Dengue Epidemic Is a Global Recurrent Crisis: Review of the Literature

Purpose: This review highlights the global scenario of dengue outbreaks, dengue pathogenesis, symptoms, immune response, diagnosis methods and preventive measures which facilitates the better understanding of the global expansion and concerns relating to the disease. Recent Findings: A recent study showed that natural killer cells of the infected person become activated soon after the infection which may help in treatment and vaccine development. A research team has also produced synthetically engineered mosquitoes that can prevent the transmission and dissemination of the dengue virus by the activation of an antibody. Furthermore, a mutation in the protein envelope of the dengue virus leads to variation in shapes, developing resistance towards the vaccine. Summary: The mosquito vectors marked their worldwide distribution through an increasing number of reported cases which was further facilitated by the growth in the shipping and commerce industries. The immune system, through activation of the innate and adaptive immune responses, facilitates the recruitment of an array of leukocytes which help neutralize the virus. Apart from the laboratory standard PRNT method, several other dengue detection methods such as ELISA, RT-LAMP and several optical, microfluidic and electrochemical methods have been developed. The existence of the 4 different viral serotypes makes the secondary infection life-threatening and also leads to difficulties in vaccine development. Since Dengvaxia® (CYD-TDV) has its own set of drawbacks and limitations, several companies have been investing for the production of more potential vaccines that are currently in trial.

The primary vector of dengue virus, Aedes aegypti from the tropical and subtropical regions of the world are mostly associated with the space occupied by humans for living and feeds on human blood [18]. Female Aedes aegypti requires blood before laying eggs. Moreover, Aedes albopictus which is an exophilic mosquito even prefers feeding on human blood. Therefore, their strong preference for human blood in order to support their survival makes them an ideal vector for DENV virus transmission [19]. The transmission of dengue virus by a vector begins as a mosquito bites an infected person, carrying dengue virus in his or her blood. The mosquito eventually gets infected and hence becomes a dengue vector which can prove to be a threat to the population since it can horizontally transmit this virus for the rest of its lifespan (two weeks to one month). Although early studies have shown no clear evidence of vertical transmission of DENV virus in Aedes mosquitoes, recent studies prove that vertical transmission of DENV virus is attainable both experimentally as well as in nature. In addition to that, some evidence affirms that Aedes albopictus are much more efficient than Aedes aegypti in terms of vertical transmission [20]. A case of vertical transmission of dengue virus was reported in Guangzhou, China. A 25 years old woman, 39 weeks pregnant was admitted in a general hospital. She experienced fever for 5 days and then gradually developed skin rash. She went into labour and delivered a girl. Results from ELISA dengue virus NS1 antigen test (Wantai, Beijing, China) and dengue virus fluorogenic quantitative PCR test (Liferiver, Shanghai, China) confirmed that both the mother and the baby suffered from dengue [21]. This indicates that vertical transmission can be an alternative mode of dengue transmission.

Entry of Virus inside Cell and Replication
Once the mosquito feeds on the blood of an infected person, the DENV develops an infection in the midgut from where it spreads and replicates in other tissues of the mosquito. It takes about 8 to 10 days and a temperature of about 25̊ C for the virus to multiply, mature, migrate and infect the salivary glands and eventually shed in the saliva [22]. While the female dengue vector bites and feeds on the blood of a healthy individual, it injects its saliva in order to prevent blood clotting of the host and facilitates feeding. Thus, infecting the individual by inoculating the virus into the dermis and epidermis as well as injecting some viruses directly into the bloodstream. It is then followed by infection in the most common cell type in the skin-keratinocyte [23]. It infects and replicates inside a specialized immune cell in the skin-Langerhans cell (a type of dendritic cell) [24]. The infection spreads to the lymph node thus activating the recruitment of leukocytes.
The replication of a flavivirus commences as it attaches to the extracellular surface receptor on the host cell (Figure 01). The virus is then taken up into the cell by endocytosis and is confined within an endosomal vacuole. Acidification of the endosome causes a change in the E-protein thus facilitating the release of the viral genome into the cytoplasm [25]. The viral genome can engage in any of the two fates-either the released genome can be transported to the ER in order to be translated in a polyprotein which undergoes post translational modification to produce structural and nonstructural protein for virus assembly and maturation. After making multiple copies of virions inside infected cells, they lyse the cells, get outside by endocytosis and continue to infect other healthy cells [26].  4.The viral RNA is translated into polyprotein in the ER. 5.After viral replication complex is synthesized, RNA synthesis starts. This takes place in two steps-positive mRNA is at first copied to negative sense mRNA and then this acts as template for synthesis of multiple stands of positive sense RNA which are then used for translation. 6.Translocation takes place. 7.Virus assembly takes place in the ER membrane. 8.Immature virus particles pass through the Golgi body where they undergo modifications. 9.Virions are released by endocytosis [27][28][29][30][31][32].
After 4 to 5 days of being bit by a dengue vector, the person develops viremia-a condition in which the level of dengue virus in blood tends to elevate due to replication of the viruses. It can last from 5 to 12 days. If a mosquito feeds on the blood of an infected individual within this time span, the mosquito becomes a dengue vector [33].
Some epidemiological studies show that the development and severity of dengue infection in humans is triggered by many factors. Age is one of them [34]. Other factors include genetic background of the host [35] [36], vector, serotype of virus [37], gender, genotype [38] [39], environmental condition, immune condition of the infected people, socioeconomic level of the population and secondary infection by heterologous serotype [40].

Immune Response of Human Body Against Dengue Virus
The immune system is the primary defense system of the body against dengue virus and consists of mainly two parts: The innate immune system and the adaptive immune system. While the innate immune response facilitates the immediate recognition and protection towards any invading pathogen, the adaptive immune response produces cells that specifically and efficiently target the pathogen or infected cell providing a long term immunity, unlike the innate immune system [41].
The cells produced by the adaptive immune system include the antibody-secreting B cells, which are capable of recognizing and binding to foreign cells with high specificity and the cytotoxic T cells which are known to attack the infected cells.
Since the keratinocytes and Langerhan cells are infected through viral replication, the Langerhans, through proper detection, display the antigens from the invading pathogens on their surfaces [42] - [45]. The display of the viral antigens triggers the innate immune response, summoning the white blood cells, monocytes and macrophages for the ingestion and destruction of the dengue virus.
The virus infects these cells instead and spreads throughout the entire body as they travel through the lymphatic system [45] [46]. As the virus spreads throughout, infecting cells of the bone marrow, lymph nodes, spleen, liver or blood, it facilitates the emergence of viremia.
While some of the infected Langerhans travel to the lymph nodes (small glands present throughout the body that are known to fight infections) to trigger the immune response, the remaining secrete proteins called interferons which disrupt the replication process of viruses through the activation of innate and adaptive immune system defenses (Figure 02) [24] [47]. They help in the recognition of The individual remains protected from the other three serotypes for about 2 to 3 months after recovery from the first dengue infection. However, this provides short term protection only and the usual observation is that a second dengue infection was much worse in individuals infected beforehand than those who were not infected earlier [46] [49]. Even though the memory B cells and memory T cells are normally known to provide protection through adaptive immune response when the virus strikes again, the mechanism fails in case of a second dengue attack. These observations were explained by Halstead through the 'antibody-dependent enhancement of infection' phenomenon which supports the idea that the antibodies present from the first dengue infection or the newly produced memory B cells facilitate the efficient viral attack of host cells instead of providing protection against the other three serotypes [49] - [54]. This happens since the antibodies from the first dengue infection form a complex with the newly attacking serotype which further binds to the Fcγ receptors (FcγR) present on circulating monocytes [55] - [59]. This results in severe dengue fever, also known as dengue hemorrhagic fever (DHF), since more cells are infected with the help of the pre-existing antibodies. This phenomenon is also active among children who received antibodies for dengue from their mothers while in the womb, putting them at a greater risk of developing severe dengue [60] [61]. Furthermore, it was also seen that the cytotoxic T cells secreted greater amounts of cytokines during the second dengue infection which are known to cause serious inflammations or tissue damage.

Symptoms and Clinical Presentation
Symptoms of mild dengue fever may not be visible among children however, if they do make an appearance, they begin 3 to 14 days after the initial infection [64]. Dengue fever (DF) results in a high-grade fever further leading to a number of other symptoms which include headache, retroorbital pain, muscle pain, vomiting and rash. The symptoms may become worse and life-threatening if the disease is moderate or severe [65] [66]. The symptomatic cases are classified into undifferentiated febrile illness (UF), dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and unusual dengue (UD) or expanded dengue syndrome (EDS) [67] [68].
Due to damaged blood vessels, blood plasma leakage and a decreased platelet count, the risk of acquiring dengue hemorrhagic fever (DHF) increases. The symptoms may also progress to massive bleeding, shock, and death; this is called dengue shock syndrome (DSS). The course of dengue infection takes off after the incubation period and is divided into 3 main phases: Febrile phase, critical phase and recovery phase (

Dengue as a Global Crisis
Dengue is an important arthropod borne viral disease that is known to affect an estimated 2.

Dengue in America
Dengue has been a major public health problem in America. A campaign by the Pan American Health Organization (PAHO) played a role in the eradication during the 1960s from the central and south American countries [111]. The discontinuation of the campaign in the 1970s further led to the reinfestation of the species. In the 1970s, Den-2 and Den-3 were present in America however, the major epidemic in 1977 was due to the Den-1 virus which had an epidemic period of 16 years. In 1981, Den-4 was introduced along with another strain of Den-2 from Southeast Asia which led to a major DHF epidemic and this new strain caused outbreaks in Venezuela, Puerto Rico, Columbia, Brazil and Suriname [112]. 14 countries in America had endemic DHF after its emergence in 1995.
The failed eradication program led to an increase in cases from 2000-2010 and therefore, over 1.7 million cases were reported, from which 50,235 were severe cases and 1,185 deaths. According to a recent report by WHO, the number of cases increased by 15 fold over the past two decades with an increase from 50,5430 cases in 2000 to 2,400,138 cases in 2010 [113]. According to a report from PAHO, America recorded the largest outbreak of dengue with about 3 million cases in the region till 2019 and the number of cases reported was 320,000 till 9 February 2020 [114].

Dengue in Australia
The

Dengue Virus Identification Techniques
The accurate and efficient diagnosis of dengue is significant in order to differentiate dengue from  (Table 02) [127].

Prevention and Treatment Strategy
The DENV is spread by a human-to-mosquito-to-human cycle by the Aedes mosquito [166] thus most strategies to prevent spread of the virus includes preventing the growth of mosquitoes and laying of eggs. The measures to be taken include proper disposal of solid waste, cleaning water storage containers every week and applying insecticides in those containers. Water storage tanks must be covered at all times and appropriate insecticides must be added to the water. Additionally, means of personal protection must also be used such as long-sleeved clothes, window screens, coils, vaporizers etc. During outbreaks, spraying insecticides inside the households and thorough monitoring must be carried out to prevent infection [113] [167] [168].
The life cycle of dengue virus involves the entry of the virus into our body, membrane fusion, RNA genome replication, assembly, and ultimate release from the infected cell. Thus, the treatment of dengue is not specific and remains to be supportive [169]. Supportive treatment includes oral fluid administration and antipyretic treatment with paracetamol along with daily full blood counts. If there is excessive vomiting or diarrhea observed, admission to hospital is necessary to avoid dehydration. The patient must be administered oral fluid as much as possible [170]. The symptoms of dengue are flu-like and so there is no specific treatment but an early diagnosis and clinical management can reduce the severity of the effects of dengue on the body [113] [171].
Currently, there is no licensed antiviral agent available against dengue as the drug needs to address all four serotypes of the dengue virus. Researchers have been trying to develop drugs by targeting several different steps in the disease pathway in order to interfere with the replication of dengue virus. For example, nucleoside analogue blocks a dengue infection by preventing synthesis of the viral RNA genome so that the dengue virus cannot replicate. Viral replication can be difficult to prevent because there is a short timeline for treatment which is why it is important to target other steps in the disease pathway to prevent dengue at later stages [172].

Vaccine for Dengue Virus
Till date, no effective direct vaccine has been discovered that can successfully combat the dengue infection. The vaccines produced must work against all four of the DENV serotypes and must provide lifelong protection. This may be done by incorporating an antigen that is common for all four serotypes. As vaccines with live, attenuated or nonliving viruses usually produce less antibody than an infection with wild-type virus, it is most likely that two doses of the virus will be required.
Several other factors need to be taken into account such as the level of symptoms seen after the vaccination, if the vaccine can be used by people of all ages and the cost of the vaccine [166] [173].
Providing lifelong protection against all four serotypes by neutralizing antibodies is difficult due to antibody-dependent enhancement (ADE) or immune enhancement. ADE observed during dengue infection due to heterologous non-neutralizing antibodies may cause dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) during secondary infection by a different serotype.
Additionally, over time, antibody responses below protective levels may increase the possibility of immune enhancement by a natural infection due to wild type DENV. Another major challenge of producing a dengue vaccine is the lack of an appropriate human like animal model for the trials of the vaccines which will allow us to observe the pathogenesis, immune response and clinical course of dengue infection in humans. Inoculation of mouse-adapted DENV strain has caused paralysis or death of that animal [174].
So far only one such vaccine for dengue has been approved, Dengvaxia® (CYD-TDV). Dengvaxia® (CYD-TDV) has been developed by Sanofi Pasteur and it is for people who have already been infected by DENV once. This works on people ages 9 to 45 and must be given three doses in intervals (113,167,175). However, a large number of vaccines are now in different phases of clinical trial in different companies (

Current Status and Future Prospects of Dengue Research
A new serotype of dengue virus known as DENV 5 was reported in October 2013 which is similar to DENV 2 and follows the sylvatic cycle rather than the human cycle that the other four serotypes follow. It has been found that DENV-5 constitutes a unique, distinct phylogenetic group, but this discovery requires further confirmation and even though this was thought to be a variant of serotype 4, several studies have shown that rhesus macaque monkeys infected with the serotype 5 produced a different set of antibodies. Developing a tetravalent dengue vaccine that is both cost effective and safe has been a challenge for scientists all over the world and several potential vaccines are being tested after Mahidol University, Thailand and Walter Reed Army Institute of Research (USA) initiated the research [190] [191]. Several other organizations such as Takeda Pharmaceuticals Company Limited, Bill & Melinda Gates Foundation, The Global Health Innovative Technology (GHIT) Fund and many more have invested millions towards dengue research [192 -195]. A study in Singapore confirmed that right after the infection, the immune cell called natural killer (NK) cells were activated in the blood and researchers hope that this knowledge may be beneficial while developing vaccines and drugs [196]. Moreover, another research team at University of California San Diego has synthetically engineered mosquitoes that will prevent the transmission of dengue virus by the activation of an antibody that prevents the replication of the virus and its dissemination [197]. Additionally, a study has shown that mutations in the protein envelope cause the dengue virus to change its shape and become resistant to vaccines and therapeutics [198].
Taking climate change into account for dengue research is important for the government and public health officials to take actions to protect the public from future dengue outbreaks. As the global climate changes, the global surface temperature will change and so will the patterns of rainfall around the world, affecting the environmental suitability for the survival and growth of dengue viruses and mosquitoes, which will eventually lead to the change in patterns of dengue globally, nationally, and locally. Having said that, several issues need to be taken into account during the research on DENV such as the sociodemographic factors e.g., travel and demographic change, and other climatic factors in some areas where temperature may not be the most vital aspect influencing the spread of dengue. Moreover, the non-climatic factors affecting the spread of A. aegypti and A.
albopictus must also be explored [199]. Scientists have been investigating dengue pathogenesis in order to gain a better understanding of dengue infection. Improving surveillance of dengue cases by databases such as DengueNet that continuously updates and shares current and historical data on dengue cases by providing early warnings prior to epidemics can help improve the preparedness of public health officials, and help reduce fatality rates [200]. Many experts have said that dengue may increase in the future due to viral evolution, climate change, globalization, travel and trade.
Settlement factors and socioeconomic factors also play a role in the spread of dengue. In light of this, it is important to follow the guidelines in WHO Global Strategy for Dengue Prevention and Control, 2012-2020 in order to prevent a huge outbreak as neither a perfect remedy nor a vaccine has been found yet [201].

Conclusion
Even though dengue is the most emerging global infection in the 21st century, the adaptation of a successfully planned control program can prove as a preventative measure. Apart from this, our body's immune system also plays a major role in the process of viral neutralization. Growth in population, frequent travels as well as urbanization has shown to affect the re-emergence of the dengue epidemic. The increasing dengue outbreak along with no specific existing treatment for the disease makes prevention our first priority. Some methods of prevention may include the use of insecticides, removal of mosquito vectors or the use of vaccines, mostly in the high burden epidemiologic areas. Rapid and more efficient dengue detection methods are required for an immediate approach towards treatment. As the factors contributing to the spreading of dengue virus are increasing with the rise in globalization and climate change, the research for dengue requires more attention in order to avoid another devastating outbreak.

Conflict of Interest
Authors declare no conflict of interest regarding the publication of the manuscript.