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Virology / Serology - Arbovirus

General Information

Historically, North Carolina has been an area endemic for several arboviruses (insect transmitted diseases), most notably Eastern Equine Encephalitis (EEE) external link and LaCrosse Encephalitis (LAC) viruses external link. These mosquito-borne viruses appear to be increasing in activity within the last decade. More recently, West Nile Virus (WNV) external link established itself in our state after first appearing in the western hemisphere in New York in 1999. For detailed distribution of WNV in the U.S., please visit the CDC's West Nile Virus Maps and Data external link page or the USGS's West Nile Virus Maps external link page.

In humans, WNV usually causes mild flu-like illness that can include fever, headache, body aches, rash, and swollen lymph glands. In more serious infections, symptoms can include a severe headache, high fever, neck stiffness, disorientation, coma, convulsions, muscle weakness, paralysis, and very rarely death. The incubation period is usually 3 to 15 days. In a minority of cases the disease may be severe, with central nervous system involvement.

Timely laboratory testing for WNV and other arboviral infections including EEE and LAC is available at the State Laboratory of Public Health free of charge via serology, viral isolation, and molecular amplification. All positive results will be promptly telephoned to the submitting physician or hospital at the number provided. A diagnostic serologic panel will be performed on all serum suspected for arbovirus that includes testing for IgG antibodies to EEE, LAC, WNV, Western Equine Encephalitis (WEE), and St. Louis Encephalitis (SLE) by immunofluorescence (IFA). All serum received will be also be tested for IgM antibodies to WNV and LAC by enzyme immunoassay (EIA). For more information about these viruses, visit CDC external link.

Specimen Acceptance Policy

Only serum and CSF may be submitted for serologic testing. Specimens submitted to the Virology/Serology Unit must be accompanied by a fully completed submission DHHS Serology Form #3445 (PDF, 385 KB). Failure to supply the requested patient information (onset date, collection date, symptoms, travel history, vaccination history) may result in significantly delayed specimen testing. Tests must be requested by name. Nonspecific requests for "viral studies" or "viral serologies" will not be accepted. Consult with the laboratory if there is a question as to which test is appropriate.

Both acute and convalescent paired serum specimens should be submitted. CSF specimens can be submitted (only with a companion serum) for molecular amplification, virus cultivation in cell culture, and/or antibody level determination. In fatal cases, submit CNS tissue specimens (spinal cord, medulla, cerebellum, and hippocampus) for virus isolation and molecular amplification, in addition to serum and CSF.

Horses: Testing of horses is also available for serum, CSF and CNS specimens for EEE and WNV. Please submit specimens through the Rollins Animal Diagnostic Laboratory external link and use the forms above for submission of these samples. Be sure to complete forms completely including vaccination history, since horse vaccines are available for EEE and WNV.

Wild Birds: When the virus first appeared in the United States, North Carolina was among many states that were able to get valuable information from the collection and testing of dead, wild birds. However, now that the virus has become established within the wild bird population, testing dead birds is no longer necessary. Therefore, North Carolina is no longer taking reports of dead wild birds or submitting any birds for testing.

Specimen Collection

To collect serum, draw blood into a red top vacuum tube allowing the tube to fill completely. Let stand for 30 minutes to ensure complete clotting of the blood. Centrifuge for 5-10 minutes at 1000-1500 x g. Transfer the serum to a plastic screw capped vial. Hemolyzed, icteric, or lipemic serum may be unacceptable for certain serologic assays.

Clearly label each vial of serum with the patient's full first and last name, either SSN or date of birth, and the date collected if paired sera are submitted. Complete a DHHS Serology Form #3445 (PDF, 385 KB) submission form specifying all required patient information and which infectious agents are suspected.

The serodiagnosis of a current or recent infection generally requires the simultaneous testing of paired serum samples, principally, acute and convalescent serum samples. The acute serum should be collected no later than 3-5 days after the onset of illness. The convalescent serum should be collected 2-4 weeks after onset. Where paired sera are advised or required, it is to the advantage of both the submitter and this laboratory if the acute serum is stored frozen by the submitter until the convalescent serum is collected. Both serum samples may be submitted with one submission form.

Serological diagnosis of arboviral infection can be made by demonstrating a four fold or greater rise in titer between acute and convalescent sera.  Additionally, single "high" antibody titers to viral, and rickettsial agents may be considered presumptive evidence of recent infection. Antibody determinations on cerebrospinal fluid may be of value in diagnosing viral encephalitis and other central nervous system diseases. Cerebrospinal fluids for serologies should always be accompanied by a serum collected the same day.

Specimen Shipping

Properly identified vials of patient sera along with the completed submission forms should be sent in the blue colored specimen mailers labeled SPECIAL SEROLOGY. For detailed shipping instructions using the double mailers, see Packing Instructions Using Outer Baggie (PDF, 1.7 MB). Ship at ambient temperature by the State Courier or U.S. Mail. Specimens should be shipped immediately and should arrive in the laboratory within 48 hours of collection. Specimens may be shipped refrigerated or at ambient temperature. If transport to the laboratory is to be delayed, specimens can be refrigerated up to seven days or frozen.

Serum transport tubes and blue colored specimen mailers are available through the NCSLPH online supply ordering system at this website. DHHS Serology Form #3445 (PDF, 385 KB) may be downloaded and printed from this website.

Specimen Reporting

The following chart lists the arboviral assays performed by this lab. A brief statement of the "normal" values for each assay is given under the heading "Negative Reference Range". The test method, specimen requirements, and turn-around-times are also listed for each assay performed.

Arboviral Assays
Test Test Method Negative Reference Range Specimen Requirement Turn-Around-Time
California Encephalitis (LAC), IgG IFA-Quant <1:16 2 mL serum/CSF; PSA 10 calendar days
LAC, IgM IFA-Quant
EIA-Qual		 <1:16
Negative 		2 mL serum/CSF; PSA 10 calendar days
Eastern Equine Encephalitis (EEE), IgG IFA-Qual <1:16 2 mL serum/CSF; PSA 10 calendar days
EEE, IgM IFA-Quant <1:16 2 mL serum/CSF; PSA 10 calendar days
St. Louis Encephalitis (SLE), IgG IFA-Quant <1:16 2 mL serum/CSF; PSA 10 calendar days
SLE, IgM IFA-Quant <1:16 2 mL serum/CSF; PSA 10 calendar days
Western Equine Encephalitis (WEE), IgG IFA-Quant <1:16 2 mL serum/CSF; PSA 10 calendar days
WEE, IgM IFA-Quant <1:16 2 mL serum/CSF; PSA 10 calendar days
West Nile Virus (WNV), IgG IFA-Quant <1:16 2 mL serum/CSF; PSA 10 calendar days
WNV, IgM IFA-Quant
EIA-Qual 		<1:16
Negative 		2 mL serum/CSF; PSA 10 calendar days


Abbreviations
EIA – Enzyme Immunoassay IFA - Indirect Fluorescent Antibody
IgG – Immunoglobulin G IgM - Immunoglobulin M
Quant - Quantitative Qual - Qualitative
PSA – Paired Sera Advised  

Additional Information

For additional information on West Nile virus and other arboviruses please visit:

See also:

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Last Modified: October 31, 2011 3:42 PM