Antiviral activity of serum from the American alligator (Alligator mississippiensis).

Abstract

Serum from wild alligators was collected and tested for antibiotic activity against three enveloped viruses using cell-based assays. Alligator serum demonstrated antiviral activities against human immunodeficiency virus type 1 (HIV-1; IC50=0.9%), West Nile virus (WNV; IC50=4.3%), and Herpes simplex virus type 1 (HSV-1; IC50=3.4%). The inhibitory concentration (IC50) is defined as the concentration of serum that inhibits 50% of viral activity. The antiviral effects of the alligator serum were difficult to evaluate at high concentrations due to the inherent toxicity to the mammalian cells used to assay viral activities. The TC50 (serum concentration that reduces cell viability to 50%) values for the serum in the HIV-1, WNV, and HSV-1 assays were 32.8, 36.3 and 39.1%, respectively. Heat-treated serum (56 degrees C, 30 min) displayed IC50 values of >50, 9.8 and 14.9% for HIV-1, WNV and HSV-1 viruses, respectively. In addition, the TC50 values using heat-treated serum were substantially elevated for all three assays, relative to untreated serum (47.3 to >50%). Alligator serum complement activity has been shown to be heat labile under these conditions. HIV-1 antiviral action was heat-sensitive, and thus possibly due to the action of serum complement, while the anti-WNV and anti-HSV-1 activities were not heat labile and thus probably not complement mediated.

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Antiviral Research 66 (2005) 35–38
Antiviral activity of serum from the American alligator
(Alligator mississippiensis)
Mark E. Merchanta,∗, Melanie Pallanschb, Robin L. Paulmanb, Jay B. Wellsb,
Aysegul Nalcab, Roger Ptakb
aDepartment of Chemistry, McNeese State University, Box 90455, Lake Charles, LA 70609, USA
bDepartment of Infectious Disease Research, Southern Research Institute, Frederick, MD 21701, USA
Received 4 October 2004; accepted 13 December 2004
Abstract
Serum from wild alligators was collected and tested for antibiotic activity against three enveloped viruses using cell-based assays. Alli-
gator serum demonstrated antiviral activities against human immunodeficiency virus type 1 (HIV-1; IC50=0.9%), West Nile virus (WNV;
IC50=4.3%), and Herpes simplex virus type 1 (HSV-1; IC50=3.4%). The inhibitory concentration (IC50) is defined as the concentration of
serum that inhibits 50% of viral activity. The antiviral effects of the alligator serum were difficult to evaluate at high concentrations due to
the inherent toxicity to the mammalian cells used to assay viral activities. The TC50(serum concentration that reduces cell viability to 50%)
values for the serum in the HIV-1, WNV, and HSV-1 assays were 32.8, 36.3 and 39.1%, respectively. Heat-treated serum (56◦C, 30min)
displayed IC50values of >50, 9.8 and 14.9% for HIV-1, WNV and HSV-1 viruses, respectively. In addition, the TC50values using heat-treated
serum were substantially elevated for all three assays, relative to untreated serum (47.3 to >50%). Alligator serum complement activity has
been shown to be heat labile under these conditions. HIV-1 antiviral action was heat-sensitive, and thus possibly due to the action of serum
complement, while the anti-WNV and anti-HSV-1 activities were not heat labile and thus probably not complement mediated.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Alligator; Antiviral; Complement; Crocodilian; Serum
Alligators often sustain serious injuries due to territorial
disputes with other alligators. These conflicts often end with
severe damage such as loss of entire limbs. However, alli-
gators exhibit remarkable ability to heal rapidly and without
infection, particularly considering the septic environment in
which they live (Merchant, unpublished observations). Re-
cent studies in our laboratory have demonstrated the wide-
ranging antibacterial and amoebacidal activities of alligator
serum (Merchant et al., 2003, 2004). More recent studies
have demonstrated that these activities are due to a potent
andbroad-actingserumcomplementsystem(Merchantetal.,
2004). This study was conducted to investigate the antiviral
effects of alligator serum.
∗Corresponding author. Tel.: +1 337 475 5773; fax: +1 337 475 5950.
E-mail address: mmerchan@mcneese.edu (M.E. Merchant).
Wildalligatorswerecapturedatnightfromaboatwiththe
use of a spotlight and a cable snare. Blood was collected via
the spinal vein using an 18ga. needle and a 60mL syringe
(Olsen et al., 1975; Zippel et al., 2003). The blood was re-
moved to a 15mL serum Vacutainer®and allowed to clot at
roomtemperatureforapproximately3h.Theserumwassep-
arated by centrifugation (3000×g, 15min) and was frozen
at −20◦C until ready for use.
Theeffectivenessofalligatorserumagainstthelaboratory-
adaptedIIIBstrainofHumanimmunodeficiencyvirus-type1
(HIV-1IIIB) was examined using a cell-based assay in which
the virus was used to infect a human T lymphoblastoid cell
line (CEM-SS). The assays were conducted as previously
described (Buckwold et al., 2004). The results represent the
means of three independent determinations for each serum
dilution and are expressed as % reduction of viral cytopathic
effects (CPE), based on virus control (cells plus virus, no
0166-3542/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.antiviral.2004.12.007

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M.E. Merchant et al. / Antiviral Research 66 (2005) 35–38
drug; 0% baseline) and cell control. The data indicate that
the alligator serum exhibits potent anti-HIV-1IIIBactivity at
20–64-fold dilutions (5 and 1.6% serum, Fig. 1A), with 100
and 89% reduction in CPE, respectively. The cell toxicity
index at these serum concentrations was 0, as 100% cell via-
bilitywasobservedintheculturedCEM-SScellstreatedwith
5.0 and 1.6% alligator serum (Fig. 1B). The anti-HIV-1IIIB
activity of the serum is difficult to assess at concentrations
above 5% due to the inherent toxicity to the CEM-SS cells
(Fig. 1B). However, it is likely that both the anti-HIV-1IIIB
activity and the CEM-SS cell toxicity are mediated by the
serum complement system, as heat treatment (56◦C, 30min)
of the serum obliterated both effects (Fig. 1A and B). Like
human complement activity, alligator serum complement is
sensitive to heat inactivation (Merchant et al., 2004). The
IC50is defined as the serum concentration that inhibits 50%
of viral activity, while the TC50is defined as the serum con-
centration that reduces the mammalian cell culture viability
to 50%. The IC50value for HIV-1IIIBantiviral activity was
determined to be 0.85% serum, while the TC50for CEM-SS
cell toxicity was 32.8% serum. In contrast, both the IC50and
TC50values were >50% serum for the heat-treated serum.
AZT was included in the assay as a positive control antiviral
compound (IC50=6.1nM; TC50>1000nM).
Fig. 1. Anti-HIV-1IIIBactivity and CEM-SS cell toxicity of alligator serum.
(A)Anti-HIV-1IIIBactivityexpressedas%reductioninviralCPE.(B)CEM-
SS cell toxicity expressed as % cell viability. HT: heat-treated.
The anti-West Nile virus (WNV) activity of alligator
serum was investigated by employing a cell-based assay
in which Vero African green monkey kidney cells were
infected with the WNV strain NY-99. The assays were
conducted as previously described (Anderson and Rahal,
2002). Similar to the HIV-1 assay, the results represent the
means of three independent determinations for each serum
dilution and are expressed as % reduction in CPE. Ribavirin
(IC50=80.6?g/mL; TC50>200?g/mL) was included as a
positive control antiviral compound. The data reveal that the
alligatorserumshowsmoderatelyhighactivityagainstWNV
at relatively low dilution (Fig. 2A). For instance, 16% alliga-
tor serum exhibited 77% of maximal anti-WNV activity. In
addition, this same serum dilution produced a 24% reduction
in Vero cell viability due to its cell toxicity (Fig. 2B). Higher
serum concentrations (50% serum) reduced cell viability by
60%, while the anti-WNV effect was reduced to 32%. The
full potential of the anti-WNV properties of alligator serum
aredifficulttoevaluateduetothetoxicityoftheserumtoward
the Vero cells. An IC50value for WNV antiviral activity was
determined to be 4.3% serum, while the TC50for Vero cell
toxicity was 36.3% serum. Moreover, it is interesting to note
that, unlike the anti-HIV effects, the anti-WNV effects and
the Vero cell toxicity were virtually identical in untreated
and heat-treated alligator serum (Fig. 2A and B). These data
indicate that the anti-WNV activity and Vero cell toxicity
Fig. 2. Anti-WNV activity and Vero cell toxicity of alligator serum. (A)
Anti-WNV activity expressed as % reduction in viral CPE. (B) Vero cell
toxicity expressed as % cell viability. HT: heat-treated.

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M.E. Merchant et al. / Antiviral Research 66 (2005) 35–38
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Fig. 3. Anti-HSV-1 activity and Vero cell toxicity of alligator serum. (A)
Anti-HSV-1 activity expressed as % reduction in viral CPE. (B) Vero cell
toxicity expressed as % cell viability. HT: heat-treated.
are not mediated by the heat-sensitive serum complement
proteinsystem.Theseactivitiesmaybeduetotheexpression
of cationic peptides, interferon-like molecules, or some
other heat stable molecule with antiviral activity.
The activity of alligator serum against herpes simplex
virus-type 1 (HSV-1) was tested via a cell-based assay in
which Vero cells were infected with HSV-1 strain HF as pre-
viously described (Buckwold et al., 2004). Again, the results
represent the means of three independent determinations for
each serum dilution and are expressed as % reduction in vi-
ral CPE. Acyclovir (IC50=7.1?M; TC50>100?M) was in-
cluded as a positive control antiviral compound. Alligator
serum exhibited moderate antiviral activity against HSV-1.
Treatment of HSV-1 with six-fold and 20-fold dilutions of
alligator serum afforded the Vero cells 76 and 61% protec-
tion relative to the virus control (16 and 5% serum, Fig. 3A).
In addition, treatment of the Vero cells with these concen-
trations of serum resulted in only modest reductions in cell
viability (15 and 7%, respectively; Fig. 3B). The IC50value
forHSV-1antiviralactivitywasdeterminedtobe3.4%serum,
while the TC50for Vero cell toxicity of 39.1% serum con-
firmedtheVerocelltoxicityobservedintheWNVassay.The
activity was not substantially lower in the heat-treated than
in the non-treated serum (p>0.05). Likewise, the cytotoxic
sensitivity of the Vero cells to the heat-treated serum is not
significantly different than the non-treated serum (p>0.05,
Fig.3B),butthecytotoxiceffectisnotcompletelysuppressed
as the heat-treated serum elicited an 18% reduction in Vero
cell CPE. These data illustrate that the mechanism of the
anti-HSV-1 activity of alligator serum is not mediated by the
serum complement system.
It is interesting to note that vertebrates as ancient as al-
ligators display natural innate immunity to viruses that they
in all likelihood have not regularly encountered during their
evolution. The fact that alligator serum was least effective
against the West Nile virus is supported by reports of iso-
latedWestNileinfectionsinalligatorpopulations(Malakoff,
2003; Miller et al., 2003). The anti-HIV-1 data presented in
this study, along with the wide spectrum of antibacterial and
antiparasitic properties of alligator serum (Merchant et al.,
2003, 2004), suggest that alligators have evolved a strong
and broad-acting serum complement system. These data are
consistent with the hypothesis that some phylogenetically
ancient vertebrates, such as certain reptiles and teleost fish,
have evolved a more complex innate immune system en lieu
of adaptive immunity (Sunyer et al., 1997). However, the ac-
tivitiesagainsttheHSV-1andWNVshowthatotherantiviral
mechanisms are active in alligator serum. To our knowledge,
this is the first report of antiviral activities against enveloped
viruses in the serum of lower vertebrates.
Acknowledgements
TheauthorswishtothankDr.MichaelBerger,WildlifeDi-
vision Director of the Texas Parks and Wildlife Department,
foraccesstopubliclandsonwhichtocapturewildalligators.
ThisresearchwassupportedbytheMcNeeseStateUniversity
Shearman Research Fund.
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Further reading
Hosoi, S., Borsos, T., Dunlop, N., Nara, P.L., 1990. Heat-labile,
complement-like factor(s) of animal sera prevent(s) HIV-1 infectiv-
ity in vitro. J. Acq. Immun. Def. Synd. 3 (4), 366–371.