*2.4.1.5. Viral isolation in cell lines*

3.6 × 101

within its limiting [13].

*2.4.1.4. Real-time PCR*

, DENV-3 (H87) 1.6 × 103

U.S. Naval Medical Research Unit Six (NAMRU-6).

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

and DENV-4 (H241) 7.6 × 101

**Figure 1.** Reverse transcriptase semi-nested reactions, using primers targeted to the C/PrM genomic region as described by Lanciotti et al. (a photo taken on April 24, 2018 in the molecular biology laboratory with the authorization of the

is time reduction to obtain results (about an hour), discriminating the amount of pathogens, and minimizing cross-contamination problems so that all reactions are carried out among a closed system. The cost of the product and a machine analyzing only a sample at a time is

It uses conventional RT-PCR principles, and it combines with fluorochromes like SYBR Green or TaqMan probes with fluorochromes capable of producing proportional fluorescence to the DNA copy samples. The strengths of this test are the same as the conventional RT-PCR, it also reduces time when releasing the results as well as the cross-contamination risk post PCR, the levels of sensitivity and specificity are higher than the conventional RT-PCR, and overall it allows quantifying the genetic material. There are different commercial kits in the market to diagnose DENV, and its sensitivity and specificity levels vary when they are compared among them [14, 15]. The CDC elaborated a real time RT-PCR in order to diagnose four serotypes in serum or human plasma samples using an ABI 7500 FAST DX thermo-cycler of Applied Biosystems and hydrolysis dual-marker TaqMan probes, and it is the first RT-PCR approved

by Food and Drug Administration (FDA) to detect DENV [16].

. The main advantage of this test

Viral isolation in cell cultures or mosquitoes followed by the virus detection using indirect immunofluorescence is considered as gold standard [8, 17]. In order to carry out the DENV viral isolation and as a general rule to any virus, it is necessary to consider the following:

	- C6/36: they are easy cell propagation, highly sensitive to DENV infection, and cultivated at 28°C that are obtained from salivary glands of *Aedes albopictus*.
	- C6/36 HT (hot temperature): they can be spread at 34°C and have a bigger sensitivity to detect DENV. This cell line maintains its high sensitivity only for some weeks due to its higher temperatures, so some researchers suggest adapting at 34°C, a C6/36 cell line growing at 28°C, and using an alternative method.
	- TRAS-284-SF cell has the main benefit of not requiring the use of fetal bovine serum (FBS) as a culture medium, and it presents a higher sensitivity to the DENV isolation [6, 9, 17].

in order to obtain the results in comparison with plaque assay. It allows processing a bigger number of samples so that it can be adapted to use 96-well culture plates in comparison with the 24-well plates which are used in the plaque assay. Another advantage is to allow the use of C6/36 cells that are highly sensitive to detect dengue virus, and they cannot be used for the

**Figure 3.** Fluorescent focus assay for DENV-2 using the C6/36 (A) and VERO-76 cells (B). The indirect immunofluorescence performed to visualize the foci was carried out on the fifth day of incubation, using the fluorescein isothiocyanate (FITC) conjugate (a photo taken on March 15, 2016 in the Serology laboratory with the authorization of the U.S. Naval Medical

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

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

65

Suckling mice were greatly used because of their easy reproduction and handling to isolate virus as well as the antigen production. About 1–3 neonatal mice and an intracranial inoculation are carried out. Then, a 21-day daily checking to observe the occurrence of neuromotor symptoms is needed. This technique is starting to cease to use due to a great variety of cell cultures that allow good sensitivity levels in DENV detection. This is why the Institutional Animal Care Committee and IACUC Committee recommend reducing this activity. One of

When using this technique, it is necessary to make sure such mice are dead as they are very resilient to lacking of oxygen so it is advisable to continue with other euthanasia techniques

The mosquitoes like *Aedes* genus can be used for dengue virus isolation when infection and disease transmission studies are carried out. The intracerebral and intrathoracic inoculations are used for mosquitoes which are immobilized at low temperatures. The mosquito infection technique is to feed them directly with the dengue-infected patient blood in the acute phase of the disease. The mosquitoes of the *Toxorhynchites* genus, which are not blood-feeding insects, can be used for the four-serotype dengue isolation, Japanese encephalitis and encephalitis of San Luis are more susceptible than cell culture isolation of dengue virus as well as the *Drosophila melanogaster* that can be inoculated by micro injection in the abdomen, and it could reach higher titers using less time in comparison with the *Aedes aegypti* inoculation [6, 25, 26].

camera.

the most common practices to carry out euthanasia on suckling mice is using a CO2

plaque assay as they do not form lytic plaques (**Figure 3**) [23].

like cervical dislocation, decapitation, etc. [6, 9, 24].

*2.4.1.8. Viral isolation in nursing mice*

Research Unit Six (NAMRU-6)).

*2.4.1.9. Inoculation in mosquitoes*

**Figure 2.** Plaque assay titration for DENV-2 using VERO-76 cells and a semi-solid method (a photo taken on March 2017, in the Virology and Molecular Biology Laboratory of the Faculty of Biological Sciences at National University of San Marcos, Peru).

#### *2.4.1.6. Plaque assay*

It is the most used test to determine viral vaccine titers so that it quantifies the virus to infect cells. It is based on the infection of a cell monolayer with different virus dilutions to evaluate. After an incubation period, the viral infection results in lytic plaques. If they are colored, they are displayed as holes in the cell monolayer. Each plaque corresponds with an infectious virus. One of the main disadvantages of this technique is that it can only use viruses being able to produce a cytopathic effect. Another one is that all native strains of DENV are not always capable of producing well-defined plaques, and the viral titers can vary depending on the cell line used. For a dengue virus case, the most used cell lines are VERO and BHK-21 (**Figure 2**) [5, 6, 23].

#### *2.4.1.7. Fluorescent focus assay*

It is a combination of plaque assay and immunofluorescence. Viruses are inoculated in different dilutions in the cell line, then a cell incubation period is fixed to plaques with any organic solvent, and an immunofluorescence is carried out. Positive cells are observed with fluorescent foci that can be counted. One of its advantages is to reduce the incubation period Laboratory Tests Used in the Diagnostic and Research of Dengue Virus: Present and Future http://dx.doi.org/10.5772/intechopen.80519 65

**Figure 3.** Fluorescent focus assay for DENV-2 using the C6/36 (A) and VERO-76 cells (B). The indirect immunofluorescence performed to visualize the foci was carried out on the fifth day of incubation, using the fluorescein isothiocyanate (FITC) conjugate (a photo taken on March 15, 2016 in the Serology laboratory with the authorization of the U.S. Naval Medical Research Unit Six (NAMRU-6)).

in order to obtain the results in comparison with plaque assay. It allows processing a bigger number of samples so that it can be adapted to use 96-well culture plates in comparison with the 24-well plates which are used in the plaque assay. Another advantage is to allow the use of C6/36 cells that are highly sensitive to detect dengue virus, and they cannot be used for the plaque assay as they do not form lytic plaques (**Figure 3**) [23].

## *2.4.1.8. Viral isolation in nursing mice*

Suckling mice were greatly used because of their easy reproduction and handling to isolate virus as well as the antigen production. About 1–3 neonatal mice and an intracranial inoculation are carried out. Then, a 21-day daily checking to observe the occurrence of neuromotor symptoms is needed. This technique is starting to cease to use due to a great variety of cell cultures that allow good sensitivity levels in DENV detection. This is why the Institutional Animal Care Committee and IACUC Committee recommend reducing this activity. One of the most common practices to carry out euthanasia on suckling mice is using a CO2 camera. When using this technique, it is necessary to make sure such mice are dead as they are very resilient to lacking of oxygen so it is advisable to continue with other euthanasia techniques like cervical dislocation, decapitation, etc. [6, 9, 24].

#### *2.4.1.9. Inoculation in mosquitoes*

*2.4.1.6. Plaque assay*

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

San Marcos, Peru).

*2.4.1.7. Fluorescent focus assay*

It is the most used test to determine viral vaccine titers so that it quantifies the virus to infect cells. It is based on the infection of a cell monolayer with different virus dilutions to evaluate. After an incubation period, the viral infection results in lytic plaques. If they are colored, they are displayed as holes in the cell monolayer. Each plaque corresponds with an infectious virus. One of the main disadvantages of this technique is that it can only use viruses being able to produce a cytopathic effect. Another one is that all native strains of DENV are not always capable of producing well-defined plaques, and the viral titers can vary depending on the cell line used. For a dengue virus case, the most used cell lines are VERO and BHK-21 (**Figure 2**) [5, 6, 23].

**Figure 2.** Plaque assay titration for DENV-2 using VERO-76 cells and a semi-solid method (a photo taken on March 2017, in the Virology and Molecular Biology Laboratory of the Faculty of Biological Sciences at National University of

It is a combination of plaque assay and immunofluorescence. Viruses are inoculated in different dilutions in the cell line, then a cell incubation period is fixed to plaques with any organic solvent, and an immunofluorescence is carried out. Positive cells are observed with fluorescent foci that can be counted. One of its advantages is to reduce the incubation period The mosquitoes like *Aedes* genus can be used for dengue virus isolation when infection and disease transmission studies are carried out. The intracerebral and intrathoracic inoculations are used for mosquitoes which are immobilized at low temperatures. The mosquito infection technique is to feed them directly with the dengue-infected patient blood in the acute phase of the disease. The mosquitoes of the *Toxorhynchites* genus, which are not blood-feeding insects, can be used for the four-serotype dengue isolation, Japanese encephalitis and encephalitis of San Luis are more susceptible than cell culture isolation of dengue virus as well as the *Drosophila melanogaster* that can be inoculated by micro injection in the abdomen, and it could reach higher titers using less time in comparison with the *Aedes aegypti* inoculation [6, 25, 26].
