**1. Introduction**

80 Current Topics in Tropical Medicine

Sterling TR, Thompson D, Stanley RL, McElroy PD, Madison A et al. 2000. A multi-state

Wilson GJ, Carter SP, Delahay RJ. 2011. Advances and prospects for management of TB transmission between badgers and cattle. *Vet. Microbiol.* 151(1-2):43-50 World Health Organization. 2011. *2011 Global Tuberculosis Control,* Geneva: WHO.

Zhao Y, Levina E, Zhu J. 2011. Community extraction for social networks. *Proc. Natl. Acad.* 

41(5):759-60

*Sci U. S. A* 108(18):7321-6

outbreak of tuberculosis among members of a highly mobile social network: implications for tuberculosis elimination. *Int. J Tuberc. Lung Dis.* 4(11):1066-73 Szell M, Lambiotte R, Thurner S. 2010. Multirelational organization of large-scale social networks in an online world. *Proc. Natl. Acad. Sci U. S. A* 107(31):13636-41 Vargas J, Gamboa C, Negrin D, Correa M, Sandoval C et al. 2005. Disseminated

Mycobacterium mucogenicum infection in a patient with idiopathic CD4+ T lymphocytopenia manifesting as fever of unknown origin. *Clin. Infect Dis.*

> The term dengue, of Spanish origin, was used to describe joint pain from an illness that attacked the British during the epidemic that affected the Spanish West Indies from 1927- 1928. Dengue was brought to the American continent to the Old World during the colonization in the late eighteenth century. However, it is not possible to say according to the historical record if the outbreaks were caused by dengue virus, as its symptoms are similar to those of several other infections, especially yellow fever (Holmes et al., 1998). The etiology of dengue has been credited to the miasma theory, bacterial or protozoan infection and finally to an ultramicroscopic agent. Similarly, transmission has been considered by respiratory airway and finally, by mosquitoes.

> The isolation of dengue virus (DENV) occurred in the 1940s during the epidemics of Nagasaki (1943) and Osaka (1944). The first known strain is coined DENV Mochizuki (Kimura & Hotta, 1944). In 1945, the Hawaii strain was isolated, and that same year other DENVs showing antigenic characteristics of different serotypes were isolated in New Guinea. The first two strains were designated serotype 1 and serotype 2. In 1956, other strains designated as serotype 3 and 4 were isolated. Thus, four dengue serotypes are known to date: DENV-1, DENV-2, DENV-3 and DENV-4 (Martinez Torres, 1990).

> Genetic variation within each serotype was first demonstrated by serological techniques. Subsequently analysis of the viral genome showed that DENV-1and DENV-2 can be classified as having five genotypes or subtypes each while DENV-3 and DENV-4having four and two respectively (Rico-Hesse, 1990, Lanciotti et al., 1994). Recently, Rico-Hesse et al (2003) reviewed the classification of DENV genotypes by the analysis and comparison of the nucleotide sequence of the complete E gene of various strains. As a result it was defined thatDENV-2 and DENV-3 have four genotypes while DENV-1 and DENV-4 have five and three genotypes respectively (Cunha & Nogueira, 2005).

#### **1.1 Etiology**

Dengue Fever, a vector-borne disease, is the most important arboviral disease worldwide. Dengue viruses (DENVs) belong to the genus *Flavivirus*, family *Flaviviridae*. These are singlestranded positive-sense RNA viruses. DENV are grouped into four antigenically related but distinct serotypes named DENV-1, 2, 3 and 4.The four serotypes of DENV are diverse and

Molecular Characterization of Dengue Virus

Vista - Roraima in 1981-1982 (Osanai, 1984).

3/DENV-4 co-infection are shown in Figure 1.

Bahia (Figueiredo et al., 2003).

Circulating in Manaus, the Capital City of the State of Amazonas, Brazil 83

DENV-1, DENV-2 and DENV-3 were common in most Brazilian cities. The DENV-4, cocirculating with DENV-1,was detected during the first epidemic reported in Brazil in Boa

In early 1998, the Laboratory of Arbovirology at the Foundation for Tropical Medicine Dr. Hector Vieira Gold (FMT/HVD) in Manaus in the state of Amazonas, started a program of monitoring and diagnosing of viral diseases transmitted by arthropods (arboviruses) to determine their etiological agents. The diagnosis was made by serologic studies using the MAC-ELISA test for detection of IgM antibodies. Sera samples from 8557 patients suspected of dengue were analyzed and 40% of the sera were ELISA positive for dengue virus. The DENV-1 was considered responsible for this epidemic (Figueiredo et al., 2004). In 2001, DENV-1 and DENV-2 were isolated in the State of Amazons and dengue hemorrhagic fever cases were registered (Figueiredo et al., 2002). In 2003, DENV-3 was isolated for the first time in the state of Amazonas from a patient coming from the state of

A variety of acute febrile diseases with and without hemorrhagic manifestations were diagnosed as dengue in Manaus in March 1998 by detection of specific IgM antibodies. Mayaro (MAYV) and *Oropouche* (OROV) viruses were also diagnosed. Infections with

In 2008, DENV-4 was first identified in Manaus (Figueiredo et al., 2008). The Amazon is situated in the Northern Region of Brazil, bordered to the north with the State of Roraima, Venezuela and Colombia. Serotype DENV-4 was endemic to Venezuela and Colombia and this may have influenced the detection of this serotype in the Amazon due to its proximity to these countries (Figueiredo, 2008). The four serotypes of dengue and cases of DENV-

Fig. 1. Agarose gel electrophoresis. Lanes 1,3, 13, 16: DENV-4; Lane 4,7 and 10: DENV-3/DENV-4; Lane 6: DENV-2, Lane 8: DENV-1; Lane 9: DNA size standards 100Kb;

Nucleotide sequence analysis of DENV-4 present in the State of Amazonas showed to be of genotype I which is only present in the Asian continent and never described in the

rubella and parvovirus were also observed (Figueiredo et al., 2004).

not phylogenetically related but strongly related to flavivirus transmitted by mosquitoes. It is reported that these viruses have emerged about 1000 years ago from a monkey virus and its transmission to humans occurred in the last 320 years. Some studies indicated its origin in Africa and others in Asia (Weaver et al., 2004).

#### **1.2 Clinical characteristics**

Dengue has two main clinical forms: classic dengue fever (DF) and dengue hemorrhagic fever (DHF) with or without shock (PAN AMERICAN HEALTH ORGANIZATION, 1994). The symptoms of DF are headache, retro-orbital pain, breaking bones sensation, muscles or joints pain, rash and leucopenia. Dengue hemorrhagic fever is characterized by high fever, hemorrhagic phenomena often with hepatomegaly and, in severe cases, signs of circulatory failure. Patients with DHF may develop hypovolemic shock resulting from plasma leakage. This clinical manifestation is called dengue shock syndrome (DSS) and can be fatal (WORLD HEALTH ORGANIZATION, 2001).

#### **1.3 Transmission**

The dengue virus is transmitted to humans through the bite of hematophagous Diptera, the mosquito *Aedes aegypti*. In the Americas, *A. aegypti* is one of the most efficient vectors of Arboviruses and is highly anthropophilic thriving in close proximity to humans and adapts very well indoors, generally in humid environment (WORLD HEALTH ORGANIZATION, 2001).

Once contracted the virus, the mosquito remains infected during its entire life and may transmit the virus to individuals during blood meals. The infected *A.aegypti* females may also transmit the virus to the next generation of mosquitoes by transovarial. Although very seldom, this means of transmission is of great epidemiological significance demonstrating that the vector play an important role in the persistence of the virus in the environment and act as reservoirs (WORLD HEALTH ORGANIZATION, 2001; Castro et al., 2004; Joshi et al., 2006).

Human beings are the main host and the virus replicates in the blood stream. Uninfected mosquitoes can contract the virus during blood meals from an infected individual. The virus multiplies in the cells of the mosquito during a period of 8 to 10 days. After this period, the vector is able to transmit the virus to humans again. In humans, the incubation period of dengue fever ranges from 2 to 7 days. Laboratory experiments showed that the mosquito *A. aegypti* may be infected simultaneously by different arboviruses and is also capable of transmitting them simultaneously (Araújo et al., 2006). According Wenming et al. (2005) it is possible that mosquitoes infected with DENV-2 and DENV-3 can transmit both in areas where two or more serotypes circulate. The poor environmental conditions of urban centers, the humidity and temperature as in Brazil associated with resistance of eggs of *A. aegypti* for long periods of desiccation favor the proliferation of mosquitoes and contribute to the spread of DENV. Dengue is currently considered the most important arbovirus and is a public health problem in tropical and subtropical countries (Guzman et al. 2006; WORLD HEALTH ORGANIZATION, 2001).

#### **1.4 Dengue in Manaus**

Infection by dengue virus in Brazil has increased significantly over the last decade, particularly after 1994, as a consequence of the spread of *A.aegypti*. The following serotypes,

not phylogenetically related but strongly related to flavivirus transmitted by mosquitoes. It is reported that these viruses have emerged about 1000 years ago from a monkey virus and its transmission to humans occurred in the last 320 years. Some studies indicated its origin

Dengue has two main clinical forms: classic dengue fever (DF) and dengue hemorrhagic fever (DHF) with or without shock (PAN AMERICAN HEALTH ORGANIZATION, 1994). The symptoms of DF are headache, retro-orbital pain, breaking bones sensation, muscles or joints pain, rash and leucopenia. Dengue hemorrhagic fever is characterized by high fever, hemorrhagic phenomena often with hepatomegaly and, in severe cases, signs of circulatory failure. Patients with DHF may develop hypovolemic shock resulting from plasma leakage. This clinical manifestation is called dengue shock syndrome (DSS) and can be fatal (WORLD

The dengue virus is transmitted to humans through the bite of hematophagous Diptera, the mosquito *Aedes aegypti*. In the Americas, *A. aegypti* is one of the most efficient vectors of Arboviruses and is highly anthropophilic thriving in close proximity to humans and adapts very well indoors, generally in humid environment (WORLD HEALTH

Once contracted the virus, the mosquito remains infected during its entire life and may transmit the virus to individuals during blood meals. The infected *A.aegypti* females may also transmit the virus to the next generation of mosquitoes by transovarial. Although very seldom, this means of transmission is of great epidemiological significance demonstrating that the vector play an important role in the persistence of the virus in the environment and act as reservoirs (WORLD HEALTH ORGANIZATION, 2001; Castro et al., 2004; Joshi et al., 2006). Human beings are the main host and the virus replicates in the blood stream. Uninfected mosquitoes can contract the virus during blood meals from an infected individual. The virus multiplies in the cells of the mosquito during a period of 8 to 10 days. After this period, the vector is able to transmit the virus to humans again. In humans, the incubation period of dengue fever ranges from 2 to 7 days. Laboratory experiments showed that the mosquito *A. aegypti* may be infected simultaneously by different arboviruses and is also capable of transmitting them simultaneously (Araújo et al., 2006). According Wenming et al. (2005) it is possible that mosquitoes infected with DENV-2 and DENV-3 can transmit both in areas where two or more serotypes circulate. The poor environmental conditions of urban centers, the humidity and temperature as in Brazil associated with resistance of eggs of *A. aegypti* for long periods of desiccation favor the proliferation of mosquitoes and contribute to the spread of DENV. Dengue is currently considered the most important arbovirus and is a public health problem in tropical and subtropical countries (Guzman et al. 2006; WORLD

Infection by dengue virus in Brazil has increased significantly over the last decade, particularly after 1994, as a consequence of the spread of *A.aegypti*. The following serotypes,

in Africa and others in Asia (Weaver et al., 2004).

**1.2 Clinical characteristics** 

HEALTH ORGANIZATION, 2001).

HEALTH ORGANIZATION, 2001).

**1.4 Dengue in Manaus** 

**1.3 Transmission** 

ORGANIZATION, 2001).

DENV-1, DENV-2 and DENV-3 were common in most Brazilian cities. The DENV-4, cocirculating with DENV-1,was detected during the first epidemic reported in Brazil in Boa Vista - Roraima in 1981-1982 (Osanai, 1984).

In early 1998, the Laboratory of Arbovirology at the Foundation for Tropical Medicine Dr. Hector Vieira Gold (FMT/HVD) in Manaus in the state of Amazonas, started a program of monitoring and diagnosing of viral diseases transmitted by arthropods (arboviruses) to determine their etiological agents. The diagnosis was made by serologic studies using the MAC-ELISA test for detection of IgM antibodies. Sera samples from 8557 patients suspected of dengue were analyzed and 40% of the sera were ELISA positive for dengue virus. The DENV-1 was considered responsible for this epidemic (Figueiredo et al., 2004). In 2001, DENV-1 and DENV-2 were isolated in the State of Amazons and dengue hemorrhagic fever cases were registered (Figueiredo et al., 2002). In 2003, DENV-3 was isolated for the first time in the state of Amazonas from a patient coming from the state of Bahia (Figueiredo et al., 2003).

A variety of acute febrile diseases with and without hemorrhagic manifestations were diagnosed as dengue in Manaus in March 1998 by detection of specific IgM antibodies. Mayaro (MAYV) and *Oropouche* (OROV) viruses were also diagnosed. Infections with rubella and parvovirus were also observed (Figueiredo et al., 2004).

In 2008, DENV-4 was first identified in Manaus (Figueiredo et al., 2008). The Amazon is situated in the Northern Region of Brazil, bordered to the north with the State of Roraima, Venezuela and Colombia. Serotype DENV-4 was endemic to Venezuela and Colombia and this may have influenced the detection of this serotype in the Amazon due to its proximity to these countries (Figueiredo, 2008). The four serotypes of dengue and cases of DENV-3/DENV-4 co-infection are shown in Figure 1.

Fig. 1. Agarose gel electrophoresis. Lanes 1,3, 13, 16: DENV-4; Lane 4,7 and 10: DENV-3/DENV-4; Lane 6: DENV-2, Lane 8: DENV-1; Lane 9: DNA size standards 100Kb;

Nucleotide sequence analysis of DENV-4 present in the State of Amazonas showed to be of genotype I which is only present in the Asian continent and never described in the

Molecular Characterization of Dengue Virus

contained in serum (Kuno et al., 1987).

reached (WORLD HEALTH ORGANIZATION, 2001).

technique is used for the identification of serotypes of dengue.

ORGANIZATION, 2001).

**2.4 Virological diagnosis** 

**3. Discussion** 

Circulating in Manaus, the Capital City of the State of Amazonas, Brazil 85

DENV-2 and DENV-3), the presence of specific IgM antibodies to dengue in the patient serum is shown by color change of the substrate that undergo enzymatic action of the conjugate. The color intensity is directly proportional to the amount of IgM antibodies

**2.3 Polymerase chain reaction (PCR) coupled with reverse transcription(RT-PCR)**  The technique of PCR can be used to detect the presence of the DENV. Viral RNA is extracted using commercial kits. The RNA is reverse transcribed to cDNA in a first step. Then specific primers for DENV nucleotide sequence are used for the amplification of targeted sequence so as to detect a small amount of RNA molecules of DENV. This method is fast and rapid compared to cell culture. Reverse transcription and PCR technique was shown for the first time as a powerful technique for the detection of the DENV during convalescence, when the antibodies would limit its detection (WORLD HEALTH

However, this method should still be considered an experimental approach. Is implementation on a large scale awaits further experiments. Moreover, a consensus on the adequate preparation of samples and for determining the sequences of bases in oligonucleotides capable of detecting all or most circulating genotypes of dengue need to be

The technique used to isolate DENV is continuous cell lines of mosquito cells C6/36 (*Aedes albopictus* clone) (Igarashi, 1978), grown in 25 cm flask with growth medium L-15 plus 5% of fetal bovine serum. To 1.5 mL of theL-15 plus 5% of fetal bovine serum containing the C6/36 cells in disposable falcon tubes (15 mL), 70 µl of serum from patients suspected of DF in acute phase was added and incubated at 28° C for two week. Culture media is changed two times per week and incubated. After 10 days of incubation, immunofluorescence (IF)

In Brazil, first cases of DHF occurred after the introduction of DENV-2 in the State of Rio de Janeiro. The cases of DHF by DENV-2 occurred after an epidemic by DENV-1 in Rio de Janeiro four years ago (Dias et al. 1991; Zagne et al., 1994). The same pattern was seen in Cuba during the 1981 epidemic with sequential infection by two serotypes (DENV-1 and DENV-2), and an interval of six months to five years or so (Kouri et al., 1986).The dynamics of epidemic of dengue in the Amazon is similar to that of other regions of Brazil and America. In Manaus – Amazonas, the first cases of dengue were registered in March 1998 by serological studies during the first epidemic of dengue (Figueiredo et al., 2004). In 2001, it was possible to identify the DENV-1 as the causative agent of that epidemic (Figueiredo et al., 2002). That same year, cases of dengue hemorrhagic fever were registered in the State of Amazonas, with the viral isolation of DENV-2 (Figueiredo et al., 2002). In 2002, DENV3 was first isolated from a patient coming from Bahia. Since then, DENV-3 was diagnosed by viral isolation from several patients with DF (Figueiredo et al., 2003).The virus DENV-4 was detected during the first epidemic reported in Brazil in Boa Vista-Roraima in 1981-1982. At

Americas (De Melo et al., 2009). The introduction of this virus in Manaus is probably due to the fact that this is a city with eco-tourism development, and possess an Industrial Center (Free Zone of Manaus), with over 450 factories of large, medium and small size (PORTAL AMAZÔNIA) and many of them are of Asian origin (Figueiredo, 2008). It is possible that the virus has been introduced from an Asian-infected visitor or vector *A.aegypti*-infected.
