Comparative West Nile Virus Detection in Organs of Naturally Infected American Crows (Corvus brachyrhynchos).Widespread deaths of American Crows (Corvus brachyrhynchos) were associated with the 1999 outbreak of West Nile West Nile may refer to:
New York City City (pop., 2000: 8,008,278), southeastern New York, at the mouth of the Hudson River. The largest city in the U.S. region. We compared six organs from 20 crow carcasses as targets for WN virus detection. Half the carcasses had at least one positive test result for WN virus infection. The brain was the most sensitive target organ target organ n. A tissue or organ that is affected by a specific hormone. target organ, n the organ or body part whose activity levels demonstrate change in the course of biofeedback. ; it was the only positive organ for three of the positive crows. The sensitivity of crow organs as targets for WN virus detection makes crow death useful for WN virus surveillance. The 1999 outbreak of West Nile (WN) virus in the New York City area (1) was associated with the deaths of thousands of American Crows (Corvus brachyrhynchos), which appeared to be highly susceptible to the virus. Local health authorities selected some of these dead birds for laboratory testing. Generally, brain tissue was targeted for virus isolation as a method of surveillance (2). Although WN virus has frequently been isolated from brain tissue, a rigorous comparison of the brain to other organs of the American Crow has not been undertaken. Accordingly, we compared the sensitivity of the brain with that of other crow organs as targets for WN virus detection by both virus isolation and 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 detection. The Study From 20 crow carcasses collected in New Jersey during September and October 1999, we removed sections of brain, liver, spleen, kidney, heart, and lung for WN virus detection by plaque assay and TaqMan reverse transcription-polymerase chain reaction (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) (3). The samples were prepared by macerating Macerating refers to softening or breaking into pieces with liquid. It can refer to a form of food preparation. Raw, dried or preserved fruit or vegetables are soaked in liquid to soften and to absorb the flavor of the liquid. approximately 0.5 [cm.sup.3] of tissue in 1.8 mL of BA-1 (composed of M-199 Hanks salts, 29.2 mg/mL L-glutamine, 0.05 M Tris-HCl, pH 7.6, 1% bovine serum albumin serum albumin n. See seralbumin. , 350 mg/L sodium bicarbonate sodium bicarbonate or sodium hydrogen carbonate, chemical compound, NaHCO3, a white crystalline or granular powder, commonly known as bicarbonate of soda or baking soda. It is soluble in water and very slightly soluble in alcohol. , 100 units/mL penicillin, 100 mg/L streptomycin streptomycin (strĕp'tōmī`sĭn), antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria (see Gram's stain), including species resistant to other , and 100 [micro]g/mL Fungizone) diluent diluent /dil·u·ent/ (dil´oo-int) 1. causing dilution. 2. an agent that dilutes or renders less potent or irritant. dil·u·ent adj. Serving to dilute. n. in a glass Ten Broeck tissue grinder Grinder A slang term for a person who works in the investment industry and makes small amounts of money at a time on small investments, over and over again. Notes: (Bellco Glass, Inc., Vineland, NJ). Virus isolation was attempted in duplicate 100-[micro]L aliquots by Vero cell plaque assay. A 5-[micro]L aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) from each sample was tested by TaqMan RT-PCR assay, which quantitates WN virus RNA. Sensitivity of each assay for detecting WN virus or RNA in each organ was determined by using only the WN virus-infected carcasses as denominator in the calculations. One hundred nineteen tissue samples from 20 crows were assayed for WN virus (Table). Positive test results for WN virus infection were obtained for 10 of the 20 carcasses. WN virus was most often isolated from brain (8 [80%] of 10) and heart (6 [67%] of 9), while WN virus RNA was most frequently detected in brain (10 [100%] of 10) and liver and kidney (each 8 [80%] of 10). The TaqMan assay identified WN virus RNA in seven tissue samples that tested negative by plaque assay, including two brain tissue samples of crows from which all other organ tissues had tested negative. Tissues from the three crows for which only brain provided positive RNA detection were confirmed positive by repeat-testing in triplicate with three different TaqMan RT-PCR primer pairs. WN virus was then isolated by plaque assay from approximately 1 g of brain tissue from one of these specimens (NJN-37, data not shown). Table. Amount of virus detected by Vero plaque assay and TaqMan reverse transcriptase polymerase chain reaction in American Crow organs Crow number Heart Kidney Liver NJN 5 (+++)(a)/3.40E+03(b) (+++)/5.90E+04 (++)/8.48E+04 NJN 6 (++)/2.36E+03 (++)/1.12E+04 -(c)/1.10E+02 NJN 7 (++)/9.57E+03 (++)/5.52E+03 -/7.61E+01 NJN 8 (+++)/2.31E+05 (+++)/5.41E+04 (+++)/5.61E+05 NJN 9 (+++)/2.94E+04 (+++)/1.96E+05 (+)/2.15E+05 NJN 11 (+++)/3.62E+04 (+++)/1.06E+04 (+++)/1.06E+03 NJN 13 NT/NT (+)/2.54E+02 -/1.50E+00 NJN 29 -/- -/- -/- NJN 30 -/- -/- -/- NJN 33 -/- -/- -/- NJN 37 -/- -/- -/- NJN 40 -/- -/- -/- NJN 41 -/- -/- -/- NJN 43 -/- -/- -/- NJN 44 -/- -/- -/- NJN 51 -/- -/- -/- NJN 57 -/- -/- -/- NJN 62 -/- -/- -/- NJN 75 -/- -/- -/- NJN 95 -/- -/- -/- Crow number Lung Spleen Brain NJN 5 (+++)/2.42E+04 (+++)/4.09E+03 (+++)/5.41E+03 NJN 6 -/4.61E+02 (+)/2.02E+02 (++)/3.12E+02 NJN 7 (+)/3.46E+03 (+)/1.26E+02 (++)/1.76E+03 NJN 8 (+++)/3.20E+04 (+++)/4.08E+04 (+++)/6.18E+04 NJN 9 (+++)/4.36E+05 (+++)/1.17E+05 (+++)/1.78E+05 NJN 11 (+++)/1.31E+04 (+++)/1.07E+03 (+++)/2.15E+04 NJN 13 -/1.68E+02 -/- (+)/9.28E+01 NJN 29 -/- -/- (++)/6.67E+00 NJN 30 -/- -/- -/- NJN 33 -/- -/- -/3.24E+00 NJN 37 -/- -/- -/2.10E-01 NJN 40 -/- -/- -/- NJN 41 -/- -/- -/- NJN 43 -/- -/- -/- NJN 44 -/- -/- -/- NJN 51 -/- -/- -/- NJN 57 -/- -/- -/- NJN 62 -/- -/- -/- NJN 75 -/- -/- -/- NJN 95 -/- -/- -/- (a) Vero cell plaque assay: (+++) = [is greater than or equal to] 100 PFU/200 [micro]L, (++) = 10-100 PFU/200 [micro]L, (+) = [is less than or equal to] 10 PFU/200 [micro]L. (b) TaqMan RT-PCR assay: PFU equivalents/5 [micro]L. (c) Negative. NT = Not tested. Conclusions The findings suggest that the brain is the most sensitive target organ (of those tested) from crow carcasses for detecting WN virus with both detection assays (p = 0.0816). However, heart, lung, liver, kidney, and spleen were all good sources of WN virus with both assays. (The liver was not a good source of detection with the plaque assay.) Using the TaqMan assay, we were able to identify WN virus RNA in several tissue specimens that were negative by Vero plaque assay. The Taqman assay may be especially useful when organs from necropsied crows no longer contain live virus. If WN virus continues to spread, rapid detection will be an important public health issue. Since WN virus attacks various internal organs in birds (4), viscera viscera /vis·ce·ra/ (vis´er-ah) plural of viscus. vis·cer·a pl.n. 1. The soft internal organs of the body, especially those contained within the abdominal and thoracic cavities. from dead crows can be used to detect the virus in a surveillance program. Our findings, consistent with those of earlier studies, indicate that the brain is the most frequently affected organ among WN virus-infected birds (4) and support the continued use of the brain as the organ of choice from dead crows for surveillance and as a target for WN virus detection in diagnostic assays. Mr. Panella is a biologist in the Arbovirus arbovirus Any of a large group of viruses that develop in arthropods (chiefly mosquitoes and ticks). The name derives from “arthropod-borne virus.” The spheroidal virus particle is encased in a fatty membrane and contains RNA; it causes no apparent harm to the Diseases Branch, Division of Vector-Borne Infectious Diseases, 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. , Fort Collins, CO, USA. He has participated in West Nile virus West Nile virus, microorganism and the infection resulting from it, which typically produces no symptoms or a flulike condition. The virus is a flavivirus and is related to a number of viruses that cause encephalitis. outbreak investigations and has contributed to studies of West Nile virus ecology in the United States. References (1.) Komar N. West Nile viral encephalitis viral encephalitis Viral meningoencephalitis Neurology, infectious disease A general term for nonpurulent–'aseptic' viral infection of the CNS Etiology Coxsackie A and B–eg, A7, enterovirus 71, herpes simplex, etc Clinical If the viral load is extreme, . Rev Sci Tech 2000;19:166-76. (2.) Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, Mostashari F, et al. Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States, 1999. Emerg Infect Dis 2001;7:615-20. (3.) Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Savage HM, et al. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a Taqman reverse transcriptase PCR RT-PCR is a one or two-step process for converting RNA to DNA and the subsequent amplification of the reversely-transcribed DNA. In the first step of RT-PCR, called the “first strand reaction,” complementary DNA (cDNA) is made from an mRNA template using assay. J Clin Microbiol 2000;38:4066-71. (4.) Steele KE, Linn linn n. Scots 1. A waterfall. 2. A steep ravine. [Scottish Gaelic linne, pool, waterfall.] MJ, Schoepp RJ, Komar N, Geisbert TW, Manduca RM, et al. Pathology of a fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City. J Vet Pathol 2000;37:208-24. Nicholas A. Panella,(*) Amy J. Kerst,(*) Robert S. Lanciotti,(*) Patricia Bryant,([dagger]) Bruce Wolf,([dagger]) and Nicholas Komar(*) (*) Centers for Disease Control and Prevention, 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; and ([dagger]) New Jersey Department of Health and Senior Services Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression Program, Trenton, New Jersey, USA Address for correspondence: Nick Panella, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO 80522, USA; fax: 970221-6476; e-mail: nap4@cdc.gov |
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