, as pre-
viously reported by others (25). Recently, sequencing and
RFLP analysis of PCR amplimers demonstrated specific char-
acteristics that distinguish B virus isolates from Japanese and
FIG. 2. Design and confirmation of recombinant B virus harboring mutations in thymidine kinase. (A) Overview of TK mutant construction:
1, orientation of the B virus genome; 2, BamHI–SalI fragment containing the complete TK open reading frame; 3, ApaI–BspEI collapse of pBVTK,
deleting 754 amino acids from the center of TK; 4, stop codon linker insertion into the BglII site to prematurely stop TK translation.
(B) Confirmation of mutants by PCR. Lanes: M, ?X174/HaeIII marker; 1, E2490; 2, BV?TK; 3, BVTKstop PCR product cut with SpeI; 4,
BVTKstop (uncut). (C) Antiviral sensitivities of E2490, BV?TK, and BVTKstop versus acyclovir, ganciclovir, and vidarabine. Error bars indicate
standard deviations of three replicates.
454 KRUG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
virus RFLP patterns were very similar to those from rhesus
macaque virus isolates, yet these isolates had distinct RFLPs
generated by digestion with SalI (Fig. 1) and other restriction
endonucleases (data not shown).
In order to determine if a B virus isolate was resistant to
ACV or GCV, we created two thymidine kinase-defective B
viruses as controls (Fig. 2). These viruses were 6-fold more
resistant to ACV and 16-fold more resistant to GCV than the
wild-type parental strain.
Sensitivity of B virus isolates to vidarabine. As previously
reported (5), B virus and HSV-1 have similar sensitivities to
VDB in cell culture, suggesting the hypothesis that the reason
B virus is less sensitive to ACV and GCV than HSV is due to
variation in the viral TKs, not the viral DNA polymerases. To
detect differences in the B virus DNA polymerases, the sensi-
tivities of the B virus isolates to VDB were determined by a
plaque reduction assay. The rationale for the use of VDB was
to determine whether mutations affecting the viral DNA poly-
merase might be present, since these mutations could poten-
tially result in an observed resistance to drugs that require
phosphorylation by the viral TK and may be mistakenly attrib-
uted to a partial or total loss of TK function.
We found that all rhesus isolates had very similar sensitivi-
ties to VDB (mean EC50, 34.3 ?M; range, 26.5 to 40.4 ?M)
(Fig. 3). As expected, the mean VDB sensitivities of the
BV?TK and the BVTKstop mutants were similar to those of
the rhesus isolates (37.8 ?M and 46.1 ?M, respectively) (Fig.
2C). While the possibility of mutations in the viral DNA poly-
merase that do not affect the sensitivity of VDB cannot be
completely ruled out, these results indicate that the DNA poly-
merase genes in these isolates are similar, suggesting that the
large differences observed with TK-activated antiviral agents
would likely be due to variations in the viral TK.
Sensitivities of B virus isolates to acyclovir and ganciclovir.
Fatalities following zoonotic infections from 1987 to 1997 oc-
curred despite pharmacologic intervention with ACV and/or
GCV. To rule out the possibility that the fatality-associated
viruses were strain variants that were “naturally” resistant to
these antiviral agents, we determined the sensitivities of our
panel of B virus isolates to these antivirals using plaque reduc-
tion assays. As controls, the TKnullmutants were used to de-
termine a standard for completely resistant virus.
Our results for ACV and GCV (Fig. 4) were similar to those
of previously published studies for rhesus B virus (38). The
EC50for each drug against each isolate was within a 2-fold
difference of the mean sensitivity of all isolates from rhesus
monkeys and humans (33.2 ?M; range, 19.3 to 45.7 ?M). The
sensitivities of Japanese macaque, cynomolgus monkey, and
pigtail macaque B virus isolates to ACV and GCV were very
similar to those of the human and rhesus B virus isolates,
suggesting that future human infections with these B virus
genotypes could be treated with these antivirals. Because the
drug sensitivities of the isolates did not approach the levels of
the TKnullmutants (Fig. 2C), our results indicate that the
isolates in this panel have wild-type antiviral drug sensitivity;
however, it is possible that the variability observed in this assay
could be due to minor differences in the TK genes.
Experimental drugs. While ACV and GCV have been
shown to be effective in some cases of B virus zoonosis, the
prognosis for patients is bleak once clinical signs indicate entry
of B virus into the CNS. In order to discover more-efficacious
B virus antiviral agents, we tested a panel of experimental
compounds with known activity against HSV. FMAU and
FEAU were found to be approximately 100 times more potent
than conventional drugs (Fig. 5, upper panel). FMAC and
FBrAC had approximately 10 times greater potency than ACV
(Fig. 5, lower panel). Each of these agents requires the viral
TK for activation, indicating that either they are activated with
higher efficiency by TK or their phosphorylated forms are
FIG. 3. Vidarabine sensitivities of B virus isolates. Plaque reduc-
tion assays were performed, and the 50% effective concentration
(EC50) of the drug against each isolate was determined. Each bar
corresponds to the mean EC50; error bars, standard deviations. Each
isolate was tested against the drug in at least two independent exper-
FIG. 4. Sensitivities of B virus isolates to conventional antivirals. Plaque reduction assays were performed, and the 50% effective concentration
(EC50) of each drug against each isolate was determined. Each bar corresponds to the mean EC50; error bars, standard deviations. Each isolate
was tested against each drug in at least two independent experiments. N.D., not done.
VOL. 54, 2010 B VIRUS ANTIVIRALS455
recognized by the viral DNA polymerase with higher affinity
than the triphosphate forms of ACV or GCV. These data
demonstrate that a more-efficacious panel of B virus antiviral
agents exists within the class of 2?-fluoro-5-substituted arabi-
Thymidine kinase heterogeneity. Most of the B virus isolates
had similar sensitivities to each drug, with a range less than
2-fold from the mean. It is possible that the observed variation
between these isolates is due to minor differences at the amino
acid level in the viral proteins that can confer resistance to
nucleoside analogs (TK or DNA polymerase). To determine
whether apparent functional mutations existed in TK, the TK
open reading frame (ORF) was sequenced from rhesus, Japa-
nese macaque, cynomolgus monkey, pigtail macaque, and hu-
man B virus isolates.
Striking conservation between B virus TK ORFs from rhesus
macaque, Japanese macaque, and human isolates was ob-
served; one nucleic acid substitution was present in 8 of 22 of
these isolates, but the difference did not affect the amino acid
sequence. Cynomolgus monkey-derived B virus isolates have
seven unique TK amino acid differences from rhesus and pig-
tail macaque B virus isolates (Fig. 6), and these differences may
underlie the increased sensitivity of the cynomolgus isolates to
FBrAC (Fig. 5, lower panel). While 27 unique amino acid
differences were present in the pigtail macaque B virus isolate
relative to rhesus and cynomolgus monkey B virus TK (Fig. 6),
the drug sensitivity of the pigtail macaque B virus fell into the
range of the rhesus B virus isolates with all tested antiviral
agents. We hypothesize that the observed amino acid differ-
ences are likely in domains that are not functional with respect
to drug interaction, or alternatively, they are in functional
domains that are sufficiently conserved to retain function.
Included in these studies were isolates from three humans
and two rhesus macaques sampled during a cluster of zoonotic
infections in Pensacola, FL (8). The three human isolates and
two monkey isolates are designated A1, A2, A3, MR1, and
MR2, respectively. While there were minor differences in drug
sensitivity among these isolates, the DNA sequences of the TK
ORF were identical (data not shown). Taken together, the
similar drug sensitivity and identical TK sequence suggest that
replication in the human host did not result in antiviral resis-
tance in these B virus isolates.
These data provide support for the value of and choices for
antiviral intervention against zoonotic B virus infections, ex-
tending data originally demonstrating the efficacy of conven-
tional drugs that target viral DNA replication. The potential of
2?-fluoroarabinofuranosyl 5-substituted pyrimidine nucleo-
sides is demonstrated for the first time; these data may serve as
the basis of compassionate use when the onset of severe mor-
bidity signals unlikely success with conventional therapeutic
approaches, as learned from the five fatal cases in recent years.
Our data further suggest that there is no immediate host pres-
sure that induces drug susceptibility-related changes in B virus
during human infection. Each isolate, whether isolated from a
macaque or from a human, was similarly sensitive to the anti-
viral agents used in these studies, suggesting that time to treat-
ment is a major factor in human survival.
The TKnullmutants had VDB sensitivities similar to those of
the wild-type isolate panel, supporting the hypothesis that the
increased requirements of ACV and GCV for B virus plaque
reduction are due to the differences between B virus and HSV
TKs. While most of the known functional domains of TK are
identical between the rhesus B virus and HSV, it is tempting to
FIG. 5. Sensitivities of B virus isolates to experimental antivirals. Plaque reduction assays were performed, and the 50% effective concentration
(EC50) of each drug against each isolate was determined. Each bar corresponds to the mean EC50; error bars, standard deviations. Each isolate
was tested against each drug in at least two independent experiments. N.D., not done.
456 KRUG ET AL.ANTIMICROB. AGENTS CHEMOTHER.
speculate that the substitution of Pro150 in the B virus TK
would place a kink in the nucleoside-binding domain that is not
present in the same position in HSV TK (Ala168). Indeed, a
change in the homologous amino acid in HSV-2 (Ser169) is
responsible for the resistance of that virus to brivudin (BVdU)
(35), and the same amino acid variation in the binding pocket
is present in cynomolgus monkey B virus TK (Ser150). Zwar-
touw and colleagues (38) demonstrated the increased resis-
tance of cynomolgus monkey B virus to BVdU almost 20 years
ago. Site-directed mutagenesis and a structure-activity rela-
tionship analysis of B virus TK would be required to determine
the roles of individual amino acids in the relative resistance of
B virus to ACV and GCV.
B virus isolates from recent zoonotic infections demon-
strated no variation in TK genes relative to isolates from rhe-
sus macaques, and all appear similar to those from rhesus
monkeys by restriction endonuclease profiles. These data val-
idate a rhesus origin of B virus associated with these specific
zoonoses. However, this is a small sample set, and it was noted
that affected individuals had been working specifically with
rhesus macaques. Thus, our data do not rule out the possibility
that B virus from macaque species other than Macaca mulatta
may share its pathogenic potential (25). Even so, B virus can be
transmitted to nonmacaque monkeys (18, 36), and other ma-
caques can harbor B virus acquired from rhesus monkeys (21,
25). Thus, regardless of the monkey species, if a monkey has
been in contact with macaques, B virus should be presumed to
be a hazard. Few cases previously reported in the literature
have been associated with specific macaques (22); in fact, many
affected individuals had histories of contact with multiple ma-
In each of the recent symptomatic cases of B virus zoonosis,
treatment has consisted of acyclovir and/or ganciclovir (3, 9,
12, 16, 20). Of 11 infected individuals, 6 survived. The data
presented here suggest that variable infection outcomes of
antiviral therapy were not associated with virus mutations. It is
possible that each individual had different host-dependent sus-
ceptibilities to B virus infection. Alternatively, since four of the
five fatally infected individuals were not treated until severe
neurological symptoms were detected, whereas three of the six
survivors were hospitalized prior to the onset of acute neuro-
logical symptoms, the time for effective intervention may have
been limited. This would underscore the importance of early
identification of zoonotic disease, which can be generally ac-
complished early after infection for less than $300 worth of
Zwartouw and colleagues (38) compared ACV and GCV
against B virus in experimentally infected rabbits, observing
100% survival for the GCV-treated group for 5 months, com-
pared to 33% survival for those treated with ACV. Interest-
ingly, one of the patients from the Michigan cluster of human
infections with B virus was given ganciclovir (5 mg/kg of body
weight intravenously) twice daily, following a lack of success
with ACV used at 15 mg/kg for 3 days, resulting in abatement
of neurological symptoms and stabilization of cerebrospinal
fluid antibody levels (12). In four other human cases, however,
GCV was unable to reverse the fatal progression of disease (9,
17, 20), substantiating the need for more-efficacious antivirals
for late-stage infection.
During the preparation of this article, a biochemical analysis
of B virus TK was published, including some overlap with the
present study (14). Our somewhat different approach was to
determine if variability existed among wild isolates of B virus,
hence our inclusion of many more virus strains, instead of just
FIG. 6. Amino acid alignment of thymidine kinase sequences from three B virus genotypes, HSV-1, and HSV-2. Thymidine kinase open reading
frames from the B virus strains E2490 (Rhesus BV), MC1 (Cynomolgus BV), and MP1 (Pigtail BV) were aligned using the Jotun Hein method.
The thymidine kinase open reading frame from HSV-1 strain F and HSV-2 strain 333 were included for comparison. Shaded amino acids indicate
identity with known functional domains.
VOL. 54, 2010 B VIRUS ANTIVIRALS457
three strains. Instead of characterizing the biochemical prop-
erties of B virus TK, we sought out compounds that had sig-
nificantly greater efficacy in cell culture against a wide scope of
B virus isolates, so that infectious-disease physicians would
have more-powerful choices to treat cases of advanced human
B virus infection. The creation of the TKnullmutant viruses as
opposed to the production of recombinant protein allowed us
to examine TK-mediated drug resistance in the context of viral
infection. The authors of the previous study could not detect
the phosphorylation of ACV or GCV with recombinant B virus
TK; however, the increased resistance of the TK mutants to
these drugs suggests that in cell culture infections, B virus TK
does activate ACV and GCV.
Our studies indicate that a new, improved group of anti-
viral agents, 2?-fluoroarabinofuranosyl 5-substituted pyrim-
idine nucleosides, should be considered for therapeutic use
to combat zoonotic B virus infection. Comprehensive anal-
yses examining the cellular toxicity and effectiveness of a
number of these compounds against HSV have been re-
ported. FEAU has been shown to be the most selective
anti-HSV antiviral in this class; it was not incorporated into
uninfected cell DNA (17) and was 10-fold less toxic than
ACV in cell culture (19). Other laboratories have demon-
strated the in vivo safety of FEAU; no negative effects were
reported in a monkey following six intravenous doses of
FEAU at 30 mg/kg (7). Further work has demonstrated the
in vivo safety and effectiveness of FEAU against simian
varicella virus in African green monkeys (26), and analysis
of FEAU in both a mouse model of HSV-2 (19) and a rabbit
model of HSV-1 (28) has also demonstrated safety and
effectiveness. In view of the impressively low doses required
to inhibit B virus replication, FEAU should be considered as
a compassionate-use treatment for human cases in which
there is progressive or severe morbidity.
We thank David Katz and Ludmila Perelygina for helpful comments
during the preparation of the manuscript.
This work was supported in part by NIH grants P40 RR05162
(J.K.H.) and 2P30-AI-50409 (R.F.S.), the Georgia Research Alliance,
and the Department of Veterans Affairs.
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