Dengue virus serotypes and genotypes and its geographical distribution.
Dengue is the most important arbovirus, many research have contributed to the diagnosis, management, prevention and control of this disease, which will be described in this chapter, for example: the importance of serotypes and genotypes for the development of the disease, the relationship of the viral load between symptomatic and asymptomatic people, the influence of antibodies on the development of the disease, co-infections with microorganisms and chronic diseases, possible reservoirs, the diagnostic assays, cross-reactions in the diagnosis, the influence of climate change on the disease and the vector, mechanisms of transmission of the disease, new drugs and plant extracts with antiviral activity, the dengue vaccine, the results of immunizations, etc. This information gives a concrete idea of the advances and challenges against this disease.
- dengue virus
Dengue is a single systematic and dynamic disease that includes severe and non-severe clinical manifestations . It is caused by any of the four dengue virus serotypes and it is transmitted by
2. Dissemination of the disease in urban areas and transmission mechanisms
Dengue is the most important and common arbovirus in more than 100 countries . This disease is caused by one of the four serotypes of dengue virus (DENV), more than one dengue serotype can be found in many geographical areas (phenomenon called hyperendemicity) . Outbreaks of this disease have been reported in America, Africa, Mid-west, Asia and the Pacific islands . Nearly 3 billion people (40% of world population) are at risk in areas where dengue occurs, and about 70% of the population at risk are in South East Asia and Western Pacific region . About 400 million people are infected with dengue annually, of which 100 million people are sick and 22,000 die of severe dengue . A virus, a vector and a sensitive population must be in the same geographic area for a dengue onset outbreak . Travelers contribute to dengue dissemination to non-endemic areas but they may also serve as sentinels for warning dissemination. Some studies indicate that dengue represents about 2% of the diseases from travelers returning to from endemic areas . Based on GeoSentinel, a data-collection network between 1997 and 2006, dengue was imported from South-east Asia (51%), South Central Asia (17%), Latin America (15%), the Caribbean (9%), parts of Africa (5%) and Oceania (2%) . The number of febrile travelers returning from the tropics and sub-tropical areas being diagnosed with dengue has increased from 2% in the 1990s to 16% by 2005 . A study of paedriatic travelers in 19 countries reported dengue and typhoid fever as the most often febrile diseases in children returning from tropical regions and sub-Saharan Africa .
3. Dengue virus serotypes and genotypes and its relation to clinical cases
The evolution of molecular biology and phylogenetic analysis has enabled molecular epidemiology studies binding DENV-phylogenetic analysis which was found in different geographic areas within a certain period of time and clinical and epidemiological data in order to establish a relation to genotypes and lineages which are found with other circulating strains as well as to identify the origin and the transmission route, the severity of the disease, etc. There are four related DENV serotypes but antigenically different DENV-1, DENV-2, DENV-3, DENV-4 and each of them generates an immune response to the infection. The nucleotide sequences show 63–68% homology within the DENV group in comparison with 44% between DENV and Yellow Fever virus (YFV), and 51% between DENV and West Nile virus (WNV), and it shares a minor homology at 80% at a level of amino acids . Previous studies based on partial (prM/E), partial or complete E gene; or complete genomic sequences recognized distinct DENV genotypes  (Table 1).
|Dengue serotype||Genotype||Geographical distribution|
|DENV-1||I||Southeast Asia, China, The Middle East|
|IV||Countries of the Pacific Rim, the Western Pacific, islands and Australia|
|V||The Americas, West Africa and Asia|
|DENV-2||Asian I||Thailand, Malaysia, Cambodia, Myanmar, Vietnam and Australia|
|Asian II||China, Indonesia, The Philippines, Taiwan, Sri Lanka, India, Honduras and Mexico|
|Southeast Asian/American||Southeast Asia, Central and South America and the Caribbean|
|Cosmopolitan||East and West Africa, the Middle East, the Indian subcontinent, Indian and Pacific Ocean Islands, Australia, Mexico|
|American||Central and South America, the Caribbean and the Indian subcontinent and the Pacific Islands|
|Sylvatic||West Africa and Southeast Asia|
|DENV-3||I||Southeast Asia, the Philippines and the South Pacific islands|
|II||Continental Southeast Asia|
|III||Asia, East Africa, the Americas|
|IV||Puerto Rico and Tahiti|
|DENV-4||I||The Philippines, Thailand, Vietnam, Myanmar, Malaysia, Sri Lanka, India|
|II||Southeast Asia (Indonesia, Malaysia, Singapore), China, islands of the Western Pacific Ocean, Australia, the Caribbean and the Americas|
The genotypes may present different lineages or clades. The genetic changes in the virus are caused by mutations or the introduction of a new variant from other region, thus genotyping of strain virus is used to identify the epidemic outbreak source and spread . Some dengue genotypes are related to a higher virulence. DENV-2 and DENV-3 Asian genotypes are associated with severe infections . In South East Asia, dengue hemorrhagic fever (DHF) and/or dengue shock syndrome (DSS) outbreaks are caused by DENV-2 southeast Asian genotype strain, meanwhile epidemics caused by DENV-2 in Latin America presents solely dengue fever (DF) in most cases. The DENV-2 genotypes circulating in the United States seem to be less virulent. Because of the introduction of the South East Asian genotype virulent, the number of cases of DHF and DSS in America has increased [1, 11].
It is thought that an infection with DENV serotype provides lifelong immunity against the same serotype infection and short-term protection against infection by a 2 or 3 month-heterologous serotype [1, 11]. Some studies contradict the hypothesis that has been accepted until now. In 2010–2011 a study in DENV-2 American/Asian genotype outbreak was conducted in Iquitos, Peru. It was carried out 15 years after the first DENV-2 American genotype in that region. The results on the study showed that protection against homologous DENV-2 may be incomplete . In another study carried out in Peru, it was found that the antibodies of a primary infection against DENV-1 neutralized more efficiently in American DENV-2 than in Asian DENV-2. It is believed that this cross-protective immunity is not so strong to inhibit viremia but it may contribute to reduce DENV-2 infection symptomatology . In 2013 evidences of a new dengue serotype appearance (DENV-5) that was identified in serum samples collected in 2007 during an epidemic in Malaysia were documented. Initially, it was thought that DENV-4 cases will circulate between primates and
4. Factors that increase dengue severity, cases and co-infection classification
Most of dengue infections are subclinical or asymptomatic. Dengue epidemics were presented in Cuba in 1981 and 1987, and most cases of dengue shock and hemorrhagic dengue were mainly found in white population than in afro-descendants . In studies carried out in Brazil and El Salvador, it was found that the African descent was a protective factor in dengue hemorrhagic manifestations . In a dengue outbreak in Santiago de Cuba in 1997, it was found that hemorrhagic dengue cases were reported more frequently in patients aged between 15 and 39 years old. Additionally, they found a history of asthma in a 16.5% of the cases . The WHO estimates that, by 2030, the diabetes mellitus will have been the 7th leading cause of death. A study carried out for evaluating the influence of diabetes mellitus and its relation to clinical manifestations of dengue, indicated higher risks of dengue symptoms potentially fatal within patients developing diabetes mellitus . Other risk factors such as sickle-cell disease, uremia, allergies, hypertension, chronic renal failure may enable disease severity . Regarding age, the lower compensation capacity of capillary plasma extravasation in children increases dengue shock risk. It has been observed that serious dengue cases were continuously presented in primary infections from breastfeeding babies whose mothers were developing immunity to some dengue serotype. The non-neutralizing antibodies produced by cross reaction during a primary infection or passively obtained from a mother to newborns are adhered to epitopes of dengue virus infections facilitating the entry of cells to Fc-receptors. This may contribute to a person’s viral load increase resulting in strong immune response that includes inflammatory mediators . Cytosines may enable plasma extravasation. In secondary infections, memory T-cells caused by previous reactions to dengue activate, replicate, produce cytosines and die of apoptosis. This can be correlated with disease severity . The patient’s background is important when ruling out other causes of this disease, for instance, other flavivirus presence like YFV, Saint Louis encephalitis virus (SLEV), Zika virus (ZIKV), WNV. Alphavirus like Chikungunya (CHIKV), Mayaro virus (MAYV), Venezuelan Equine Encephalitis virus (VEE), Bunyavirus like Oropouche virus (OROV), group C virus, Guaroa virus (GROV), Influenza virus, arenavirus, filovirus as well as other microorganisms can cause the disease with symptoms with similar symptoms such as malaria, Leptospirosis, typhoid fever, rickettsia diseases, etc. . Dengue infections with different serotypes as well as the sequence of these infected patients may influence on the severity of the disease. In a study carried out in Singapore, between 2005 and 2011, febrile adult patients found in DENV-1 cases that were associated with dengue hemorrhagic fever (adjusted RR = 1.74) and severe dengue (adjusted RR = 2.1) were assessed, while DENV-2 had a low risk of dengue hemorrhagic fever (adjusted RR = 0.5) . A special attention should be given to the clinical features of this disease in order to learn differences that lead to the identification of microorganisms causing the disease. It was found that there were no elevated hematocrit or shock cases in CHIKV infection as compared to dengue cases with high hematocrit in 40–69% of cases and shock in 10–39% of cases when comparing the clinical data and laboratory features within dengue infections and chikungunya . The arthralgias were more frequent in CHIKV in 70–100% of cases as compared to at least 10% of dengue cases. The Zika infections may present Oedema in limbs as compared to dengue and chikungunya which is low or nil. The presence of this type of differences does not exclude atypical complications and presentations of the disease, thus the laboratory diagnosis plays a crucial role .
DENV, ZIKV and CHIKV are transmitted by
In 1970, dengue disease was classified as non-classical dengue fever, classical dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Sometime after this, various studies reported lack of correlation between classification and disease severity . This classification showed a high specificity in the identification of hemorrhagic dengue; however, its sensitivity was low when detecting severe dengue cases requiring medical care and/or hospitalization. Thus, the WHO proposed a new classification according to the level of clinical severity by dividing dengue disease in two categories, severe dengue and non-severe dengue in 2008 . This classification makes possible to establish the management and improve notifications for a better epidemiological surveillance so that warning signs and early warning of severe cases requiring hospitalization are proposed . Regarding a study conducted to compare both classifications, it was found that the sensitivity of the new classification to detect severe cases was 65%, and the old classification was 30%. The new classification had 72% of sensitivity to detect patients needing advanced medical services and the old classification only had 32%. Further studies alike indicate that the WHO classification in 2009 has brought benefits in epidemiology and clinical use, some others have proposed to include other variables on severe dengue categories in order to increase sensitivity in a case detection that may require advanced medical care .
5. Dengue vectors and mechanisms of infection
Dengue virus is mainly transmitted by
6. Dengue virus jungle cycle and reverse zoonosis
DENV can circulate in jungle cycles where virus can remain in place using
7. Problems of dengue diagnosis
A series of multiple variant testing have been carried out for dengue diagnosis depending on a person’s infection phase, these tests are conducted with different immunological targets for virus recognition, a part of its structure or a reaction in an infected person or an animal’s body indicating an exposure to DENV  (Table 2).
|Diagnostic test||Immunological target|
|Polymerase chain reaction (PCR)||RNA detection|
|Rapid tests||NS1, IgM, IgG|
|Immunofluorescence (IF)||Virus, IgM, IgG|
|Plaque assay (PA) and fluorescent focus assay (FFA)||Virus (titer)|
|Enzyme-linked immunosorbent assay (ELISAS)||NS1, IgM, IgG, IgA|
|Neutralization tests||Neutralizing antibodies (IgG)|
In a dengue study, diagnostic tests display numerous problems which we will describe briefly. The presence of false positive as a result of contamination and different variants of RT-PCR such as endpoint PCR, nested PCR, real-time PCR as well as the use of different primers and enzymes lead to variations in levels of sensitivity and specificity. Some commercial test enables the identification of different microorganisms by utilizing RT-PCR, PCR tests and the detection of the product in about an hour. The RUO Film Array Global Fever Panel tests are utilized for the detection of 6 bacterias, 4 protozoans and 9 viruses like Chikungunya virus, Crimean-Congo hemorrhagic fever virus, Ebola virus, Lassa virus, Marburg virus, West Nile virus, Yellow fever virus, Zika virus and Dengue virus. Whole blood (EDTA) is used as a sample. The use of automation equipment can help to reduce contamination problems and deliver results in a short time as well as providing diagnosis for 19 pathogens. Regarding to improvements for this type of systems the equipment can only process one sample at a time, and the study is expensive. The rapid tests do not need sophisticated equipment or qualified staff, it is feasible in situ and they are inexpensive. Nonetheless, its sensitivity and specificity are not as higher as other techniques. Cell cultures are often used for dengue virus isolation. Many cell lines are used for virus isolation; however, their sensitivity can vary depending on the type of cell line, the clone, the system used for viral isolation and the sample type used for isolation [35, 36]. In order to isolate DENV, it is preferable to use C6/36 cell line obtained from
8. Animal models for dengue
It is not yet found a suitable animal model expressing all characteristics for dengue disease . Among the most common models used for research are mice showing drawbacks with low-level virus replication of clinical samples, and non-human primates showing a problem for not expressing the clinical disease in the same manner as humans . The use of nursing mice used in intracranial inoculations with high viral load inducing neurological diseases and paralysis are still been used; however, its use continues more for the production of biological supplies. DENV can slightly replicate in mice A/J, BALB/c and C57BL/6. Mice A/J and BALB/c can die of paralysis [39, 40]. For animal studies, it is considered that an infection with DENV 104–106 PFU (plaque-forming units) concentrations imitate inoculum concentrations of a mosquito bite . The mouse model is generally used initially to assess vaccines being the most suitable the immunocompetent mice but when it exhibits low viremia may result in the underestimation of the results. The use of nursing mice is a good way to assess possible candidate vaccines inducing neurovirulence. Although the NHP do not develop the disease, the antibody seroconversion produced is human-like [39, 40].
9. Antivirals for treating dengue
At the moment, there is no antiviral therapy for DENV treatment. It is believed that the compounds working as inhibitors of RNA-dependent RNA polymerase have shown low efficiency. Moreover, there is a possibility that viral epitopes of DENV may trigger an immune cell response, preceded by development of severe disease. Thus, these epitopes are examined as targets for antiviral productions and they are known as DENV entry inhibitors and can be used in combination with inhibitors of the virus replication in order to increase efficiency.
Inhibition of DENV attachment and entry into the host cell can inhibit immune activation.
Various compounds as peptide entry inhibitors, DN59 and 1OAN1 may inhibit the antibody-dependent enhancement (ADE) in vitro. The doxorubicin antibiotic SA-17 is structured similarly to tetracycline and it has an antiviral activity against DENV serotype 1, 2 and 3 in Vero and C6/36 cells interfering with viral entry by binding to the hydrophobic pocket of the E-protein without exhibiting virucidal activity. The glycosidase inhibitors are unpopular due to its toxicity and low specificity but may help understand E-protein glycosylation processes. The binding agents to carbohydrates occur only during the stage of virus adsorption to the host cell. Concanavalin and agglutinins of wheat germ can bind N-acetylglucosamine residues and the percentage of DENV-lytic plaques in BHK cells can be reduced. Plant lectins such as
10. Dengue vaccine
In May 2019, the U.S. Food and Drug Administration (FDA) approved the use of Dengvaxia®, a vaccine against DENV that may be used in people aged from 9 to 45 years old . This vaccine utilizes a live attenuated chimeric yellow fever/dengue virus based on a Yellow fever 17D vaccine virus backbone chimerized with prM and E proteins from DENV1–4 replacing the YF prM and E, and 3 doses are administered every 6 months. In phase III trials the overall protective efficacy was 56.7% and 60.8% in South East Asia and Latin America countries, respectively. In 2017, the vaccine manufacturer, Sanofi Pasteur, announced that some people getting the vaccine without having been previously infected by DENV may be at risk for developing severe dengue if the disease is acquired after immunization . The vaccine candidates utilize strategies such as vaccines of Live-attenuated virus, Inactivated virus, Recombinant protein, DNA vaccine, Viral vector vaccine, Virus like particles and others. Two vaccine candidates (DENVax and TV003/TV005) are being tested in efficacy trials in both Asia and Latin America. DENVax is a tetravalent recombinant live-attenuated dengue vaccine licensed to Takeda. The live virus vaccine utilizes chimerization with DENV-2 PDK-53 as the backbone with DENV-2/-1, -2/-3, and -2/-4 chimeras are created by replacing the DENV-2 prM and E genes with the respective genes from the other DENV serotypes. Other vaccine candidate is TV003/TV00 with a whole attenuated virus for three of four serotypes (attenuated by deletion of 30 nucleotides from 3′ UTR of DENV-1, DENV-3, DENV-4, and a chimeric DENV-2/DENV-4) . In spite of having already a vaccine available to prevent dengue, there is a need to further research in order to improve the vaccine for dengue or to produce a new vaccine which enables improvements in certain aspects, for example, a single dose that may immunize for extended periods of time even lifelong, the vaccine effectiveness no longer relying on previous exposure to flavivirus or DENV, the age of group to provide protection being the broadest possible including children under 9 years old, and its administration decreasing the chances of getting the most aggressive dengue disease at no point post vaccination.
Dengue is still a major public health concern worldwide. Several natural and social factors have contributed to the number of cases increased in recent decades. The efforts attained in the search of new antivirals and vaccines, and prevention and control strategies for this disease have not proved sufficient. However, the science and knowledge development acquired up to now provide us the tools we need to, and mark the way to follow, in order to achieve a control of this disease.
Conflict of interest
The authors declare no conflict of interest.
The views expressed in this chapter are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government.
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