Rapid field immunoassay for detecting antibody to Sin Nombre virus in deer mice.We developed a 1-hour field enzyme immunoassay Immunoassay An assay that quantifies antigen or antibody by immunochemical means. The antigen can be a relatively simple substance such as a drug, or a complex one such as a protein or a virus. (EIA (Electronic Industries Alliance, Arlington, VA, www.eia.org) A membership organization founded in 1924 as the Radio Manufacturing Association. It sets standards for consumer products and electronic components. ) for detecting antibody to Sin Nombre virus The Sin Nombre virus (literally "unnamed virus" in Spanish) (SNV) is the prototypical etiologic agent of hantavirus cardiopulmonary syndrome (HCPS). It was first isolated from rodents collected near the home of one of the initial patients with hantavirus pulmonary syndrome in deer mice deer mice Peromyscus maniculatus Public health The murine vector for Hantavirus. See Hantavirus. (Peromyscus maniculatus). The assay specificity and sensitivity were comparable to those of a standard EIA. This test will permit identification of rodents with antibody to this other hantaviruses. ********** Hantaviruses (family Bunyaviridae, genus Hantavirus hantavirus, any of a genus (Hantavirus) of single-stranded RNA viruses that are carried by rodents and transmitted to humans when they inhale vapors from contaminated rodent urine, saliva, or feces. There are many strains of hantavirus. ) are rodentborne or insectivoreborne viruses; some are recognized causes of human hemorrhagic fever with renal syndrome hemorrhagic fever with renal syndrome n. See epidemic hemorrhagic fever. or hantavirus pulmonary (or cardiopulmonary) syndrome (HPS See Seer*HPS. ) (1). The normal transmission cycle is rodent to rodent, without arthropod arthropod Any member of the largest phylum, Arthropoda, in the animal kingdom. Arthropoda consists of more than one million known invertebrate species in four subphyla: Uniramia (five classes, including insects), Chelicerata (three classes, including arachnids and horseshoe intermediate hosts. Each hantavirus has a single principal reservoir host reservoir host n. A host that serves as a source of infection and potential reinfection of humans and as a means of sustaining a parasite when it is not infecting humans. , which suggests a coevolutionary relationship (2). In North America, the principal cause of HPS is Sin Nombre virus (SNV SNV Synovus Financial Corp. (stock symbol) SNV Schweizerische Normenvereinigung (Swiss standards body) SNV Stichting Nederlandse Vrijwilligers (Netherlands Development Organization) ) because of the geographically widespread nature of its rodent host, the deer mouse (Peromyscus maniculatus), the most common mammal in North America. As with other rodent reservoirs that harbor unique hantaviruses, most, if not all, deer mice become persistently infected without discernible pathologic consequences (3,4), which makes distinguishing infected from uninfected deer mice by simple observation impossible. Development of a field-relevant technique for detection of antibody to SNV would be of value; the technique could be exploited for further investigations of the virus-reservoir host interactions and characteristics and to determine whether experimental infections of deer mice with SNV accurately parallel natural infections (3,4). Commonly used serologic tests for deer mice require a minimum of 3-5 hours to complete (2,5,6) and thus are impractical to use in the field in a single day without putting the rodents at risk for death from heat, cold, dehydration, trap injuries, and other hazards while tests are being conducted. We modified a previously described protein-A/G horseradish peroxidase horse·rad·ish peroxidase n. An enzyme used in immunohistochemistry to label the antigen-antibody complex. enzyme-linked immunosorbent assay enzyme-linked immunosorbent assay n. ELISA. Enzyme-linked immunosorbent assay (ELISA) A diagnostic blood test used to screen patients for AIDS or other viruses. (PAGEIA) to detect antibodies to SNV in deer mice (7). The test can be completed in [approximately equal to] 1 hour under relatively primitive field conditions. The assay has advantages over more laborious assays used for similar purposes and, because it is mammal-specific rather than species-specific, we expect this assay will be applicable to serologic tests of mammals of many other species. The Study A fragment of the S segment (nt 43-394) encoding part of the nuclecapsid was cloned into pET21b with a C-terminal His tag to produce a 15-kDa truncated antigen (8) for use in the assay. Deer mice were trapped near Fort Lewis, Colorado, and blood was collected as previously described (9); whole blood was diluted in (1:100) 1 mL of phosphate-buffered saline (PBS PBS in full Public Broadcasting Service Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural, ) in 96 deep-well plates (P-DW-11-C, Axygen, Union City, CA, USA) at time of collection to expedite sample loading. The remainder of the blood was frozen on dry ice and returned to the laboratory for additional testing. Wells of 96-well polyvinyl chloride plates (Falcon 353912, BD Biosciences, San Jose, CA, USA) were coated with 100 [micro]L of 2 [micro]g/mL recombinant nucleocapsid nucleocapsid /nu·cleo·cap·sid/ (noo?kle-o-kap´sid) a unit of viral structure, consisting of a capsid with the enclosed nucleic acid. nu·cle·o·cap·sid n. in PBS and blocked (0.25% gelatin gelatin or animal jelly, foodstuff obtained from connective tissue (found in hoofs, bones, tendons, ligaments, and cartilage) of vertebrate animals by the action of boiling water or dilute acid. in PBS) a week in advance. Wells were washed in the field 3x with 200 [micro]L of PBS (pH 7.0) by using an 8-channel pipettor, and blood in PBS was added from the deep well plate; positive and negative (1 : 100) controls (diluted in PBS) were included. Plates then were incubated at ambient temperature (range [approximately equal to] 23[degrees]C-29[degrees]C) for 30 min. After 3 more washes with PBS/0.5% Tween-20, 100 [micro]L of pretitrated staphylococcal staphylococcal pertaining to Staphylococcus spp. staphylococcal clumping test used as a means of measuring the quantity of fibrinogen-split products in a sample of blood. protein-A/streptococcal protein-G horseradish peroxidase conjugate conjugate /con·ju·gate/ (kon´jdbobr-gat) 1. paired, or equally coupled; working in unison. 2. a conjugate diameter of the pelvic inlet; used alone usually to denote the true conjugate diameter; see (Pierce Biotechnology, Inc., Rockford, IL, USA) diluted 1:1,000 in PBS was added for 30 min. Plates again were washed 3x with PBS-Tween-20, and 100 [micro]L of activated ABTS ABTS American Board of Thoracic Surgery ABTS ASCII Block Terminal Services ABTS Arbin Battery Test System ABTS Abusive Tax Shelter ABTS Advanced Business Technology Services (Edwardsville, IL) ABTS Abort Basic Link Service ABTS Abort Sequence substrate was added to each well. After 15 min of incubation at ambient temperature, wells were scored by using a 0-4+ system, with 0 indicating no reaction (i.e., clear, no color) and 4+ representing the strongest signal (i.e., dark green color). Samples deemed 1+, 2+, 3+, or 4+ were considered positive (very weak, weak, strong, very strong, respectively). Samples were retested under laboratory conditions with PAGEIA and standard Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. (CDC See Control Data, century date change and Back Orifice. CDC - Control Data Corporation ) enzyme immunoassay (EIA) (5). Blood samples from 222 deer mice were collected during 3 trapping sessions in the summer of 2006, and 39 samples were scored as positive in the field by PAGEIA; 183 were negative by the field PAGEIA, repeat laboratory PAGEIA, and the standard EIA in the laboratory. One sample (HA-2564) was scored negative by field and laboratory PAGEIA, but (low) positive (optical density [OD] of 0.327) by conventional EIA (Table). Of the 39 samples that were scored positive in the field, 5 discrepancies between these and laboratory tests were found (Table). One sample (TS-0830-7) scored as 1+ in the field was determined to be negative on subsequent laboratory testing by both PAGEIA and conventional EIA. The other 4 samples (HB-2628, HA-2609, HA-2616, HB-2710) were scored as positive by field and laboratory PAGEIA but negative by conventional EIA. In the field, each of these samples was scored as 1+ or 2+ and had ODs of 0.331-0.664 by laboratory PAGEIA. However, ODs ranged from 0.076 to 0.228 by conventional EIA. The PAGEIA results were similar to results of conventional EIA, with a specificity of 82.9% (184 negatives/222 total rodents) versus 84.7% (188/222) for conventional EIA. The sensitivity of the PAGEIA was 97.1% (34 positive by PAGEIA/35 positive by conventional EIA). Conclusions We have modified an existing serologic se·rol·o·gy n. pl. se·rol·o·gies 1. The science that deals with the properties and reactions of serums, especially blood serum. 2. assay so that it is suitable for use in the field. The assay relies on a staphylococcal protein-A and streptococcal streptococcal /strep·to·coc·cal/ (-kok´al) pertaining to or caused by a streptococcus. Streptococcal (Streptococcus) Pertaining to any of the Streptococcus bacteria. protein-G horseradish peroxidase conjugate (10). Each protein has the capacity to bind to to contract; as, to bind one's self to a wife s>. See also: Bind the Fc portions of antibodies, including immunoglobulin M (IgM) and IgA for protein A (11,12), but has highest affinity for IgG subclasses of many mammalian species. All samples scored 3+ or 4+ were also positive in laboratory tests when results were read by using a spectrophotometer spectrophotometer, instrument for measuring and comparing the intensities of common spectral lines in the spectra of two different sources of light. See photometry; spectroscope; spectrum. . Thus, we are confident that such samples in the field will indicate seropositive seropositive /se·ro·pos·i·tive/ (-poz´i-tiv) showing positive results on serological examination; showing a high level of antibody. se·ro·pos·i·tive adj. animals. Because we are suggesting that this assay be used for identifying seropositive rodents and not for determining seroprevalence seroprevalence Immunology The proportion of a population that is seropositive–ie, has been exposed to a particular pathogen or immunogen; the seropositivity of a population is calculated as the number of individuals who produce a particular antibody divided (although it appears to be adequate for those studies as well) and to be conservative, we considered only samples that appeared dark green (3+ and 4+) in the field assay to be positive with relative certainty. To minimize the complexity of the PAGEIA under field conditions, we did not use a negative control antigen to assess nonspecific nonspecific /non·spe·cif·ic/ (non?spi-sif´ik) 1. not due to any single known cause. 2. not directed against a particular agent, but rather having a general effect. nonspecific 1. reactivities of serum samples. Use of this test will allow deer mice with antibody to SNV to be identified. Deer mice are the population most likely to be naturally infected with that virus, and those rodents can be retained for further testing and for studies of tissues, live cells, and body fluids to be used for subsequent laboratory investigations, such as for determining cellular immunologic responses, viremia viremia /vi·re·mia/ (vi-re´me-ah) the presence of viruses in the blood. vi·re·mi·a n. The presence of viruses in the bloodstream. levels, viruria levels, and virus shedding in excreta excreta /ex·cre·ta/ (eks-kret´ah) excretion (2). ex·cre·ta pl.n. Waste matter, such as sweat or feces, discharged from the body. and secreta secreta /se·cre·ta/ (se-kre´tah) [L., pl.] secretion (2). se·cre·ta n. Substances secreted by a cell, a tissue, or an organ; the products of secretion. secreta [L. . Additional limitations of the PAGEIA are similar to those of other serologic tests. PAGEIA can detect only seropositivity Seropositivity is the presence of a certain antibody in a blood sample. A patient with seropositivity for a particular antigen or agent is termed seropositive. , which is not necessarily indicative of current infection or of current shedding of virus. It also binds only with high affinity to IgG; thus, it is not useful for discriminating other immunoglobulin classes, such as IgM, the presence of which usually indicates recent infection. Because of the broad mammalian species specificities of a protein-A and protein-G conjugate, the rapid PAGEIA likely can be used to test for antibodies to other antigens in other mammals. Lee et al. (7) characterized the reactivities of protein A and protein G with IgG from rodents of several species. They found that serum specimens from both sigmodontine rodent species (deer mice and hispid cotton rats, Sigmodon hispidus) they tested were recognized by protein-A and/or protein-G conjugates. Similar laboratory-based PAGEIAs have also been used to detect antibody to antigens of agents causing other infectious diseases, including severe acute respiratory syndrome Severe Acute Respiratory Syndrome (SARS) Definition Severe acute respiratory syndrome (SARS) is the first emergent and highly transmissible viral disease to appear during the twenty-first century. coronavirus-like viruses and Nipah virus in bats (13-15). Acknowledgments We thank Brian Hjelle for helpful discussions on the manuscript. Funding was provided by NIH "Not invented here." See digispeak. NIH - The United States National Institutes of Health. contract AI25489 (to T.S., C.H.C., and B.J.B.) and grant AI054461 (to T.S.). C.H.C. was also partially funded by CDC, contract US3/CCU 813420-09. Beta Beta Beta Biological Honor Society and the University of Northern Colorado  It has been suggested that this article or section be merged with and ()University of Northern Colorado (Northern Colorado) also provided support (to A.A.R. and T.S., respectively). References (1.) Schmaljohn C, Hjelle B. Hantaviruses: a global disease problem. Emerg Infect Dis. 1997;3:95-104. (2.) Childs JE, Ksiazek TG, Spiropoulou CF, Krebs JW, Morzunov S, Maupin GO, et al. Serologic and genetic identification of Peromyscus maniculatus as the primary rodent reservoir for a new hantavirus in the southwestern United States. J Infect Dis. 1994;169:1271-80. (3.) Botten J, Mirowsky K, Kusewitt D, Bharadwaj M, Yee J, Ricci R, et al. Experimental infection model for Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus). Proc Natl Acad Sci U S A. 2000;97:10578-83. (4.) Botten J, Mirowsky K, Kusewitt D, Ye C, Gottlieb K, Prescott J, et al. Persistent Sin Nombre virus infection in the deer mouse (Peromyscus maniculatus) model: sites of replication and strand-specific expression. J Virol. 2003;77:1540-50. (5.) Feldmann H, Sanchez A, Morzunov S, Spiropoulou CF, Rollin PE, Ksiazek TG, et al. Utilization of autopsy RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic for the synthesis of the nucleocapsid antigen of a newly recognized virus associated with hantavirus pulmonary syndrome hantavirus pulmonary syndrome An often fatal RTI caused by a hantavirus; the first cluster occurred in the Four Corners region of Southwestern US Epidemiology Mean age 32, 61% ♀, 72% Native American Case definition Unexplained bilateral interstitial . Virus Res. 1993;30:351-67. (6.) Hjelle B, Jenison S, Torrez-Martinez N, Herring B, Quan S, Polito A, et al. Rapid and specific detection of Sin Nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for field diagnosis. J Clin Microbiol. 1997;35:600-8. (7.) Lee BH, Yoshimatsu K, Araki K, Ogino M, Okumum M, Tsuchiya K, et al. Detection of antibody for the serodiagnosis serodiagnosis /se·ro·di·ag·no·sis/ (-di?ag-no´sis) diagnosis of disease based on serologic tests.serodiagnos´tic se·ro·di·ag·no·sis n. pl. of hantavirus infection in different rodent species. Arch Virol. 2003;148:1885-97. (8.) Jonsson CB, Gallegos J, Ferro P, Severson W, Xu X, Schmaljohn CS. Purification and characterization of the Sin Nombre virus nucleocapsid protein expressed in Escherichia coli. Protein Expr Purif. 2001;23:134-41. (9.) Calisher CH, Sweeney W, Mills JN, Beaty BJ. Natural history of Sin Nombre virus in western Colorado. Emerg Infect Dis. 1999;5: 126-34. (10.) Harlow E, Lane D. Antibodies: a laboratory manual. Cold Spring (NY): Cold Spring Harbor Press; 1988. (11.) Patrick CC, Virella G, Koistinen J, Fudenberg HH. Differential binding of IgA proteins of different subclasses and allotypes to Staphylococcal protein A. Z Immunitatsforsch Immunobiol. 1977;153: 466-9. (12.) Graille M, Stura EA, Corper AL, Sutton B J, Taussig MJ, Charbonnier JB, et al. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity. Proc Natl Acad Sci U S A. 2000;97:5399-404. (13.) Lau SK, Woo PC, Li KS, Huang Y, Tsoi HW, Wong BH, et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci U S A. 2005;102:14040-5. (14.) Leroy EM, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba P, et al. Fruit bats as reservoirs of Ebola virus. Nature. 2005;438: 575-6. (15.) Reynes JM, Counor D, Ong S, Faure C, Seng V, Molia S, et al. Nipah virus in Lyle's flying foxes, Cambodia. Emerg Infect Dis. 2005;11:1042-7. Tony Schountz, * Charles H. Calisher, ([dagger]) Tiffany R. Richens, ([dagger]) Audrey A. Rich, * Jeffrey B. Doty, ([dagger]) Mark T. Hughes, ([dagger]) and Barry J. Beaty ([dagger]) * University of Northern Colorado, Greeley, Colorado, USA; and ([dagger]) Colorado State University Colorado State University, at Fort Collins; land-grant with state and federal support; chartered 1870, opened 1879 as an agricultural college, assumed present name in 1957. There is a veterinary teaching hospital, an agricultural campus, and a research campus. , Fort Collins, Colorado The City of Fort Collins, a home rule municipality situated on the Cache la Poudre River along the Colorado Front Range, is the county seat and most populous city in Larimer County, Colorado. , USA Address for correspondence: Tony Schountz, School of Biological Sciences, Box 92, University of Northern Colorado, Greeley, CO 80639, USA; email: tony.schountz@unco.edu The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated. Dr Schountz is an assistant professor of microbiology in the School of Biological Sciences at the University of Northern Colorado. His research interest is the immunologic basis of persistence of zoonotic Zoonotic A disease which can be spread from animals to humans. Mentioned in: Zoonosis agents.
Table. Comparison of results of PAGEIA and standard EIA for
detection of antibody to Sin Nombre virus (SNV) in blood
samples from 40 deer mice captured in southwest Colorado,
2006 *
Laboratory
Field PAGEIA PAGEIA OD Laboratory
Accession score ([double PAGEIA score
no. ([dagger]) dagger]) ([section])
HA-2548 3+ 1.254 Pos
HA-2552 4+ 2.406 Pos
HA-2554 4+ 1.788 Pos
HA-2558 4+ 2.383 Pos
HA-2560 4+ 1.913 Pos
HA-2564 0 0.001 Neg
HA-2565 1+ 0.236 Pos
HA-2567 4+ 2.123 Pos
HB-2604 4+ 2.065 Pos
HB-2608 2+ 0.855 Pos
HB-2612 1+ 0.282 Pos
HB-2616 1+ 0.311 Pos
HB-2617 2+ 0.517 Pos
HB-2618 2+ 0.494 Pos
HB-2622 3+ 1.254 Pos
HB-2628 1+ 0.493 Pos
HB-2630 3+ 1.609 Pos
HA-2570 4+ 1.970 Pos
HA-2578 4+ 2.101 Pos
HB-2642 4+ 2.784 Pos
HA-2601 4+ 2.699 Pos
HA-2609 2+ 0.608 Pos
HA-2612 4+ 2.482 Pos
HA-2616 1+ 0.331 Pos
HB-2681 3+ 0.977 Pos
HB-2682 3+ 1.095 Pos
HA-2634 4+ 3.010 Pos
HA-2647 4+ 2.824 Pos
HA-2666 4+ 2.682 Pos
HB-2706 1+ 0.836 Pos
HB-2710 2+ 0.664 Pos
HB-2712 3+ 1.599 Pos
HB-2717 1+ 1.098 Pos
HB-2720 3+ 2.581 Pos
TS-0830-6 2+ 0.889 Pos
TS-0830-7 1+ 0.000 Neg
TS-0830-8 3+ 2.014 Pos
TS-0830-9 4+ 1.949 Pos
TS-0830-18 4+ 2.112 Pos
TS-0830-20 3+ 1.427 Pos
Standard
Accession EIA OD Standard EIA
no. ([paragraph]) score (#)
HA-2548 0.903/0.066 Pos
HA-2552 1.083/0.113 Pos
HA-2554 1.395/0.058 Pos
HA-2558 1.462/0.055 Pos
HA-2560 1.378/0.086 Pos
HA-2564 0.327/0.055 Pos
HA-2565 0.715/0.046 Pos
HA-2567 1.485/0.080 Pos
HB-2604 1.161/0.067 Pos
HB-2608 0.653/0.095 Pos
HB-2612 1.136/0.071 Pos
HB-2616 0.458/0.001 Pos
HB-2617 0.819/0.008 Pos
HB-2618 1.085/0.009 Pos
HB-2622 1.519/0.029 Pos
HB-2628 0.220/0.082 Neg
HB-2630 0.681/0.008 Pos
HA-2570 0.389/0.024 Pos
HA-2578 1.185/0.017 Pos
HB-2642 1.294/0.063 Pos
HA-2601 0.921/0.121 Pos
HA-2609 0.228/0.042 Neg
HA-2612 1.072/0.085 Pos
HA-2616 0.076/0.059 Neg
HB-2681 1.392/0.048 Pos
HB-2682 1.326/0.042 Pos
HA-2634 0.863/0.014 Pos
HA-2647 0.720/0.023 Pos
HA-2666 0.477/0.028 Pos
HB-2706 0.324/0.032 Pos
HB-2710 0.155/0.035 Neg
HB-2712 0.345/0.033 Pos
HB-2717 0.293/0.027 Pos
HB-2720 0.630/0.039 Pos
TS-0830-6 0.799/0.097 Pos
TS-0830-7 0.030/0.033 Neg
TS-0830-8 0.800/0.024 Pos
TS-0830-9 0.884/0.054 Pos
TS-0830-18 1.180/0.021 Pos
TS-0830-20 0.820/0.072 Pos
* PAGEIA, protein-A/G horseradish peroxidase enzyme-linked
immunosorbent assay; EIA, enzyme immunoassay; OD, optical
density; Pos, positive, Neg, negative.
([dagger]) Field scores were based upon visual inspection
without instrumentation, with 0 as negative, 1+ as very weak,
2+ as weak, 3+ as strong, and 4+ as very strong, relative to
positive and negative control samples.
([double dagger]) Field-collected samples were retested by
PAGEIA under laboratory conditions and the OD reported here.
The instrument was blanked on the negative control sample.
([section]) OD >0.200 above the negative control was considered
positive.
([paragraph]) For laboratory EIA, OD was recorded for diluted
(1:100) blood with both SNV antigen (numerator) and control
antigen (denominator).
(#) A sample tested with SNV antigen and having an OD [greater
than or equal to] 0.300 was considered positive if the OD of
that sample with the control antigen was [less than or equal
to] 0.150. In regard to sample HBV-2717, the OD of the laboratory
CIA with antigen was 0.293, very near the acceptable minimum, and
the background was 0.027, which is very low; this sample was
considered provisionally positive.
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