DETECTION OF DENGUE AND YELLOW FEVER FLAVIVARIIDS IN AEDES (DIPTERA: CULICIDAE) POPULATIONS IN ZARIA AND ENVIRONS

ABSTRACT

Dengue virus (DENV) and Yellow fever virus (YFV) are arboviruses transmitted by Aedes mosquitoes. The preponderance of Aedes mosquitoes in Zaria has been ascertained in some studies that reported on breeding of mosquitoes. However, the incidence of these viruses in the mosquito population is not known. This study investigated the incidence of the two arboviruses in the mosquito vectors in Zaria and its surroundings. Four locations were selected: Dumbi, Kufena, Hanwa and Bomo. Mosquitoes belonging to the genus Aedes were collected outdoor and indoor using mouth aspirators and pyrethroid-based insecticides knock-down method, from July to October 2013. Mosquitoes collected from the locations were pooled according to species, locations and whether they were collected outdoor or indoor. Viral RNA were extracted from the pools using RNA isolation kit (Shanghai ZJ Bio Technology). Real time reverse transcriptase polymerase chain reaction was used to detect dengue and yellow fever virus from the pools of mosquitoes. The target region for the four serotypes of dengue virus was the 3 UTR and the target region for yellow fever virus is the NS 5 gene. A total of 257 Aedes mosquitoes were collected. The result shows that Aedes vittatus(59.46%) was the most abundant species outdoor while Aedes aegypti(0.9%) was the least. Similarly, Aedes furcifer(50%) and Aedes aegypti(47.95%) were more preponderant indoor. Accordingly, Kufena (38.52%) had the highest collection of Aedes mosquitoes while Hanwa (17.12%) had the least collection. The fluorimeter channel (FAM) detected Ctvalues for 25 pool mosquito sample each for dengue and yellow fever virus. The Ct values ≤38 were positive as detected in the FAM. The Ct values show thatAedes vittatus from Dumbi, Hanwa and Kufena locations were negative for Dengue virus while Aedes furcifer, Aedes aegypti, Aedes taylori and Aedes metallicus were positive for Dengue virusin all the locations except in Kufena. Similarly, Aedes furcifer, Aedes aegypti, Aedes vittatus, Aedes taylori and Aedes metallicus were all positive for Yellow fever virus in all the locations. In all, 40%of the reaction samples were positive for Dengue virus while 60% were positive for Yellow fever virus from the four locations. Bomo recorded the highest incidence (60%) for Dengue virus detected in Aedes mosquitoes while Hanwa had the least record (10%). Similarly, Bomo had the highest incidence (33.33%) of Yellow fever virus while Dumbi and Hanwa each had the least incidence (20%). Based on the findings of this study, it is evident that dengue and yellow fever viruses are co-circulating in the Aedes population around Zaria and the mosquito vectors are active vectors in the four locations. In conclusion, this study has shown thatAedes furcifer and Aedes aegyptiare the most active vectors of Dengue and Yellow fever virus, whereas,Aedes vittatus is an active vector of Yellow fever in Kufena and Hanwa. This surveillance study has therefore brought to the fore, the urgent need for mosquito control in view of the emerging and re-emerging viral diseases.

CHAPTER ONE

1.0 INTRODUCTION

1.1       Background of the study

Arboviruses are important human and veterinary viruses that are maintained in nature through biological transmission between susceptible vertebrate hosts by blood feeding arthropods such as female mosquitoes.They are important emerging pathogens world-wide. Dengue, yellow fever, and Japanese encephalitis viruses,infect hundreds of millions of people and animals each year (Grubaugh, 2013). The feeding activities of the vectors have made them effective in viral transmission to vertebrate hosts (Grubaugh, 2013).Dengue virus (DENV) and Yellow fever virus (YFV) are enveloped arboviruses whose genome is composed of a single, positive polarity RNA molecule (Henchal and Putnak, 1990).They are single-stranded RNA viruses belonging to the Flaviviridae family, Genus Flavivirus. Dengue virus has been classified into four serotypes, DENV-1, DENV-2, DENV-3, and DENV-4, which are antigenically different viral serotypes; infection with one serotype confers a lifelong immunity only to that particular serotype (Beasley and Barrret, 2008). All the four serotypes of Denguevirus have been reported to circulate in Asia, Africa and America (Nathan and Dayal-Drager, 2006). In America, Dengue virus affected more people during the 2002 outbreak than the entire decade of 1980s (Guzman and Kouri, 2003). The epidemic of Dengue in Nicaragua in 1998, showed that of the 1,027 patients examined, 614(60%) were confirmed positive, out of which 268(44%) were classified as Dengue fever, 267(43%) as Dengue fever with haemorrhagic manifestations, 40(7%) as Dengue haemorrhagic fever and 20(3%) as Dengue shock syndrome (Harris et al., 2000). In

Asia, the epidemic of Dengue in 2007 in Singapore, resulted to 24 deaths giving an incidence of 192.3 cases per 100,000 and a case fatality of 0.27% (Ler et al., 2011). In Africa, the burden of Dengue remains poorly understood. Travellers to African countries and military personnel visiting or stationed in Africa have been identified as having laboratory-confirmed Dengue infections, indicating that the virus is circulating (Sharp, 1995; CDC, 2005). Symptoms range from a mild, febrile illness to severe Dengue haemorrhagic fever (Sanchez-Seco etal., 2006). Dengue fever and its more serious forms, Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS) have become important public health problems and were formally included within the disease portfolio of the United Nations Development Programme/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases by the Joint Coordination Board in June 1999 (WHO, 1999). Given the scope of the disease and the large numbers of persons with symptomatic infection, Dengue infection may have a tremendous impact on the health-care systems of the countries affected and on household and labour economies, especially during epidemics (Suaya et al., 2006). The history of Dengue in Africa is poorly documented buta retrospective sero-survey by Kokernot and co-workers suggests that Dengue existed in Africa as far back as 1926-1927, when it caused an epidemic in Durban, South Africa (Kokernot et al., 1956).With poor surveillance for Dengue in Africa, it is clear that epidemic Dengue fever caused by all four Dengue serotypes has increased dramatically since 1980 with most epidemics occurring in Eastern Africa and, to a lesser extent, in Western Africa (Sang, 2006).Whilst Dengue appears to be spreading in Africa, the funding received for surveillance and other research activities pertaining to Dengue has been very limited. This has mainly been due to the assumption that Dengue is not a significant health problem on the continent, perhaps due to the rarely reported cases andsevere forms of

Dengueillness. Most Dengue infections are subclinical in nature or present as Dengue fever, resulting in wrong diagnoses and treated as malaria or other endemic fevers such as, typhoid and leptospirosis. This has resulted in an underestimation of the magnitude of the Dengue problem in Africa (Sang, 2006).

In Nigeria, DENV-1, DENV-2 and DENV-3 were first isolated around the Jos plateau, during the 1960s from samples taken from humans (Carey, 1971).Subsequently, all the four serotypes were also reported to have been isolated in other parts of Africa (Gubler and Clark, 1995). Furthermore,Flavi viruses infection have been recorded in Nigeria among 1,816 children and adults from urban and rural areas in samples obtained mainly during the early 1970s. Virus-specific haemaglutination inhibition tests have revealed the prevalence of immunity to DENV-1 as(38%), DENV-2(45%) Yellow fever viral infection (43%) and the West Nile viral infection (49%) (Fagbami et al., 1977).These four serotypes of Dengue virus (DENV) and Yellow fever virus are primarily transmitted from infected humansto susceptible humans by the female Aedes mosquitoes during a blood meal (Halstead, 1988).

Yellow fever virus is an important re-emerging arboviral disease and a cause of severe illness and death in South America and Africa (Deubel et al., 1997).In South America, all reported cases are considered the result of endemic transmission of jungle yellow fever by tree-hole breeding Haemagogus spp., mosquitoes that had acquired infection by feeding on an infected monkey in the forest canopy. The annual incidence of endemic jungle yellow fever in South America has varied from 46 to 524 cases, from 1990 to 2004 for an average incidence of 168 cases (Monath, 2006).

Yellow fever epidemics in Africa, caused by transmission of the virus by Aedes aegypti continue to occur and involve human populations both in towns and in rural villages (Nasidi et al., 1989). Unpredictable and explosive urban outbreaks are largely

observedin West Africa (Ellis and Barret, 2008). Itcauses severe haemorrhagic symptoms like those of Dengue although a suitable vaccine is available. The virus has however gained considerable notoriety in the last few years because of the resurgence in epidemic activity of the vector (Monath, 1997; 2001).Infections occur unnoticed during periods between epidemics, but the endemic disease burden in Africa is difficult to quantify due to insensitive surveillance. In 1986 over 5000 people died of yellow fever in eastern Nigeria; while in 1987 there were over 400 deaths from yellow fever in Western Nigeria, and over 1500 deaths were estimated to have occurred in Northern Nigeria. The most striking aspect of the incidence data was the highpeak experienced between 1987 and1994, which reflects a series of concurrent annual epidemics in West Africa, principally Nigeria (Nasidi et al., 1989;Monath, 2006).Official reports of yellow fever between 1965 and 2004 in South America and Africa number over 33,000 cases with overall incidence in Africa nearly 5 times that in South America (Monath, 2006). Yellow fever has two types of transmission cycles in Africa: Urban and Sylvatic. It can be transmitted from infected humans to susceptible humans by the urban mosquito vector Aedes aegypti,while the sylvatic cycle involves transmission from infected non-human primates to humans by mosquitoes such as Aedes africanus, Ae.furcifer, Ae.metallicus, Ae.vittatus, Ae.simpsoni and Ae.taylori. Clinical forms range from an asymptomatic form or mild febrile illness to fatal infections (Digoutte et al., 1995).It is difficult to differentially diagnose yellow fever with the presence ofjaundice from viral hepatitis,arboviral, haemorrhagic fevers, bacterial, parasitic diseases and diseases due to toxic substances that also cause jaundice. The virus is generally not detected in the blood during the late phase of the disease due to an increase in the neutralizing antibody titre (Deubel et al., 1997).

Aedes mosquitoes have been incriminated in various yellow fever epidemics in Nigeria (Banget al., 1980; 1981; Savage et al., 1992).Through virus isolation, a number of Aedes species have been associated with Dengue transmission in West Africa including:Aedes taylori, Aedes furcifer, Aedes luteocephalus, Aedes vittatus and Aedes aegypti (Diallo, 2003).In Zaria, studies have shownthe abundance ofAedesbreeding in rock pools.(Adebote et al., 2004 and 2008).The laboratory diagnosis of Dengue virus andYellow fever infection can reliably be made by the detection of specific virus, viral antigen, genomic sequence, and/or antibodies (Gubler, 1998; Guzman and Kouri, 2003). There are three basic methodsused by most laboratories for the diagnosis of Dengue virus and Yellow fever virus infections, these are viral isolation and characterization, detection of the genomic sequence by a nucleic acid amplification technology assay, and detection of Dengue virus specific antibodies (WHO, 1997).Dengue haemorrhagic fever has been related to historical re- infection with different serotypes, detection and typing of DENV in the species of Aedeshas provided crucial information for prevention and control.Since both Dengue and yellow fever viruses are transmitted by the species ofAedes,co-circulation in a given region is possible and differential detection isvery helpful (Sanchez-seco et al., 2006). The species of Aedesare in theFamily Culicidae, Sub family Culicinae, Order Diptera and Sub order Nematocera. They breed in transient water collections such as tree holes (Phytotelmata), domestic containersaround homes and inselbergs(Adebote et al., 2008). The femalesare diurnal, mostly exophagic and exophilic, transmitting the virus during a blood meal (Adebote et al., 2008;Afolabi and Ndams, 2010).

1.2 Statement of Research Problem

Dengue and Yellow fever are amongst the emerging and resurging diseases vectored by several Aedesspecies in Africa, Asia and America (Savage and Smith, 1995, Gratz and Knudsen, 1996). Bang et al. (1980; 1981) reported on Aedesspecies implicated in various yellow fever epidemics in Southern parts of Nigeria. But so far, in Zaria Northern part of Nigeria, there isno report, to our knowledge, on the detection ofYellow fever and Dengue viruses in mosquito species,even though various studies conducted in Zaria and its environs have shown the preponderance of the species of Aedes (Aedebote et al., 2008 and Afolabi and Ndams, 2010).This poses a great danger and a precursor for outbreak or epidemic should the viruses be present in the vector. Reported incidence of Dengue has increased worldwide in the past few years but little is known about its incidence in Nigeria and particularly in Zaria where the vector is abundant (Afolabi and Ndams, 2010).Yellow fever is a major threat in Nigeria, 100,000 cases with 40% case mortality were recorded in a Yellow fever outbreak in 1969. Though, this figure is regarded as an underestimation due to the fact that most cases are not reported in hospitals (WHO, 1986). Nonetheless, between the year 1985 and 2000, yellow fever outbreak have plagued various parts of Nigeria (Onyido et al., 2009).There is also a good reason to believe that the 2.5 billion people who live in regions at risk of Dengue infection are also at risk of yellow fever (Reiter, 2010).Despite a safe and effective vaccine, there are approximately 200,000 cases, including 30,000 deaths due to yellow fever virus each year, of which 90% are in Africa (Ellis and Barrett, 2008).An estimate of 50 to 100 million cases of Dengue fever had been reported to occur annually, resulting in 24,000 deaths corresponding to an incidence rate of 2.5–5.0% from 2.5 billion people potentially at risk (Rigau-Perez and Gubler, 1999; Gibbons and Vaughn,

2002; WHO, 2002). Hundreds of thousands of hospitalizations each year have been associated with these cases (Gubler and Meltzer, 1999).Major demographic changes resulting from factors such as rapid urbanization, failure to control mosquito vectors and environmental changes due to the global climate change particularlyin tropical regions such as here in Zaria in particular,present enormous future challenges from infectious diseases including Dengue and Yellow fever.

1.3 Justification

Adult mosquito abundance is a key factor contributing to the risk of disease transmission. Monitoring the abundance of adult mosquito populations provides information on the size of the vector population and thesurveillance of arboviruses in field-collected mosquitoes. This is an important tool for detecting emerging viruses (Roiz etal., 2012). In Zaria and its environs, these vectors of arboviruses abound hence,molecular-based assays and the use of real time reverse transcriptase PCR which are suitable tool for effective detection will be critical in the monitoring of associated diseases in mosquito vectors. This study will help provide important information for prevention and control of arboviral diseases. Cross reaction between flaviviruses is well known and thus viral detection techniques for diagnosis are supplemented by more specific assays for precise identification (Guzman and Kouri, 2004). There could be the possibility of co-transmission of both viruses byAedesmosquitoes in Zaria,hence, differential detection of Dengueand Yellow feverviruses would be very helpful in the designing of control and prevention strategies. This study will provide information that is crucial in surveillance of Dengue and Yellow fever virusesinAedes mosquitoes circulating, since previous infections with one of the four Dengue serotype can be an

important risk factor for developing DHF and DSS upon infection with heterotypic serotypes (Halstead, 1998).

1.4 Aim 

To detect Dengue and Yellow fever viruses in Aedes populations in Zaria and environs.

Objectives

Specific objectives are

i. To determine the relative abundance of Aedes mosquito species in Zaria and environs.

ii. To detect Dengue virus and Yellow fever virus in the abundant Aedes species in the area. 

iii. To determine the prevalence of Dengue virus and Yellow fever virus in the Aedes species in their locations.

1.5 Research Questions

i. What are the abundant species of Aedes in the areas?

ii. Are Dengue and Yellow fever flavivariids found in Aedes species in the study area?

iii. What are the prevalence of dengue and yellow fever viruses in Aedes species in study area?

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