Mosquito-Borne Arboviruses

Mosquito-borne arboviruses are transmitted to humans through the bite of an infected mosquito, and infections typically occur during the summer months when insects are most active. In the United States, the most common mosquito-borne illness is West Nile virus (WNV), a neurotropic human pathogen capable of spreading to the brain via hematogenous dissemination. Other neurotropic mosquito-borne arboviruses, including St. Louis encephalitis (SLE) virus and Japanese encephalitis virus, are less common but may cause similar symptoms. 

Emerging arboviruses, including dengue, Zika, and chikungunya, have similar epidemiologies, transmission cycles, and clinical symptoms at onset (although complications vary substantially).  Laboratory testing options for mosquito-borne arboviruses include serology and nucleic acid testing (NAT). The appropriate methodology depends on the suspected infection and duration of symptoms.

Quick Answers for Clinicians

What are the CDC’s current testing recommendations for Zika virus?

Given the decline in Zika virus outbreaks, the CDC has updated its testing guidance  for persons who live in or have recently traveled to areas with active dengue transmission and a risk of Zika virus infection. Zika virus testing is not advised in nonpregnant individuals.  Refer to the table titled “Summary of Laboratory Testing Recommendations for Pregnant Women and Infants” for a full list of indications.

Are Eastern equine encephalitis (EEE) cases on the rise, and when should laboratory testing for EEE be considered?

Eastern equine encephalitis (EEE) has been in the news lately because of the unusually high number of cases reported thus far in 2019. As of October 1, 31 cases in the United States, including nine deaths, were reported to the CDC. Most of these cases occurred in the northeastern U.S. By contrast, an average of seven human cases of EEE were reported in the years between 2009 and 2018.  Despite the rise in reported cases, EEE is relatively uncommon; only 5% of people bitten by an infected mosquito will develop the disease. Of those, roughly 30% who develop severe EEE die from the disease, whereas others may experience ongoing neurologic complications. 

As with other mosquito-borne arboviruses, testing may be indicated in symptomatic individuals who have either lived in or traveled to areas where EEE is circulating. A panel that tests for related and/or cocirculating arboviral illnesses may be considered; if the patient presents with encephalitis, cerebrospinal fluid (CSF) testing is warranted. 

Why is it important to test for related viruses in persons suspected of having a mosquito-borne illness?

Many mosquito-borne viruses cocirculate in some areas of the world and can cause similar clinical presentations. Moreover, serology testing can be complicated by cross-reactivity with related arboviruses. Therefore, adequate history of travel and vaccinations, especially if an individual has been in areas where viruses are cocirculating or where immunizations (eg, for yellow fever) are routine, can help determine the best strategy for further testing to identify the infecting virus. Plaque-reduction neutralization testing (PRNT) is recommended for confirmation because it can resolve nonspecific reactivity and, in some cases, identify the infecting virus.

Where do mosquito-borne arboviruses circulate?

Refer to the following sites for national and global maps of mosquito-borne virus circulation trends:

  • CDC ArboNET : up-to-date provisional reporting of arboviruses circulating in the United States, including West Nile virus (WNV), St. Louis encephalitis (SLE) virus, Eastern equine encephalitis (EEE) virus, and dengue, chikungunya, and Zika viruses
  • Zika virus travel information : global map of countries and territories with Zika virus cases
  • DengueMap : global map of dengue virus risk throughout the world
  • Chikungunya geographic distribution : map of countries and territories where chikungunya virus cases have been reported
Where can I find the most up-to-date testing guidance for mosquito-borne viruses?

Neurotropic Mosquito-Borne Viruses

West Nile Virus

In the U.S., the most common mosquito-borne illness is WNV. WNV can lead to a wide range of symptoms, from asymptomatic disease to severe meningitis and encephalitis. The milder version of WNV, referred to as West Nile fever, can be characterized by an abrupt onset of fever, headache, and possibly rash, among other symptoms. The more severe neuroinvasive disease affects one in 150 infected individuals and presents with meningitis, encephalitis, or flaccid paralysis syndrome. 

Indications for Testing

Testing for WNV is indicated for patients who either live in or have traveled to areas where WNV is circulating during mosquito season and who present with acute, unexplained febrile illness, rash, and/or meningitis/encephalitis. 

Diagnostic Laboratory Testing

Serology

Acute WNV infection is diagnosed by testing serum or cerebrospinal fluid (CSF) for WNV-specific immunoglobulin M (IgM) antibodies. WNV IgM antibodies are detectable 3 to 8 days after illness onset, but in some cases can persist for 30 to 90 days. IgG is detectable approximately 3 weeks postinfection and persists for years. Therefore, the presence of IgG only indicates previous exposure. False-positive results can occur due to cross-reactivity with other arboviruses (eg, Eastern equine encephalitis [EEE] virus, Western equine encephalitis [WEE] virus, SLE virus) and dengue virus. Plaque-reduction neutralization testing (PRNT) may be required to determine the specific infecting virus. 

Nucleic Acid Testing

NAT is not recommended for diagnosis and is typically reserved for immunocompromised patients or for clarifying equivocal serology results. 

Other Neurotropic Arboviruses

Although less common than WNV, the following arboviruses can cause meningitis and encephalitis:

These viruses are diagnosed in a similar fashion as WNV. ARUP Laboratories offers an arboviral serology panel that tests the arboviruses endemic to the U.S., and specific serology tests are available for Japanese encephalitis virus.

Nonneurotropic Mosquito-Borne Viruses

Zika Virus

Zika virus is a member of the Flavivirus genus and is transmitted to humans by Aedes spp mosquitos. Transmission is also possible during sexual contact, blood product transfusions, and pregnancy (from mother to fetus). Notably, infection with Zika virus during pregnancy has been linked to congenital microcephaly and other serious brain defects in fetuses and infants. Human to human transmission apart from sexual transmission has been described, but the mechanism of transmission is uncertain.  The majority of individuals infected with Zika virus are asymptomatic. In symptomatic individuals, infection is usually mild and self-limiting and may last for several days to a week. Zika viral infection symptoms include fever, headache, retro-orbital pain, conjunctivitis, maculopapular rash, myalgia, and arthralgia. These symptoms are not unique to Zika virus and may be seen in patients infected with other arboviruses, such as dengue and chikungunya.

Diagnostic tests for Zika virus include NAT and IgM serology. The test of choice depends on when the patient presents in relation to symptom onset or last possible exposure to Zika virus. Due to cocirculation and cross-reactivity, additional testing for dengue and chikungunya viruses should be considered.

Indications for Testing

Zika virus outbreaks have declined significantly since 2016 and are now outnumbered by reported dengue virus cases. Given this arboviral epidemiologic shift, Zika testing is not recommended for nonpregnant individuals; symptomatic nonpregnant individuals are advised to consider dengue virus testing.   In the event that a country reports an outbreak of Zika virus, the CDC’s dengue and Zika virus diagnostic testing for patients with a clinically compatible illness and risk for infection with both viruses  should be followed. The CDC’s current Zika virus testing guidance by population is detailed in the table below.

Summary of Laboratory Testing Recommendations for Pregnant Women and Infants
Population Characteristic(s) Recommendation(s)
Pregnant women Symptomatic Perform dengue and Zika virus NAT testing on a serum specimen, and Zika NAT on a urine specimen

Perform dengue IgM testing

Refer to the CDC’s Zika and Dengue Testing Guidance (updated November 2019)  for more information

Asymptomatic, residence in or recent travel to the U.S. and its territories Routine Zika testing is NOT recommended
Asymptomatic, residence in or recent travel to the U.S. and its territories Routine Zika testing is NOT recommended, but NAT testing may still be considered
Prenatal ultrasound findings consistent with congenital Zika virus infection; residence in or travel to areas with a risk of Zika virus during pregnancy

Perform Zika virus NAT and IgM testing on maternal serum and NAT on maternal urine

If Zika NAT is negative and IgM is positive, confirmatory PRNT should be performed against Zika and dengue

If amniocentesis is performed as part of clinical care, Zika virus NAT testing of amniocentesis specimens should also be performed; refer to the CDC’s Zika and Dengue Testing Guidance (updated November 2019)  for more information

Infants With birth defects consistent with congenital Zika syndrome and born to mothers with possible Zika virus exposure during pregnancy, regardless of the mother’s Zika virus testing result

Testing recommended

Perform serum and urine NAT and serum IgM serology concurrently

If CSF is obtained for other purposes, perform NAT and IgM antibody testing on CSF

Cord testing is not recommended; refer to the CDC testing algorithm for infants with possible congenital Zika virus infection  for more information

Without birth defects consistent with congenital Zika syndrome but born to mothers with laboratory evidence of possible Zika virus infection during pregnancy
Without birth defects consistent with congenital Zika syndrome and born to mothers without laboratory evidence of possible Zika virus infection during pregnancy No testing recommended
Source: CDC, 2019  

Diagnostic Laboratory Testing

Nucleic Acid Testing

The CDC recommends molecular testing by NAT on serum and urine in symptomatic pregnant women who meet the criteria in the table above during the acute phase of the disease (<14 days since symptom onset).  Refer to testing guidance for dengue virus if patients are symptomatic and not pregnant. Zika testing is not currently recommended for this group based on the current epidemiology of the virus. 

NAT tests may not detect Zika virus RNA in an infant who had a Zika virus infection in utero or in a child if the period of viremia has passed. 

Serology

Zika virus-specific IgM and neutralizing antibodies typically develop toward the end of the first week of illness. IgM levels are variable but are generally positive beginning about 4 days after symptom onset and can persist for up to 12 weeks after symptom onset or exposure (but may persist longer). False-positive Zika virus IgM serology results may occur in patients with a recent closely related arboviral infection (eg, dengue virus) or with recent vaccinations to related arboviruses (eg, Japanese encephalitis or yellow fever). Therefore, concurrent evaluation for other possible cocirculating arboviruses, especially dengue and chikungunya, should be considered.     Note that the CDC currently does not advise the use of Zika virus-specific serology testing in nonpregnant individuals. 

Clinical decisions regarding patient management cannot be based on a positive IgM result alone. Confirmatory PRNT for Zika virus neutralizing antibodies is performed by the CDC or a CDC-designated laboratory on all presumptive positive Zika IgM results.  ARUP provides these test results in the final patient chart.

Zika IgM Serology Testing

Result Interpretation

Presumptive Zika Possible Zika Flavivirus Positive Negative Zika
Zika IgM antibody detected

Confirmatory PRNT by CDC or CDC-designated laboratory required

Testing for related viruses with cocirculation should also be considered

Zika IgM antibody detected, suggesting possible recent infection

Other arbovirus IgM antibodies with Zika virus cross-reactivity may be present

Confirmatory PRNT by CDC or CDC-designated laboratory required

Testing for related viruses with cocirculation should also be considered

Presumptive positive for other flaviviruses

Perform dengue and WNV IgM testing

No evidence of IgM antibodies to Zika virus

Perform NAT testing to exclude false-negative IgM result for specimens collected <14 days after symptom onset

No further testing required for specimens collected ≥14 days after symptom onset

Source: CDC, 2019 

Dengue Virus

Dengue virus and Zika virus are closely related arboviruses with similar symptoms and distribution patterns throughout the tropics; however, dengue virus is currently causing large outbreaks in many areas of the world, while Zika virus cases have declined.  Dengue virus has four distinct serotypes (1, 2, 3, and 4) and is endemic throughout the tropics and subtropics. Although transmission most commonly occurs from the bite of an infected Aedes spp mosquito, dengue virus can also be transmitted through blood, breast milk, and during childbirth. Clinical manifestations range from the more common self-limited dengue fever to dengue hemorrhagic fever with shock syndrome, the most serious manifestation. The risk of progression to severe disease is much higher in secondary dengue infection than in primary infection. 

Diagnostic tests for dengue virus include serology, NS1 antigen testing (not currently performed at ARUP Laboratories), and NAT. The test of choice depends on when the patient presents in relation to symptom onset or last possible exposure to dengue virus. Additional testing for other arboviruses, particularly Zika virus, if an outbreak is occurring or has occurred, is recommended.  

Indications for Testing

Testing for dengue virus should be considered in patients with appropriate clinical manifestations (eg, fever, headache, hemorrhage, retro-orbital pain) and recent travel to or residence in an area with known dengue transmission.  

Diagnostic Laboratory Testing

Nucleic Acid Testing

NAT or NS1 antigen and IgM antibody testing are recommended during the first week of illness.   NAT is the preferred method for diagnosis because it can confirm infection and identify the specific virus, distinguishing between dengue and Zika viruses that often cocirculate, for example. A negative NAT or NS1 result should be followed with an IgM antibody test.  Refer to the CDC for more information about differentiating between dengue and Zika virus. 

Dengue Virus by PCR Testing

Result Interpretation

Detected Not Detected
Indicative of current infection

No further testing indicated

Does not exclude dengue virus infection

If test was performed during the first 5 days of illness, follow with dengue IgM serology

PCR, polymerase chain reaction

Source: CDC, 2019 

Serology

Although generally the preferred method of diagnosis, NAT can only detect dengue virus infection for a short period of time. Therefore, serology is usually used to diagnose dengue virus infection, and after 7 days of illness, only serology testing is recommended. In primary dengue infection, IgM antibodies are reliably detectable after 7 days from symptom onset and can remain detectable for 3 months or longer.  Although IgM antibody testing is important for diagnosis, false-positive dengue enzyme-linked immunosorbent assay (ELISA) results can occur due to cross-reactivity in patients infected with other flaviviruses such as Zika virus, WNV, and SLE virus.   Therefore, concurrent evaluation for other possible cocirculating arboviruses, such as Zika virus, should be considered, and PRNT is recommended.    Detailed vaccine, travel, and possible exposure history is key for determining the need for additional laboratory testing to identify the infecting virus. A positive dengue IgM-only result (negative for NAT and NS1) is considered presumptive dengue infection only if no other arboviruses are circulating in the areas where the patient was exposed. 

Dengue Virus IgM and IgG Serology Testing

Result Interpretation

IgM IgG Interpretation
Positive Positive Presumptive current or recent dengue virus infection
Negative Negative No evidence of dengue virus infection
Positive Negative Current or recent dengue virus infection; consider repeat testing to demonstrate seroconversion
Negative Positive Past dengue virus infection
Source: CDC, 2019 

Chikungunya Virus

Chikungunya virus is primarily transmitted to humans by Aedes spp mosquitos. Bloodborne transmission is possible, and rare in utero and intrapartum transmissions have been documented.  Unlike individuals with dengue and other mosquito-borne arboviruses, most individuals infected with chikungunya virus become symptomatic. Acute onset of fever and polyarthralgia are the most common clinical symptoms associated with chikungunya virus. Joint pain can be severe and may be persistent or relapsing in the months following acute illness. 

Indications for Testing

Testing for chikungunya should be considered in patients with acute onset of fever and polyarthralgia and recent travel to or residence in an area with known chikungunya virus transmission. 

Diagnostic Laboratory Testing

Nucleic Acid Testing

NAT testing is appropriate for all individuals suspected of having chikungunya virus who present in the early stages of disease. A positive result indicates current infection,  and no further testing is indicated. A negative PCR result does not exclude infection and should be followed by virus-specific IgM serology. 

Chikungunya Virus by PCR Testing

Result Interpretation

Detected Not Detected
Indicative of current infection

No further testing indicated

Does not exclude chikungunya virus infection

If test was performed during the first 5 days of illness, follow with IgM serology

Source: Johnson, 2016 
Serology

IgM serology is appropriate for persons suspected of having chikungunya virus who present later in the course of illness (beyond the first week of illness). Virus-specific chikungunya IgM antibodies are detectable toward the end of the first week of illness and remain detectable for 3-4 months after infection, whereas IgG antibodies to chikungunya virus remain detectable for years.

As with Zika virus, false-positive chikungunya virus IgM serology results may occur in patients with closely related infections or with recent vaccinations to related viruses. Therefore, concurrent evaluation for other possible cocirculating viruses, especially Zika and dengue, should be considered.   

Chikungunya Virus IgM and IgG Serology Testing

Result Interpretation

IgM IgG Interpretation
Positive Positive Current or recent chikungunya virus infection
Negative Negative No evidence of chikungunya virus infection
Positive Negative Current or recent chikungunya virus infection; consider repeat testing to demonstrate seroconversion
Negative Positive Past chikungunya virus infection
Source: Johnson, 2016 

ARUP Laboratory Tests

Neurotropic Mosquito-Borne Viruses
West Nile Virus

Order when WNV is suspected and patient presents with encephalitis

Order when WNV is suspected and patient does NOT present with encephalitis

Typically not a first-line test

Can be used to confirm positive WNV antibodies test result or to clarify equivocal serologic test results

Not a preferred test

Other Neurotropic Arboviruses

Order when arboviral illness is suspected and patient presents with encephalitis

Panel components: WNV, SLE, California encephalitis, EEE, and WEE viruses

Order when arboviral illness is suspected and patient does NOT present with encephalitis

Panel components: WNV, SLE, California encephalitis, EEE, and WEE viruses

Detect presence of Japanese encephalitis-related IgG and IgM antibodies in individuals with evidence of acute encephalitis syndrome who have recently traveled to or resided in an endemic country in Asia or the western Pacific

Patient’s travel history is necessary to aid in test interpretation

Recommend cotesting for other flaviviruses (eg, dengue fever virus) due to coexistence and antibody formation interference in individuals previously infected with flavivirus

Not recommended as a standalone test

Nonneurotropic Mosquito-Borne Viruses
Zika Virus

Recommended test for patients presenting in the acute phase of infection (<14 days after symptom onset or exposure)

Paired serum and urine specimens are preferred

3002525

3002263

Recommended screening test for patients with symptoms for ≥14 days

Follow-up test for patients with negative serum and urine NAT

Dengue Virus

Test detects and differentiates dengue subtypes 1-4

Can be used to diagnose dengue virus during acute phase of disease (>5 days after symptom onset)

In endemic regions, cotesting is recommended for other arthropod-borne viruses, including chikungunya and Zika, due to overlapping symptoms

May aid in diagnosis of dengue virus when timing of infection is uncertain

In endemic regions, cotesting is recommended for other arthropod-borne viruses, including chikungunya and Zika, due to overlapping symptoms

Aid in detecting past dengue infection

In endemic regions, cotesting is recommended for other arthropod-borne viruses, including chikungunya and Zika, due to overlapping symptoms

Chikungunya Virus Testing

Detect chikungunya virus

May aid in diagnosis of chikungunya viral infection during acute phase of disease (>5 days after symptom onset)

In endemic regions, cotesting is recommended for dengue and Zika viruses because clinical picture of these diseases is similar

Aid in detecting past chikungunya viral infection

In endemic regions, cotesting is recommended for dengue virus because clinical picture of these diseases is similar

Medical Experts

Contributor

Slev

Patricia R. Slev, PhD
Associate Professor of Clinical Pathology, University of Utah
Section Chief, Immunology; Medical Director, Immunology Core Laboratory, ARUP Laboratories

References