*2.4.3.1. Plaque reduction neutralization test (PRNT)*

This test is considered the gold standard to detect neutralizing IgG antibodies because they have high sensitivity but can have a cross-reaction among members of flavivirus group. It is based on the binding of antibodies present in the sample which contains a known virus load (working dilution). The mixture of both is incubated and inoculated in a cell line until forming lytic plaques that are observed when coloring the cell monolayer. All samples neutralizing and avoiding the forming of certain number of plaques (being the most commonly used for 50–90% reduction) are with the presence of neutralizing antibodies indicating exposure to dengue. Using 1 in 30 diluted serum samples allowed discriminating between DENV-1 and DENV-2 serotypes in collected serum in Cuba, before and after the 1981 epidemic caused by DENV-2. There are many different variants of a PRNT test that could produce a variation in the results when being compared with using different used reduction rates, different PRNT methods (solid or semi-solid), and different cell lines. The most used for dengue are VERO cells that are recommended by World Health Organization (WHO), and BHK-21 that are used by other laboratories like Pedro Kouri Institute of Cuba. The use of different genotypes can alter the antibody titer results. Kochel et al. [30] evaluated Peruvian samples infected with DENV-2 American genotype. Antibody titers were found in higher levels when using the same genotype rather than using the Asian genotype. PRNT can be used to differentiate dengue infection to yellow fever infection. It has been found that the lowest serum dilution capable of distinguishing between both infections is 1 in 5. In a dengue secondary infection, PRNT can have a cross-reaction with other serotypes. In infected populations with sequentially different serotypes of dengue, the highest antibody titer pertains to the first infection (the "original antigenic" sin phenomenon) (**Figure 4**) [5–7, 30–32].

#### *2.4.3.2. Focus reduction neutralization test (FRNT)*

It combines the PRNT test with immunofluorescence or ELISA to count fluorescent foci or spots and calculate their reduction rate in the samples. Among its advantages, this allows reducing the days to obtain the results and undertaking studies with native strains so that

**Figure 4.** Plaque reduction neutralization test for DENV-2 using BHK-21 clone 15 cells. Dilutions from 1:20 to 1: 160 were used. Sample A and C do not present antibodies against the DENV2, and sample B is positive up to a dilution greater than 1: 160 (a photo taken on March 23, 2012 in the PRNT laboratory with the authorization of the U.S. Naval Medical Research Unit Six (NAMRU-6)).

some of them produce tenuous plates that are difficult to count but they make easier to detect fluorescent foci. Moreover, more sensitive lines can be used to detect dengue, not forming C6/36 plates, and they can be adapted to plates with a higher number of wells enabling to process a higher number of samples and saving materials. And, fluorescent foci can be counted with an ultraviolet light microscope or using computerized readers capable of reading fluorescent foci and reducing time to obtain results [33, 34].

the actual virus to a sample that can be infected cells with a dengue virus antibody joined to a fluorescent marker named conjugated (direct immunofluorescence). One of the most used fluorochromes for this technique is the fluorescein isothiocyanate (FITC). First, a specific antibody is to bind to a specific virus in the indirect immunofluorescence (the used antibody can be monoclonal or polyclonal, and it is bind to a conjugated). Observing the samples under ultraviolet light of the microscope, the fluorescent cells indicate the presence of the virus in the cell. This test is quite cost effective, and its sensibility and specificity can vary depending on the antibody quality used for virus identification. The polyclone antibodies are produced by sensitizing mice with the specific virus to detect. If an inactivated and structurally complete virus is used, the mice will produce antibodies against virus proteins resulting in a high cross-reaction within the virus of the same genome as it is the case of DENV, YFV, ZIKV, SLV, WNV, etc. Monoclonal antibodies produced in hybridomas can eliminate or reduce crossreactions (cell lines are able to produce antibodies against one or various virus epitopes) with bigger specificity. There are antibodies produced in hybridomas capable of identifying and differentiating flavivirus group, a general dengue virus (dengue complex), and to each den-

**Figure 5.** Enzyme-linked immunosorbent assay-format microneutralization test for DENV-2. The samples were inoculated in triplicate in VERO-76 cells using dilutions from 1:40 to 1: 320, and the ELISA test was performed on day 5 post inoculation (a photo taken on April 24, 2018 in the serology laboratory with the authorization of the U.S. Naval

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69

There are rapid tests based on lateral flow chromatographic immune assays produced by different laboratories. Proteins such as NS1, IgM, IgG in serum, blood or plasma samples can be detected through these tests, and they can simultaneously identify antibodies or proteins produced by ZIKV, DENV, and CHIKV. Assessing four rapid tests for NS1, its sensibility was in 71.9–79.1% range and its specificity in 95–100% antibodies compared to the viral isolation. These tests have the benefit of being cost effective, not requiring qualified personnel to be

gue virus serotype (**Figure 6**) [5, 6].

Medical Research Unit Six (NAMRU-6)).

*2.4.3.5. Rapid tests*

#### *2.4.3.3. Microneutralization-ELISA*

It is a variable in the PRNT test and uses the same immunological basis with the advantage of being worked on 96-well plates different from the PRNT that generally uses 24-well plates. This allows saving materials and handling a higher number of samples. This technique is used to detect the presence of neutralizing antibodies in the sample which is mixing the patient's sample that is previously inactivated in different dilutions with the dengue virus serotype to be evaluated, once the antigen-antibody binding is inoculated in the specified cell line as VERO-76 cells. If there were neutralizing antibodies in the sample, these ones would block the entry from the virus to the cell and would not produce the infection. The presence of the virus in the cell is revealed by an ELISA procedure. Samples exposing the color change in a substrate will be considered as negatives to an IgG antibody against the dengue serotype under study, and the samples that do not produce color change in the substrate will be positive for the presence of IgG antibodies (**Figure 5**). When this technique was evaluated with serum samples from patients with primary infection, the results were very similar to the ones in PRNT. However, the result correlation was very poor in comparison with this technique using samples from patients with secondary infections [35].

#### *2.4.3.4. Immunofluorescence test*

This test can be direct or indirect, and it is the most commonly used to identify the infected cells deriving from cell lines, salivary glands of mosquitoes, etc. It is based on the binding of Laboratory Tests Used in the Diagnostic and Research of Dengue Virus: Present and Future http://dx.doi.org/10.5772/intechopen.80519 69

**Figure 5.** Enzyme-linked immunosorbent assay-format microneutralization test for DENV-2. The samples were inoculated in triplicate in VERO-76 cells using dilutions from 1:40 to 1: 320, and the ELISA test was performed on day 5 post inoculation (a photo taken on April 24, 2018 in the serology laboratory with the authorization of the U.S. Naval Medical Research Unit Six (NAMRU-6)).

the actual virus to a sample that can be infected cells with a dengue virus antibody joined to a fluorescent marker named conjugated (direct immunofluorescence). One of the most used fluorochromes for this technique is the fluorescein isothiocyanate (FITC). First, a specific antibody is to bind to a specific virus in the indirect immunofluorescence (the used antibody can be monoclonal or polyclonal, and it is bind to a conjugated). Observing the samples under ultraviolet light of the microscope, the fluorescent cells indicate the presence of the virus in the cell. This test is quite cost effective, and its sensibility and specificity can vary depending on the antibody quality used for virus identification. The polyclone antibodies are produced by sensitizing mice with the specific virus to detect. If an inactivated and structurally complete virus is used, the mice will produce antibodies against virus proteins resulting in a high cross-reaction within the virus of the same genome as it is the case of DENV, YFV, ZIKV, SLV, WNV, etc. Monoclonal antibodies produced in hybridomas can eliminate or reduce crossreactions (cell lines are able to produce antibodies against one or various virus epitopes) with bigger specificity. There are antibodies produced in hybridomas capable of identifying and differentiating flavivirus group, a general dengue virus (dengue complex), and to each dengue virus serotype (**Figure 6**) [5, 6].

#### *2.4.3.5. Rapid tests*

some of them produce tenuous plates that are difficult to count but they make easier to detect fluorescent foci. Moreover, more sensitive lines can be used to detect dengue, not forming C6/36 plates, and they can be adapted to plates with a higher number of wells enabling to process a higher number of samples and saving materials. And, fluorescent foci can be counted with an ultraviolet light microscope or using computerized readers capable of reading fluo-

**Figure 4.** Plaque reduction neutralization test for DENV-2 using BHK-21 clone 15 cells. Dilutions from 1:20 to 1: 160 were used. Sample A and C do not present antibodies against the DENV2, and sample B is positive up to a dilution greater than 1: 160 (a photo taken on March 23, 2012 in the PRNT laboratory with the authorization of the U.S. Naval Medical

It is a variable in the PRNT test and uses the same immunological basis with the advantage of being worked on 96-well plates different from the PRNT that generally uses 24-well plates. This allows saving materials and handling a higher number of samples. This technique is used to detect the presence of neutralizing antibodies in the sample which is mixing the patient's sample that is previously inactivated in different dilutions with the dengue virus serotype to be evaluated, once the antigen-antibody binding is inoculated in the specified cell line as VERO-76 cells. If there were neutralizing antibodies in the sample, these ones would block the entry from the virus to the cell and would not produce the infection. The presence of the virus in the cell is revealed by an ELISA procedure. Samples exposing the color change in a substrate will be considered as negatives to an IgG antibody against the dengue serotype under study, and the samples that do not produce color change in the substrate will be positive for the presence of IgG antibodies (**Figure 5**). When this technique was evaluated with serum samples from patients with primary infection, the results were very similar to the ones in PRNT. However, the result correlation was very poor in comparison with this technique

This test can be direct or indirect, and it is the most commonly used to identify the infected cells deriving from cell lines, salivary glands of mosquitoes, etc. It is based on the binding of

rescent foci and reducing time to obtain results [33, 34].

using samples from patients with secondary infections [35].

*2.4.3.3. Microneutralization-ELISA*

Research Unit Six (NAMRU-6)).

68 Dengue Fever - a Resilient Threat in the Face of Innovation

*2.4.3.4. Immunofluorescence test*

There are rapid tests based on lateral flow chromatographic immune assays produced by different laboratories. Proteins such as NS1, IgM, IgG in serum, blood or plasma samples can be detected through these tests, and they can simultaneously identify antibodies or proteins produced by ZIKV, DENV, and CHIKV. Assessing four rapid tests for NS1, its sensibility was in 71.9–79.1% range and its specificity in 95–100% antibodies compared to the viral isolation. These tests have the benefit of being cost effective, not requiring qualified personnel to be

an immunological and physiological result similar to the human ones. It is known that it is susceptible to a flavivirus such as the Japanese encephalitis, YFV, and Murray valley encephalitis. Studies about infected porcine models administered subcutaneously with DENV-1 (107

Laboratory Tests Used in the Diagnostic and Research of Dengue Virus: Present and Future

http://dx.doi.org/10.5772/intechopen.80519

resulting to developed viremia, and IgM and IgG antibodies without symptoms are carried out. The secondary infection with the same serotype produced extensive macular and papular rashes similar to the ones affecting humans. When infecting the swine model with a DENV-1

Throughout a dengue research, the use of laboratory tests play a fundamental role in identifying the virus (serotype, genotype and lineage), its genetic material, viral proteins, the presence of antibodies against the virus or assessing the patient's condition, and for instance, hematocrit, platelet count, white blood cell count, blood count, etc. together with patient's signs and symptoms and some epidemiological criteria, it allows categorizing the disease for dengue with or without warning signs or severe dengue. This classification assists in deciding what therapy to choose and preventing from a severe dengue development. Besides the above mentioned techniques in this chapter, there are laboratory techniques which are not commonly used at present because they are expensive and/or they require sophisticated equipment but they could be used for DENV studies. Among them, there is the transmission electron microscopy (TEM) which is a gold standard technique for the absolute quantification of particles. However, it does not allow differentiating an infectious virus from a noninfectious one. The high performance liquid chromatography (HPLC) where the virus load is quantified through the UV analysis of fractions produced during HPLC. Flow cytometry (FC) can be used to quantify viral proteins being present on the surface of the infected cells. The next-generation whole genome sequencing is the most advanced sequencing technology which allows learning the complete and detailed genome sequence of an organism in a short

intravenous line, a rash skin and dermal edemas appeared on the animal [36, 37].

period of time (days), though its cost is still quite higher to some researches [27, 38].

endemic areas obtaining the same immunity level regardless the disease prevalence.

knowledge like:

certain conditions [40].

Dengue researches in the future will try to respond to different problems, needs, and gaps in

The production and improvement of the vaccine against DENV like Dengvaxia® (CYD-TDV), produced by Sanofi Pasteur, is a prophylactic, tetravalent, live attenuated recombinant viral vaccine which uses a 3-dose vaccination schedule, and it is recommended for people ranged from 9 to 45, or 9 to 60 year old (depending on the license) [39]. Nonetheless, it is necessary to continue with searches to develop a vaccine which can be used from the first year of life enabling to achieve the maximum immunity with a single dose and can be used in different

Today, it is acknowledged that the immunity against dengue after a natural infection can be for a lifetime [5]; however, some studies indicate that a dengue reinfection may occur under

The relation between chronic diseases and dengue has been studied as risk factors in order to determine the severity of dengue disease in individuals. In recent decades, the increase of

**2.6. The future of laboratory tests and its connection with dengue studies**

PFU)

71

**Figure 6.** Indirect immunofluorescence test performed on C6/36 cells. In (A), uninfected red cells are observed and in (B), infected green cells are observed (a photo taken on April 24, 2018 in the serology laboratory with the authorization of the U.S. Naval Medical Research Unit Six authorization (NAMRU-6)).

done nor using sophisticated equipment. Within its limitations, it is not possible to identify the circulating serotype in a virus, the band intensity is not related to the antibody titers nor the actual NS1 antigen in the sample, cross-reactions with other flavivirus are common, a negative result in these tests does not exclude that the patient has been exposed to the virus, and some samples present high titers of rheumatoid factor affecting the results. The positive samples for these results should be verified with other alternative methods [28].

#### **2.5. The animal models for dengue virus**

Studies to evaluate antivirals against dengue, vaccines, plant extracts, etc. mainly use the mouse model and nonhuman primates (NHPs). However, each of them presents a series of constraints that impair research developments. The infections of the mouse model are used for pre-clinical development of vaccines. In this model, a neurotropic disease can occur; however, this does not usually happen in humans. Some mice like C57BL/6, BALB/c and A/J enable viral replication, but A/J and BALB/c mice develop paralysis. Some studies show that C57BL/6 mice can have hemorrhages when being infected with a DENV2 strain 16681 using a virus with a titer of 3 × 109 PFU/mL. Moreover, an infection and endothelial cell damage as well as hemorrhages in tissues can occur without showing signs of disease. The DENV replication in mice is low, but suckling mice inoculated by an intracranial via at high DENV doses can induce neurological illnesses and paralysis that is used to measure the effectiveness of the vaccines. They can also be used to test a virus neurovirulence or an attenuation to produce vaccines. NHPs are used to research an immune reaction against DENV, an evaluation of candidates' vaccines, a replication kinetic, etc. This model has a major constraint because it does not produce signs of clinical disease. Nonetheless, DENV can infect some cells in the body. An inoculation with 10<sup>5</sup> PFU DENV concentrations enables a lower viral replication in humans, and it is restricted to lymphoid-rich tissues producing lymphadenopathy, lymphocytosis, and leukopenia. Rhesus macaques can produce antibodies against DENV and a similar viral load to the humans. The inoculation of high titer DENV via intravenous in rhesus macaques can produce hemorrhage. The physiology of pigs is similar to the human one. Yucatan miniature swine models present an immunological and physiological result similar to the human ones. It is known that it is susceptible to a flavivirus such as the Japanese encephalitis, YFV, and Murray valley encephalitis. Studies about infected porcine models administered subcutaneously with DENV-1 (107 PFU) resulting to developed viremia, and IgM and IgG antibodies without symptoms are carried out. The secondary infection with the same serotype produced extensive macular and papular rashes similar to the ones affecting humans. When infecting the swine model with a DENV-1 intravenous line, a rash skin and dermal edemas appeared on the animal [36, 37].
