Emerging Infectious Diseases
[Volume 5 No.1 / January - February 1999]

Suggested Citation

Hantavirus

The following articles on hantavirus contain preliminary results of 3 years
of longitudinal mark-release-recapture studies in the southwestern United
States, provide an analysis of the sensitivity of the field techniques and
statistical analyses for detecting changes in rodent population densities,
and discuss the role of longitudinal studies in understanding reservoir
host ecology as it relates to human disease. Although the data presented in
the articles are overall standardized, methods used to extract information
vary by research institution.

Long-Term Studies of Hantavirus Reservoir Populations in the Southwestern
United States: Rationale, Potential, and Methods

James N. Mills, Terry L. Yates, Thomas G. Ksiazek, C.J. Peters, and James
E. Childs
Centers for Disease Control and Prevention, Atlanta, Georgia, USA

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      Hantaviruses are rodent-borne zoonotic agents that cause
      hemorrhagic fever with renal syndrome in Asia and Europe and
      hantavirus pulmonary syndrome (HPS) in North and South America.
      The epidemiology of human diseases caused by these viruses is
      tied to the ecology of the rodent hosts, and effective control
      and prevention relies on a thorough understanding of host
      ecology. After the 1993 HPS outbreak in the southwestern United
      States, the Centers for Disease Control and Prevention
      initiated long-term studies of the temporal dynamics of
      hantavirus infection in host populations. These studies, which
      used mark-recapture techniques on 24 trapping webs at nine
      sites in the southwestern United States, were designed to
      monitor changes in reservoir population densities and in the
      prevalence and incidence of infection; quantify environmental
      factors associated with these changes; and when linked to
      surveillance databases for HPS, lead to predictive models of
      human risk to be used in the design and implementation of
      control and prevention measures for human hantavirus disease.

Hantaviruses (genus Hantavirus, family Bunyaviridae) are rodent-borne
zoonotic agents that cause mild to severe hemorrhagic fevers throughout
most of Europe, Asia, and the Americas. The epidemiology of these
hemorrhagic fevers is largely defined by the distribution and ecology of
the rodent hosts of the viruses. Hantaviruses have been identified at a
dramatically increased rate in recent years; some 30 hantaviruses are now
recognized throughout the world (1,2). With very few exceptions, each virus
is associated with a single primary rodent host of the family Muridae. The
rodent, in which the virus establishes a chronic infection, sheds
infectious virus into the environment in urine, feces, and saliva (3,4);
these characteristics are key to the transmission of the virus, both to
humans (most frequently by inhalation of infectious aerosols [5]) and among
rodents (frequently by aggressive encounters and biting [6,7]).

Human diseases due to hantaviruses, which have been recognized at least
since World War I and probably occurred much earlier (8), were unknown in
the Americas until recently. Although Rattus norvegicus infected with Seoul
virus is common in many cities throughout the Americas (9), human disease
associated with a rat-borne hantavirus was not documented in a U.S. city
until 1994 (10). Prospect Hill virus, an indigenous North American
hantavirus, was isolated from the meadow vole (Microtus pennsylvanicus) as
early as 1982 (11) but has never been associated with human disease.

Since 1993, when hantavirus pulmonary syndrome (HPS) was recognized and its
etiologic agent, Sin Nombre virus (SNV), was isolated and associated with
the deer mouse (Peromyscus maniculatus) (12,13), at least 20 New-World
hantaviruses, all associated with the same group of indigenous American
rodents (family Muridae, subfamily Sigmodontinae) have been described, and
HPS has been diagnosed from Canada to Patagonia. The severity
(approximately 50% death rate) and wide geographic distribution of this
rodent-borne zoonotic disease has prompted intensive collaboration between
public health investigators and ecologists to elucidate the ecologic and
epizootiologic features of infection in host populations and the factors
that lead to human infection.

Because no specific treatment is yet available, prevention measures are
essential in decreasing HPS-related illness and death. Developing effective
preventive measures requires a detailed knowledge of the ecology and
epizootiology of hantavirus infection in reservoir populations and the
specific situations and mechanisms that result in the transfer of
hantaviruses from hosts to humans.

Reservoir Studies

Reservoir studies, whose role in understanding, controlling, and preventing
human disease has been reviewed, have resulted in a series of research
goals that may facilitate the collection of data concerning reservoir
ecology, a subject pertinent to human health (14). The first goal is to
identify the reservoir host; others are a) to determine the area in which
the disease may be endemic by identifying the geographic range of the host
and the range of infection by the pathogen within the host range; b) to
more precisely define relative risk to humans by determining the
distribution of the host and pathogen among distinct habitats regionally;
c) to investigate potential mechanisms of pathogen transmission within host
populations by conducting cross-sectional surveys to define the prevalence
of infection among various subpopulations of the host (e.g., male versus
female, juvenile versus adult); d) to conduct long-term prospective studies
to explain the temporal patterns of infection in host populations; and e)
to integrate the data from these studies in a predictive model that will
allow early identification of specific times and places where conditions
can increase rodent populations or infection in rodent populations and
elevate the risk for human disease. This model could be used to minimize
the incidence of human disease through public education, habitat
modification, or reservoir control.

Investigations of HPS cases in the United States have resulted not only in
studies of the deer mouse and SNV but also in the identification of three
additional host-virus relationships that maintain hantaviruses responsible
for human disease (New York virus carried by the white-footed mouse,
Peromyscus leucopus [Figure 1] [15]; Black Creek Canal virus carried by the
cotton rat, Sigmodon hispidus [16]; and Bayou virus carried by the rice
rat, Oryzomys palustris [17]). Most HPS cases in the United States have
been caused by SNV.

Intensive studies of deer mouse       
populations have addressed most of
the proposed goals. One of the most   
common and most extensively studied   [fig 1]
small mammals in North America, the   Figure 1. White-footed mouse
deer mouse has a well-known           (Peromyscus leucopus). Photo by R.B.
geographic distribution (18).         Forbes, Mammal Image Library of the
Antibody screening of deer mouse      American Society of Mammalogists.
populations throughout North America
has provided evidence of SNV infection throughout most of the species'
range (Ksiazek et al., unpub. data). Regional studies have shown
differences in the prevalence of hantavirus infection among deer mouse
populations in different habitats and helped define the varying disease
risk to humans in these habitats (7). Finally, studies of the age- or
size-specific prevalence of hantavirus infection among reservoir
populations have shown that SNV, and other hantaviruses, are transmitted
horizontally within reservoir populations, and that one important specific
mechanism of transfer may be aggressive encounters and bites, most
frequently among male animals (6,7,19). Although these cross-sectional
studies have increased our understanding of host-virus ecology as it
relates to human disease, they have not explained the temporal dynamics of
host-virus ecology nor have they identified the environmental factors
associated with these dynamics; only long-term prospective studies can
provide this additional information.

Long-Term Studies

Long-term studies, widely regarded by ecologists as indispensable for
understanding the temporal dynamics of vertebrate communities (20), are
especially useful for assessing the effects of rare events (e.g., El Nio
southern oscillation) and for detecting and observing processes that unfold
slowly in communities or populations (e.g., establishment or disappearance
of a reservoir species from part of its range; changes in reservoir
population density; changes in community composition; introduction or
extinction of a pathogen in a specific host population; and changes in the
incidence or prevalence of infection within the host population).

Long-term studies of reservoir populations have helped elucidate the
temporal dynamics of hantavirus infection in host populations for Seoul and
Prospect Hill viruses (21) and identify characteristics of reservoir
ecology associated with outbreaks of human disease. The numbers of cases of
hemorrhagic fever with renal syndrome due to Puumala virus in Scandinavia
and Argentine hemorrhagic fever due to Junn virus (an arenavirus with many
epidemiologic similarities to hantaviruses) were correlated with cyclic
changes in the density of reservoir host populations (22,23). Increases in
population density are associated with improved reproductive success and
survivorship that may be due to improved habitat. Changes in the
environment may be associated with favorable weather patterns, accelerated
vegetation growth, and availability of plant and small-animal foods
(14,23). The 1993 HPS outbreak in the southwestern United States may have
resulted from improvements in the quality of deer mouse habitat caused by
the 1991-92 El Nio southern oscillation (24). When the environmental
variables associated with increasing reservoir population densities are
identified and quantified, a key component of a predictive model of human
risk will be in place.

Despite their importance and utility, long-term studies of reservoir
populations associated with zoonotic agents are rare. By definition, they
require stable funding for many years, they are labor intensive, expensive,
and may not produce significant results in the short term. The periodic
shifts in environmental conditions that change host populations and
increase risk for human disease may take many years.

The most common method for conducting long-term studies of small-mammal
populations is the mark-release-recapture (MRR) technique. Animals trapped
live on permanent trapping plots are measured, sampled (blood or oral
swab), identified with a permanent mark or number, and released at the
exact site of capture. The trapping plots are operated at predetermined
intervals for several days. Animals recaptured in subsequent trappings are
measured and sampled again so that changes in numbers of animals, body
growth rates, movement, reproductive condition, and infection status can be
monitored. Environmental variables such as weather conditions and
vegetative cover also may be monitored on the trapping plots. Control
plots, where invasive procedures are minimized, may be necessary for
determining or correcting for the influence of sampling methods on animal
survival or population size.

After the 1993 HPS outbreak, the Centers for Disease Control and Prevention
(CDC) established a network of hantavirus and rodent monitoring sites in
the southwestern United States to 1) monitor and quantify the seasonal and
year-to-year changes in host population density and the prevalence and
incidence of hantavirus infection, 2) identify and quantify the biotic and
abiotic environmental factors associated with and likely influencing these
dynamics, 3) identify mechanisms of virus transmission within reservoir
populations, and 4) identify and measure any effects of infection on
individuals and populations of the host.

The studies should lead to predictive models of human risk for hantavirus
infection and should facilitate prevention and control of human hantavirus
disease.

Study Sites

						  Arizona, Colorado, and New Mexico
                                      were chosen as the general study
						  area because of their high numbers
 [fig 2]                              of HPS cases at the time the study
 Figure 2. Geographic locations of    was being designed. In addition,
 nine sites where                     four sigmodontine rodent species
 mark-release-recapture webs are beingidentified as hantavirus reservoirs
 operated to study rodent reservoirs  inhabit at least parts of the
 of hantaviruses in a three- state    three-state area (P. maniculatus, P.
 area of the southwestern United      boylii, Reithrodontomys megalotis,
 States. PCMS=Pinyon Canyon Maneuver  and S. hispidus). Longitudinal MRR
 Site (U.S. Army).                    studies are being conducted on 24
                                      trapping webs at nine sites in the
three states (Figure 2): 10 webs at four sites in New Mexico, operated by
the University of New Mexico; six webs at three sites in Colorado, operated
by Colorado State University; four webs at one site in northern Arizona,
operated by Yavapai College; and four webs at one site in southern Arizona,
operated by the University of Arizona. Site selection criteria included
presence of populations of Peromyscus spp. and evidence of infection by SNV
or related viruses in these populations. The location of each trapping web
was fixed precisely by global positioning system technology. A sketch of
each trapping web site, including a description of the vegetation, was
prepared.

Placing Permanent Trapping Webs

Small-mammal populations were monitored through the use of permanent
trapping webs (25) (Figure 3A). Each web covered 3.14 ha and contained 12
100-m transects radiating from a central point and resembling the spokes of
a wheel (Figure 3B). Each web contained 148 Sherman (8 x 9 x 23 cm; H.B.
Sherman Trap Company, Tallahassee, FL) and 24 Tomahawk (14 x 14 x 40 cm;
Tomahawk Live Trap Company, Tomahawk, WI) live-capture traps, at 12 trap
stations along each radiating spoke. The first four trap stations were at
5-m intervals and the remaining eight at 10-m intervals. Four Sherman traps
were placed around the central point. In addition to a Sherman trap, one
Tomahawk trap was placed at trap stations 7 and 12 in each radiating arm
(Figure 3B). Two to four webs were located at each sampling site. At least
one web at each site was designated a control web. At this web, rodent
populations were monitored but not sampled by blood and oral swab
collection so that the effects of sampling on small-mammal survivorship
could be assessed. At the remaining webs, virus activity in small-mammal
populations was monitored through monthly blood and oral swab samples from
captured animals. After the second year of the study, the purpose of the
control webs was achieved, so sampling of captured small mammals from these
webs was initiated (25,26).

Trapping Schedules                            
                                     
All trapping web sites were visited  
monthly, except those in Colorado,   [fig 3]
which were visited every 6 weeks,    Figure 3. A. Characteristics of
as weather permitted. Webs were      landscape and vegetation near Fort
operated for 3 consecutive nights on Lewis trapping web "A," southwestern
each trapping occasion, generally    Colorado. Photo courtesy of C.
coinciding with the new moon. Traps  Calisher.  B. Schematic representation
were set out in the evening of the   of a trapping web showing the relative
first day and baited with peanut     locations of the 148 trap stations.
butter and rolled oats, cracked      Small circles indicate the location of
corn, or mixed grain. In cold        one Sherman trap, larger circles, one
weather, cotton or polyester         Sherman plus one Tomahawk trap.
fiberfill was placed in the traps to Diameter of the web was 200 m. After
provide nesting material and reduce  Parmenter et al. (25).
trap-associated deaths.

Captured rodents were collected, transported, and sampled according to
standardized procedures (27,28). Briefly, traps were checked for captures
early each morning. Investigators wearing rubber gloves collected the traps
containing captured animals, labeled them with the web and trap station
number, and placed them in double plastic bags for transport to a
centralized outdoor processing station. Before opening the bags containing
captured small mammals, investigators donned protective clothing, including
latex gloves, disposable surgeon's gowns, and respirators fitted with HEPA
filters. Each captured animal was processed individually. The animal was
first shaken from the trap into a plastic bag containing cotton or gauze
soaked with inhalant anesthesia (methoxyflurane, Pitman-Moore, Mundelein,
IL; or isoflurane, Abbott Laboratories, North Chicago, IL). To prevent
potential cross-infection between animals, each was anesthetized in a clean
plastic bag, and the anesthesia-soaked cotton was contained in a tea
strainer that allows diffusion of the anesthesia, yet between animals can
be wiped with a disinfectant. In one case, investigators used a specially
adapted "nose cone" for anesthesia (Abbott et al., this issue, pp.
102-112). After being anesthetized, the animal was removed from the bag and
placed on a clean surface. A standardized form was used at all trapping
sites to collect the following data (28): unique capture number; date of
capture; exact location of capture on the trapping web (trap station
number); ear tag number; fate (first capture, recapture [different trapping
session], or repeater [within same 3-day trapping session]); species; age
(juvenile, subadult, or adult); mass; lengths of tail plus body, tail only,
ear, and right hind foot; reproductive status including position of the
testes (scrotal or abdominal) for males and condition of the vagina (closed
or perforate) and description of the nipples (enlarged or small, lactating
or not) for females; and the presence or absence of scars or wounds. For
animals from the sampling webs, oral swabs were taken with Dacron-tipped
applicators cut with scissors at the level of the Dacron and inserted into
0.5 ml of virus medium (phosphate-buffered saline containing 20% fetal
bovine serum, 2% penicillin and streptomycin, and 0.1% Fungizone) in a 2-ml
cryovial. Approximately five drops of whole blood were collected into a
second cryovial by a capillary tube inserted into the retro-orbital
capillary plexus. Whole blood and oral swab samples were immediately placed
in liquid nitrogen or on dry ice until transferred to -70C freezers for
storage. Animals recaptured on days 2 or 3 of the trapping session were not
bled a second time to avoid trauma. Animals newly captured were marked with
a uniquely numbered ear tag (some smaller animals were marked by toe
clipping). The animal was then replaced in the original trap or in a clean,
ventilated one-quart, screw-capped jar, and was allowed to recover fully
from the effects of anesthesia and was released at the exact site of
capture. A clean, baited trap was replaced at the site, and the original
trap was returned to the processing site to be decontaminated before reuse.
Animals from the control webs were treated similarly, except that blood and
oral swab samples were not taken.

Investigators recorded environmental data, including a general description
of the vegetation and depending on resources available, more detailed
descriptions of vegetation at individual webs and weather conditions during
the trapping session (detailed rainfall and temperature data were available
from meteorologic stations near each trapping site).

Laboratory Analysis

Serologic testing was conducted at CDC, Atlanta, or at the Arthropod-Borne
and Infectious Diseases Laboratory, Colorado State University, Fort
Collins, CO, USA. Samples of whole blood were tested for antibody reactive
with SNV recombinant nucleocapsid protein antigen by enzyme-linked
immunosorbent assay according to a standardized protocol (29). Briefly,
blood specimens were initially diluted 1:25 in 5% skim milk in 0.01 M
phosphate-buffered saline with 0.5% Tween-20 and subsequently diluted to
1:100 through 1:6,400 in fourfold dilutions in microtiter plates. Samples
were tested against the recombinant nucleocapsid antigen and a recombinant
control antigen (29). A conjugate mix of anti-Rattus norvegicus and
anti-Peromyscus leucopus (heavy and light chains) immunoglobulin G (IgG)
(Kirkegaard and Perry, Gaithersburg, MD) was used to detect bound
immunoglobulin. Adjusted optical densities (OD) for each dilution were
calculated by subtracting the OD(sup 410) of the control antigen from the 
OD(sup 410) of the SNV antigen. Titers were assigned on the basis of an 
adjusted OD value exceeding 0.20 for each dilution. A second measure 
consisting of the sum of the adjusted OD values for all four dilutions was 
also calculated. Serum specimens were considered SNV-positive if their titer 
was 1:400 or their sum-adjusted OD was 0.95. The cut-off values were determined 
by assessment of rodents found to be SNV-positive by several serologic tests
during the initial investigation of the 1993 outbreak (13) and have been
reassessed periodically among large populations of rodents collected in
North and South America. Antibodies to other North American hantaviruses
are cross-reactive with SNV antigen. This assay would detect (but not
distinguish among) infections by New York virus (from the white-footed
mouse [15]), Prospect Hill-like viruses (from arvicoline rodents [30]), El
Moro Canyon virus (from the Western harvest mouse [31]), Black Creek Canal
virus (from the cotton rat [16]), and Bayou virus (from the rice rat [17]).

At this writing, analyses on oral swab specimens (antigen or antibody
detection, polymerase chain reaction) have not been conducted. Blood and
oral swab samples are archived in -70C freezers at CDC, Atlanta, and at
the Museum of Southwestern Biology, Albuquerque, New Mexico.

Acknowledgments

      We are indebted to many investigators at CDC for laboratory analyses
and database management. Special thanks to R. Meyer, M.L. Martin, V.
Semenova, G. Gallucci, M. Curtis, A.J. Williams, and P. Stockton in the
laboratory; B. Farrar in the stock rooms; and L. Morgan and K. Schmidt at
the computer. K. Wagoner, K. Colbert, and B. Ellis prepared the figures. K.
Abbott, C. Calisher, B. Ellis, and M. Morrison provided helpful comments on
an earlier version of the manuscript.

      Dr. Mills is chief of the Medical Ecology Unit, Special Pathogens
Branch, Division of Viral and Rickettsial Diseases, CDC. His research
interests include zoonotic diseases, specifically host-pathogen evolution
and interactions.

      Address for correspondence: James N. Mills, Centers for Disease
Control and Prevention, 1600 Clifton Road, Mail Stop G14, Atlanta, GA
30333, USA; fax: 404-639-1118; e-mail: jum0@cdc.gov.

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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

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