MOSQUITO-BORNE SURVEILLANCE AT A CATTLE MARKET IN OGUN STATE, NIGERIA

BY

Oresegun, Olanrewaju Adekunle

Matriculation Number: RUN/MCB/14/5835

JULY, 2016

MOSQUITO-BORNE ARBOVIRUS SURVEILLANCE AT A CATTLE MARKET IN OGUN STATE, NIGERIA

BY

ORESEGUN, OLANREWAJU ADEKUNLE Matriculation Number: RUN/MCB/14/5835

A Dissertation Submitted to the Department of Biological Sciences, College of Natural Sciences, Redeemer‟s University, Ede, Nigeria in partial fulfilment of the requirements for the award of Master of Science degree (M.Sc.) in Microbiology

JULY, 2016

CERTIFICATION

I certify that this research study was carried out by Oresegun, Olanrewaju Adekunle with

Matriculation Number RUN/MCB/14/5835, of the Department of Biological Sciences,

Redeemer‟s University, Ede, Osun State, Nigeria, under my supervision.

…………………………………. ………………………….

Professor I.O.O Komolafe Date

Project Supervisor/Head, Department of Biological Sciences

……………………………………. …………………………

Dr. Deborah U. Ehichioya, Date

Project Supervisor

…………………………………. ………………………….

Professor I.O.O Komolafe Date

Head, Department of Biological Sciences

………………………………… ………………………….

External Examiner Date

DEDICATION

I dedicate this work to the Almighty God for His grace, provision, wisdom and understanding.

ACKNOWLEDGEMENT

All glory to the Alpha and Omega, giver of all good things, the all sufficient One for His mercies, provisions, goodness, tender love, faithfulness, kindness and blessings.

A lot of thanks to my parents, Mr. and Mrs. S.A. Oresegun and my siblings, Olabanji and

Oyindamola, and my amazing Mokesoluwa Akinyede, for their unfailing love, prayers, financial support, parental care and interest in my success in life.

I express my profound gratitude to my amiable, energetic, inspirational and motivating supervisor, Dr. Deborah Ehichioya, who played a major role in the success of this work. I appreciate your untiring efforts, guidance, tutelage, and thorough supervision during the planning, experiment and writing stages of this work. I also wish to appreciate Dr. Onikepe

Folarin for her guidance, tutelage and assistance. I give special thanks to the Dean of College of

Postgraduate studies, Professor Christian Happi, the Head, Department of Biological Sciences;

Professor I.O.O Komolafe, all lecturers and staff in the Department of Biological Sciences.

This work was supported by grants from African Centre of Excellence for Genomics of

Infectious (ACEGID), World Bank (ACE019) and the National Institutes of Health

(grant 5U01HG007480-03).

I appreciate the contributions of the following families; Mr. and Mrs. S. O. Savage, Pastor and

Mrs. S. A. Adedokun, Professor K.I.T.Eniola, Dr. J. A. Adetoso, Dr. O. A. Igbalajobi, Mrs. A.

Fatiregun, the Head and staff, Department of Biological Sciences, Joseph Ayo Babalola

University, Dr. Soji Fakoya, my colleagues at work, my course-mates for their support, patience and encouragement. Lastly, I appreciate the executives and people of Kara cattle market, especially Mr. Rasheed, for their support during sampling for this work

ABSTRACT

A surveillance study of mosquito-borne in a cattle market was undertaken at Kara,

Mowe-Ibafo area of Ogun State between March and April 2016.The study aimed to assess arbovirus distribution in cattle-market areas (Kara, Mowe-Ibafo area of Ogun State); assess the risk of human exposure to arbovirus diseases; possible identification of novel arboviruses or variants of existing ones; identification of associated mosquito vectors.Five CDC CO2 – baited light traps were set daily at locations A to E. Each trap was left for twelve (12) hours between the hours of 7:00pm and 7:00am. The trapped mosquitoes were kept in the freezer and left to die at cold temperature below 00C. The frozen mosquitoes from individual trap were then transferred into petri dishes, sealed, labelled and stored at temperature below 00C.The mosquitoes were identified and pooled into species (, , Mansoni and Anopheles).

The mosquitoes were homogenised and RNA was extracted from the pools using QIAGEN

QIAamp Viral RNA Mini Kit. All samples were screened for four different arbovirus families, namely Pan-; Pan-; Pan-rhabdovirus and Mesonivirus using SuperScript®

III One-Step reverse-transcriptase PCR Kit (Invitrogen).A total of 1,650 mosquitoes were collected, of these, 1,626 were successfully identified and pooled by species and sex. Of the

1,626 successfully identified mosquitoes, 89.85% (N = 1,461: 1,435 female; 26 male) were

Culex species, 3.51% (N = 57 females) were Aedes species, 3.57% (N = 58 females) were

Mansonia species and 3.08% (N = 50: 1 female; 49 male) were Anopheles species. Of the 50 mosquito pools screened, only six (6) were positive for Pan-Flavivirus, none was positive for

Pan-phlebovirus, twenty-seven (27) pools were positive for Pan-rhabdovirus, while five (5) pools seemed to be positive for Mesonivirus.Arboviruses contribute greatly to mortality all over the world. The result also established that the prevalence of Rhabdoviruses is quite high in the study area, as 54% of the samples were positive.

TABLE OF CONTENTS

Title Page i

Certification ii

Dedication iii

Acknowledgements iv

Abstract v

Table of Content vi

CHAPTER ONE

Introduction 1

Literature review 4

Aim of Study 2

Objectives of Study 2

Common vectors 5

Mosquitoes 5

Biting midges 8

Ticks 10

Virus family associated with arboviruses

12Flaviviridae

12Bunyaviridae

15

Togaviridae 19

Reoviridae 21

Asfarviridae 22

Rhabdoviridae 22

Mesoniviridae 23

Statement of problem 24

Rationale 25

Broad objectives 25

Specific objectives 25

CHAPTER TWO

Materials and methods 26

Study area 26

Mosquito trapping 30

Mosquito identification 32

Mosquito homogenization 32

Extraction of ribonucleic acid (RNA) 32

Rehydration of primers 33

PCR mastermix 35

Pan-flavivirus 35

Pan-phlebovirus 35

Pan-rhabdovirus 35

Mesonivirus 36

Thermal profile 36

Agarose gel electrophoresis 37

CHAPTER THREE

Results 38

Mosquito abundance 38

Sum of mosquitoes trapped by species per location 40

Sum of mosquitoes trapped by location 42

Sum of mosquitoes trapped by weather 44

Sum of mosquitoes trapped by day and weather 46

Sum of mosquitoes trapped by weather condition per location 48

Evaluation of the rt-pcr assay analysing mosquito samples 50

RT-PCR for pan-flavivirus 50

RT-PCR for pan-phlebovirus 50

RT-PCR for pan-rhabdovirus 50

RT-PCR for mesonivirus 51

CHAPTER FOUR

Discussion 56

Limitations 59

Conclusion and recommendation 59

References 60

LIST OF PLATES

Plate 1 A female Anopheles mosquito having a blood meal 7

Plate 2 Adult biting midge 9

Plate 3 Adult deer () 11

Plate 4 Structure of a typical Bunyaviridae virion 17

Plate 5 Structure of a typical Togaviridae virion 20

Plate 6 Cattle and sheep kept and sold at Kara market 27

Plate 7 A canoe transporting passengers on the Ogun River 28

Plate 8An overview of environmental conditions at Kara market/abattoir 29

Plate 9Set-up of Carbon dioxide baited light traps 31

LIST OF FIGURES

Figure 1 Chart showing sum of mosquito species trapped per location 41

Figure 2 Sum of mosquitoes trapped by location 43

Figure 3 Sum of mosquitoes trapped by weather condition 45

Figure 4 Sum of mosquitoes trapped per day, with its prevailing weather condition 47

Figure 5 Sum of mosquitoes trapped by weather condition per location 49

Figure 6 Gel result for Pan-flavivirus showing ladder and positive band at260 bp 52

Figure 7 Gel result for Pan-phlebovirus showing ladder and positive band at370 bp

53

Figure 8 Gel result for Pan-rhabdovirus showing ladder and positive band at 470 bp 54

Figure 9 Gel result for Mesonivirus showing 100 bp ladder 55

LIST OF TABLES

Table 1 Primers Sequence for this project 34

Table 2 Identified Mosquitoes 39

CHAPTER ONE

INTRODUCTION

Arboviruses are a heterogeneous group of transmitted by blood-feeding , such as mosquitoes, ticks, midges, and sand (Ochieng et al., 2013). In the last decades, the appearance of new infectious diseases and invasion/introduction of diseases into new areas have brought about increase in emerging and re-emerging pathogens. Most of these which, are zoonotic, can cause infection in both man and animals (Pfeffer and Dobler, 2010).

Viruses transmitted by haematophagous arthropods have been indicted to be responsible for some of the major emerging infectious problems in the world. Arboviruses constitute a large biological group of viruses (Sang et al., 2006). They play a vital role in the

emerging and re-emerging of viral diseases. Arboviruses multiply within the tissues of the arthropod hosts thereby producing high viral titre in the salivary glands. Upon bites from arthropods during blood meals, the viruses are introduced into the vertebrate hosts (human and/or animals). Most arboviruses are maintained in zoonotic cycles from arthropods to susceptible vertebrate hosts (humans and animals) by infected arthropods bite; usually having humans usually as the dead-end . The viruses multiply rapidly within the tissues of the arthropod, thereby yielding high titres of in the salivary glands and are then passed on to susceptible (Pfeffer and Dobler, 2010).

Three factors that are essential for the establishment and maintenance of arbovirus infection are the virus, the arthropod and the vertebrate. These factors play major roles for successful establishment of cycle of arbovirus. The must have competence for the particular virus, the vertebrate host should be susceptible to the virus and the virus should have the capability of producing high-level viraemia, in to enable transmission to the vectors.

For continuous transmission cycle, all factors that interplay must be sufficiently available at the time and place (Pfeffer and Dobler, 2010).

Studies have shown that arboviruses are widespread in Nigeria, due to the abundance of the mosquito vectors (such as Aedes, Culex, Mansonia) responsible for the transmission of viral infections, such as dengue, yellow , , Zika, , Dugbe virus,

Igbo-Ora virus and others (Moore et al., 1975; Fagbami, 1978 and Baba et al., 2013). Arbovirus co-infections, is not uncommon and was recently described in Nigeria (Baba et al., 2012).

Dengue fever virus infection shows signs and symptoms which are very similar to those of some tropical disease fever, such as and typhoid (Baba et al., 2012). Most times, in

Nigeria, febrile patients are usually treated for malaria, because it is highly endemic in the country (Amexo et al., 2004). Consequently, this may result in the slow identification of an arboviral disease outbreak and its potential high morbidity and mortality (Baba et al., 2013).

Investigation and diagnosis of arboviruses are not systemically and critically done; they are usually considered by clinicians, when patients‟ samples test negative for malaria and typhoid

(Sow et al., 2016). Arboviral diseases are usually uncontrolled, and the viruses responsible for these infections are emerging and reemerging throughout the world (Conway et al., 2014).

These arboviruses, as a result of their evolution, have the ability to cause infections both in arthropods and mammalian hosts (including man), causing widespread infections in man

(Conway et al., 2014). It is therefore very important to understand the role of vectors in the spread or transmission of these arboviral infections, and this understanding can be used in the development of novel strategies in controlling the spread of diseases.

Currently, (DENV) is being indicted as the most problematic arbovirus to human population. Reports show that about 100-390 million people are infected globally every year, causing about 96 million symptomatic infections and accounting for 12,500 deaths annually - mostly among young children (Bhatt et al., 2013; Guzman et al., 2010). As a consequence of

2 the increasing range of Aedes species mosquito vectors, DENV is considered a reemerging pathogen. A number of factors, such as, global warming, , , ease of travel, are responsible for the increasing spread of these mosquito species around the world

(Kilpatrick and Randolph, 2012).

Mosquitoes, while having blood meal from infected host(s) become infected with arboviruses.

The viruses, when picked, travels into the midgut along with the blood meal, after breaching the midgut barrier. Some arboviruses, known to infect only arthropods, may have been introduced into the arthropods from the environment or during a sugar or blood meal. Whereas, other arboviruses are known to infect both arthropods and (Conway et al., 2014).

The establishment of an enzootic transmission cycle requires the selection of viral proteins that can interact with diverse molecules which include cell surface receptors and entry factors, immune components, protein translation machinery, and protein export machinery in cells of both arthropods and mammals. It is assumed that many mutations are required for an arthropod- only virus to transit to an enzootic transmission cycle that involves a mammalian host (Conway et al., 2014).

Mosquito species are known to feed on either nectar juice from flowering plants, vertebrate blood, or both, using their mouth-part known as proboscis. Mosquitoes have different means of sourcing for nectar juice or blood meals, which include – movement, body heat, carbon dioxide (CO2), and compounds released from host skin and normal bacterial flora (Smallegange et al., 2010; Bohbot et al., 2013). Culex species feed mainly on avian host (American robins), but can feed on humans if their preferred host is unavailable. Although, the genetic alterations required for host-preference are not known, insects detect attractive cues (attractants) through several molecules, which include odorant receptors and an obligate co-receptor called “orco”

(DeGennaro et al., 2013).

3 Over 530 arboviruses have been described, of which about a hundred are known to be pathogenic to humans (CDC, 2015). Humans are not necessarily the primary host in the transmission cycle, and may only be incidental hosts, as in the case of West Nile virus.

Arboviruses that are pathogenic to humans mainly belong to four distinct genera of RNA viruses – Flavivirus (e.g., Dengue virus, virus, and West Nile virus);

Alphaviruses (e.g., Chikungunya virus and Easter equine encephalomyelitis); Phlebovirus

(e.g., virus); (e.g., California virus) (Lequime et al., 2016).

LITERATURE REVIEW

Vector-borne infections are diseases caused by pathogens (viruses, parasites, bacteria and fungi) that are transmitted by insects such as mosquitoes, ticks, sand flies and midges. These vector-borne diseases have long impacted human lives, having devastating impacts on humans and livestock. A remarkable event in history is the bubonic plagues referred to as “the Black

Death” that killed millions of people worldwide. This was caused by another insect-flea, carrying bacteria. The scourge of vector-borne infections is still greatly felt in recent times; malaria is still endemic in sub-Saharan , affecting millions of people. Vector-borne diseases poses threats to the health and livelihood of human and livestock population in the tropics where their impact is greatest (MosquitoZone, 2015).

Arthropod-borne diseases are one of the deadliest and unpredictable diseases on earth. The insect vectors are difficult to control; they multiply rapidly, thereby making it difficult to control them and the diseases they transmit. Vectors bridge the gap between pathogens and animal hosts (humans, monkeys, rats, birds, pigs, dogs, cattle, goats, sheep, etc.). Vector-borne diseases are generally characterised by:

4  High disease transmissibility

 Explosive, unpredictable spread of disease

 Resilient to control and prevention because of vector‟s small size and sheer numbers

 Larger range versus diseases that require direct contact.

Generally, vectors are usually not affected adversely as a result of carrying any of their pathogens (virus, protozoa, nematodes and bacteria). Although, damage may be done to their tissues, which usually makes them more suitable to transmit the pathogens, thereby causing infections. Once a vector carries a pathogen, it remains infected throughout its entire live.

Mosquitoes and ticks have been reported to be responsible for the transmission of majority of the most important vector-borne diseases in the world (Ochieng et al., 2013). Some other close associates that have also been indicted are the sand flies and black flies. Each of these arthropod vectors is unique in its feeding behaviour, habitat, and time of activity. While some are active only at nights, some others are active and bite only at dusk and dawn. Some can breed in sunlight; others breed in the dark or shady environment away from light. While high salinity is lethal to some, others thrive optimally in saline habitat. Deep knowledge of specific habits of different vectors can help in devising means of controlling or preventing their spread of infections (MosquitoZone, 2015).

COMMON ARTHROPOD VECTORS

MOSQUITOES

Mosquitoes belong to the family Culicidae. They are ectoparasites with tube-like mouthparts called proboscis. The females feed on blood of a wide range of hosts, including mammals, birds, reptiles and amphibians. When mosquitoes bite, their saliva often cause irritating rash on the host‟s skin. Mosquitoes use exhaled carbon dioxide, body odours and temperature emitted, and movement find their victims. Only female mosquitoes have the mouthparts necessary for

5 sucking blood (Plate 1). When biting with their proboscis, they stab two tubes into the skin: one to inject an enzyme that inhibits blood clotting; the other to suck blood into their bodies.

Female mosquitoes do not feed on blood for their own nourishment but use it as a source of protein for their eggs. For food, both males and females eat nectar and other plant sugars

(National Geographic, 2016). Surprisingly and interestingly, mosquitoes are regarded as the deadliest animal on earth. This is because mosquitoes serve as vectors for transmitting several harmful infections that include malaria, yellow fever, west Nile virus, , Zika virus, filariasis, Chikungunya, , Murray Valley encephalitis and other arbovirus diseases (Michigan Organization, 2013).

6

Plate 1: A female Anopheles mosquito having a blood meal (University of Kentucky

College of Agriculture (UKCA), 2013)

7 BITING MIDGES

Biting Midges are a family of small flies, about 1-4mm long (Figure 2). They are also known as midges and sand flies. They are found throughout the world in aquatic or semi-aquatic habitats and mountain areas. There are over 4,000 species of biting midges in the

Ceratipongonidae family. The genus- Culicoides has over 1,000 species. Midges are distributed worldwide; present in the tropics, sub-tropics, and the Caribbean (Gillett-Kaufman,

2013). As with the mosquitoes, the females are adapted to haematophagous mode of nutrition; they feed on vertebrate hosts such as humans, cattle and horses. However, some species, such as Dasyhelea, feed only on plant nectar, while species in the other genera are predators of small insects. Midges lay their eggs in damp environment, such as beneath the bark of decomposing tree, mud, stream margins, or water-holding plants (phytotelmata). The blood-sucking midges species serve as vectors of disease-causing viruses, protozoa, or filarial worms. Midges in the genus Culicoides cause allergic response in horses when they bite. However, when they bite humans, it results in intense itching, red welts, as a result of localized allergic reaction to the proteins in their saliva.

8

Plate 2: Adult biting midge, Culicoides sonorensis Wirth and Jones (Schmidtmann, E.T.

(cited in Connelly, R.C., 2013).

9 TICKS

Ticks are tiny animals (arachnids) closely related to mites, scorpions and spiders. Ticks differ from insects, in that their bodies are divided into two parts, whereas, insects have three body parts. An adult ticks have eight legs and have no antennae and wings. Some species of ticks have eyes on their hind-body. Haller‟s sensory organs, containing chemoreceptors are used for scanning the environment, are present on the front legs. Ticks are also haematophagous and detect their hosts by vibration, warmth, carbon dioxide, moisture and body odour. On detecting the hosts, they attach to feathers, fur, skin or clothing, then they pierce through the host‟s skin with hypostomes, for blood meals. There are about 840 species of ticks throughout the world, classified into three families:

 Argasidae: Argasids or soft ticks, with a tough, leathery skin and a concealed ventrally

projecting capitulum (approximately 170 species). There is no scutum in adult animals.

A scutum is a dorsal shield. Examples include species in the genera Argas,

Ornithodoros, Otobius, Antricola, Nothoaspis

 Ixodidae: Ixodids or hard ticks have a rigid scutum and a capitulum with mouthparts

projecting forwards (approximately 670 species). This capitulum is visible when

viewed from dorsal. Examples include species in the genera Amblyomma, Aponomma,

Boophilus, Cosmiomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes,

Margaropus, Nosomma, Rhipicentor, Rhipicephalus

 Nutalliellidae: This family has only one species.

Of the three families, only two families of ticks, Ixodidae (Figure 3) and Argasidae (soft ticks), are known to transmit diseases or illness to humans. Ticks act as vectors of pathogens that cause diseases such as tickborne encephalitis, Lyme disease, tickborne relapsing fever and

Crimean Congo haemorrhagic fever.

10

Plate 3: Adult deer ticks (Ixodes scapularis) (National Geographic, 2016)

11 VIRUS FAMILIES TRANSMITTED BY ARTHROPODS

Arboviruses of humans, livestock and wildlife include members of the following virus families:

Flaviviridae, Bunyaviridae, Togaviridae, , Asfaviridae and . Most arbovirus families have ribonucleic acid (RNA) genome with the exception of Asfarviridae

(African swine fever virus) (Johnson et al., 2012).

FLAVIVIRIDAE

Flaviviruses are positive (+) sense single-stranded RNA viruses with linear non-segmented genomes (CDC, 2014). Their , measuring about 40-50 nm in diameter, are icosahedral in shape and surrounded by spherical envelope (Kronen, 2008). Flaviviridae has 4 genera, namely: Hepacivirus, Flavivirus, Pegivirus, and Pestivirus. Viruses in the Flavivirus genus are arboviruses. Humans and other mammals are the primary hosts of these viruses, and they are spread primarily by arthropod vectors (most commonly mosquitoes and ticks) (ViralZone,

2016). Some of the common mosquitoes-transmitted viruses are yellow fever virus, Zika virus,

Dengue fever virus, Japanese encephalitis virus and West Nile virus. Examples of viruses transmitted by ticks include -borne encephalitis (TBE), Kyasanur forest disease and

Alkhurma disease (CDC, 2014).

West Nile Virus (WNV) belong to the virus family- Flaviviridae and genus- Flavivirus. It is enveloped and non-segmented, having single stranded ribonucleic acid (ssRNA) as its genome.

WNV is principally transmitted by ornithophilic mosquito- , although other mosquito species have been shown to support replication and transmission of the virus. The virus causes disease in humans, horses and some avian species. Common symptoms include fever, ataxia, and paresis, paralysis of limbs, recumbence, muscle tremor and rigidity. The

12 disease is common throughout Africa and Asia, also present in and the Americas

(Johnson et al., 2012).

Dengue fever virus (DFV) belong to the virus family- Flaviviridae and genus- Flavivirus. It is enveloped and non-segmented, having single stranded ribonucleic acid (ssRNA) as its genome

(Johnson et al., 2012). Dengue fever is a mosquito-borne disease transmitted by the Aedes species (mostly and less often, ). There are four of the virus (DENV 1, 2, 3, 4), any of which is capable of causing disease. Infection with one offer life-long immunity against that particular serotype, but does not offer immunity against the other three serotypes. Therefore, it is possible for one to have dengue fever virus infections four times, each caused by a different serotype (CDC, 2009). Dengue fever is characterised by symptoms such as frontal , retro-orbital pain, , arthralgia, haemorrhagic manifestations, rash, and low count. A severe and sometimes fatal form of dengue fever may result, this form is known as dengue haemorrhagic fever (DHF).

Warning signs include severe abdominal pain, persistent vomiting, marked change in temperature (from fever to hypothermia), haemorrhagic manifestations, or change in mental status (irritability, confusion). Other symptoms are early signs of , including restlessness, cold clammy skin, rapid weak pulse, and narrowing of the pulse pressure (CDC, 2009; WHO,

2015). The disease is presently endemic in more than 100 countries in the WHO regions of

Africa, the Americas, the Eastern Mediterranean, South-East Asia and the Western Pacific. The

America, South-East Asia and Western Pacific regions are currently the most seriously affected

(WHO, 2015).

Yellow fever virus (YFV) belong to the virus family- Flaviviridae and genus- Flavivirus. It is enveloped and non-segmented, having single stranded ribonucleic acid (ssRNA) as its genome

(Johnson et al., 2012; CDC, 2015). The virus is transmitted through the bite of an infected mosquito vector. Mosquitoes of the Aedes species and Haemagogus species have been

13 identified as vectors of yellow fever virus. The mosquitoes either breed around houses

(domestic), in the jungle (wild) or in both habitats (semi-domestic). The virus causes disease in humans and other ; anthroponotic, that is, human-to-vector-to-human transmission could also occur. There are 3 transmission cycles for yellow fever: sylvatic (jungle), intermediate (savannah), and urban. The sylvatic (jungle) cycle involves transmission of the virus between nonhuman primates (e.g. monkeys) and wild mosquito species found in the tropical rainforest canopy. The virus is transmitted via mosquitoes from monkeys to humans when the humans encroach into the jungle during occupational (lumbering or research) or recreational activities. In humid or semi-humid parts of Africa, an intermediate (savannah) cycle involves transmission of YFV from tree hole-breeding Aedes species to humans living or working in jungle border areas or villages. In this cycle, the virus may be transmitted from monkeys to humans or from human to human through bites from these mosquitoes. The urban cycle involves transmission of the virus between humans and peridomestic mosquitoes, primarily A. aegypti, in densely populated areas. This results in large scale (WHO,

2014; CDC, 2015).

When humans become infected with yellow fever virus, the level is usually very high.

As a result, blood-borne transmission can occur through blood-to-blood products (e.g. transfusion, needles, etc.). The disease is characterised by symptoms such as fever, muscle pain with prominent backache, headache, shivers, loss of appetite, and nausea or vomiting during the first phase. The disease could also progress to the more toxic second phase which is characterised by jaundice (from where the “yellow” in yellow fever is derived) and complains of abdominal pain with vomiting. Bleeding can occur from the mouth, nose, eyes or stomach, blood-stained faeces and vomits (WHO, 2014). Up to 50% of patients that progress into the second phase die within 10-14 days. The disease is endemic in Africa and Latin America, about

44 countries affected. About 31 countries in Africa with an estimated population of 508 million

14 people are at risk of yellow fever. While the remaining 13 countries are in Latin America, with

Bolivia, Brazil, Colombia, Ecuador and Peru at highest risk (WHO, 2014).

Wesselsbron Virus (WSLV) belong to the virus family- Flaviviridae and genus- Flavivirus. It is enveloped and non-segmented, having single stranded ribonucleic acid (ssRNA) as its genome. WSLV is transmitted by mainly by mosquito- Aedes species (e.g. ). The virus causes disease in sheep and goats; common symptoms include abortion, congenital abnormalities, fever and anorexia (Johnson et al., 2012). WSLV can also cause disease in humans; showing symptoms such as influenza-like illness. The virus is present across Africa; it has been isolated in countries like Nigeria, Mauritania and Senegal (Baba et al., 1995; Diallo et al., 2005).

Japanese encephalitis virus (JEV) belong to the virus family- Flaviviridae and genus- flavivirus. It is enveloped and non-segmented, having single stranded ribonucleic acid (ssRNA) as its genome (Johnson et al., 2012). JEV is transmitted mainly by Culex tritaeniorrhyncus

(which favours breeding in rice paddies); C. gelidus complex aid transmission to birds which maintains the virus in the environment (Erlanger et al., 2009). JEV causes disease in adult pigs, which could be asymptomatic, but results in abortion, still-birth, and birth defects. The virus also affects humans, causing encephalitis (Gould and Solomon, 2009). JEV is present across

Asia.

BUNYAVIRIDAE

Bunyaviridae are negative single-stranded and enveloped ribonucleic acid (RNA) viruses.

They have three nucleocapsids that are elongated and have helical symmetry (Virus Pathogen

Database and Analysis Resource (ViPR), 2016). They are common in arthropods or rodents, however, some viruses in this family sometimes cause infections in humans and plants

(Plyusnin and Elliott, 2011). Bunyaviridae are generally transmitted by arthropod vectors such

15 as mosquitoes, ticks and ; with the exception of Hantaviruses- transmitted through contact with faeces of deer mice (Plyusnin and Elliott, 2011). This virus family are spherical

(sometimes pleomorphic) in structure, having diameters ranging between 80-120nm. The virions are covered with glycoprotein projections that are embedded in lipid bilayer envelope

(ViPR, 2016). Bunyaviridae has 5 genera namely: Hantavirus, Nairiviris, Orthobunyavirus,

Phlebovirus, Tospovirus (plant viruses). Some diseases associated with this virus family include California encephalitis virus, Hantavirus, Crimean-Congo haemorrhagic fever, Rift

Valley fever and Dugbe virus (ViralZone, 2010).

16

Plate 4: Structure of a typical Bunyaviridae virion

17 Rift Valley Fever Virus (RVFV) belongs to the virus family Bunyaviridae and genus

Phlebovirus. It is enveloped and segmented, having single stranded ribonucleic acid (ssRNA) as its genome. RVFV is transmitted principally by the Aedes mosquitoes; Culex species have also been associated with transmission of the virus. The virus affects ruminants such as sheep, goats, and camels. Age is a very important factor that influences susceptibility to the virus; new-born animals are more susceptible than the adults. Adult animals show less severe disease.

The virus causes disease characterized by fever, recumbency, high abortion rates and haemorrhagic diarrhoea. The virus can also cause disease in humans, having symptoms such as influenza, haemorrhagic fever associated with liver damage and sometimes encephalitis. The virus is endemic in Africa, especially East Africa (Kenya and Tanzania) (Johnson et al., 2012).

Akabane virus belong to the virus family Bunyaviridae and genus Orthobunyavirus. It is enveloped and segmented, having single stranded ribonucleic acid (ssRNA) as its genome. The virus causes Akabane disease in ruminants such as cattle, sheep and horses. The disease is usually without symptoms in adults. Akabane disease results in abortions, stillbirths, and congenital abnormalities in pregnant animals. The virus is transmitted by midges- Culicoides brevitarsis and C. wadei in Australia; C. milnei and C. imicola in Africa, C. oxystoma in Japan.

Some mosquito species including Aedes vexans, and Anopheles funestus can also transmit the virus. Serological studies suggest the presence of the virus throughout Africa, Asia, and the northern Australia (Johnson et al., 2012).

Crimean-Congo haemorrhagic fever (CCHF) virus belongs to the virus family Bunyaviridae and genus Nairovirus. It is enveloped and segmented, having single stranded ribonucleic acid

(ssRNA) as its genome (Johnson et al., 2012). Ticks of genus Hyalomma transmit the virus. It is the most widespread disease caused by tickborne viruses. It causes disease mainly in humans and common symptoms include fever, , myalgia, temporary hair loss, poor vision

18 and loss of appetite. The virus can be spread from infected humans to other humans through close contacts, which may result in community or nosocomial outbreaks (Aradaib et al., 2010;

Naderi et al., 2010). The virus is endemic in Africa, Asia, Middle and Far East.

Nairobi sheep disease virus (NSDV) belong to the virus family- Bunyaviridae and genus-

Nairovirus. It is enveloped and segmented, having single stranded ribonucleic acid (ssRNA) as its genome (Johnson et al., 2012). The virus is mainly transmitted by the three-host Ixodid tick Rhipicephalus appendiculatus, which is common in Southern, Eastern and Central Africa.

The livestock host for this tick include cattle, sheep, goats and horses. Common features of the disease include high fever, diarrhoea, nasal discharge, conjunctivitis, abortion in pregnant animals, dullness, loss of appetite and depression (Marczinke and Nichol, 2002). The disease is enzootic in Kenya; it has also been detected in countries of East, Southern, and Central

Africa.

TOGAVIRIDAE

These viruses have linear, non-segmented, single-stranded positive sense RNA as their genome. They are enveloped and virions are spherical in shape, having diameter of about 65-

70 nm. Their capsids are icosahedral in shape (Murray et al., 2005). This virus family has only two genera, which are: and Rubivirus. The are mainly associated with arthropod vectors. Diseases associated with these genera include: Alphavirus: arthritis, encephalitis (e.g. Eastern Equine encephalitis, Western equine encephalitis, Venezuelan

Equine encephalitis), and Chikungunya fever; Rubivirus: congenital rubella syndrome caused by the sole member of this genus- rubella virus (ViralZone, 2010; ViPR, 2016). The natural hosts include human, mammals, marsupials, birds and mosquitoes.

19

Plate 5: Structure of a typical Togaviridae virion

20 REOVIRIDAE

This family of virus derived its name from the Latin word “orphan”. Reoviruses are non- enveloped and have icosahedral capsids with diameter of about 80-82 nm. The inner core is about 60 nm wide. The virions contain segmented double-stranded RNA genome. Natural hosts of these viruses include vertebrates, invertebrates, plants and fungi. They are the double- stranded RNA virus family that causes infection in humans (ViPR, 2016). The family-

Reoviridae, is further divided into two sub-families, which are: and

Sedoreovirinae (Carstens, 2010). The Spinareovirinae are characterized by presence of spikes on the surface of the core particles, while the lack spikes on the core particles

– they have relatively smooth surface (Attoui and Mertens, 2007). Spinareovirinae has about 9 genera, which are: Aquareovirus, , Cypovirus, Dinovernavirus, Fijivirus,

Idnoreovirus, Mycoreovirus, Orthoreovirus, and Oryzavirus. Sedoreovirinae on the other hand has 6 genera, which are , , , , and (ViralZone, 2008). However, not all of these genera are arboviruses. The genera that have been indicted as arboviruses include Orbivirus, Seadornavirus, Coltivirus,

Mimoreovirus, and Cardoreovirus. Diseases associated with the arbovirus genera include , African horse fever, , , Epizootic haemorrhagic fever.

Equine encephalitis virus (EEV) belong to the virus family Reoviridae and genus Orbivirus. It is non-enveloped and segmented, having double stranded ribonucleic acid (dsRNA) as its genome. The virus causes an acute disease in horses characterized with high fever and depressed appetite. Other symptoms of the disease include swelling of the lips and eyelids, neurological disease and abortion. EEV is transmitted be midges- Culicoides species. The virus was originally isolated in South Africa and Israel; seven serotypes have been reported within

South Africa (Johnson et al., 2012).

21 Bluetongue virus (BTV) belong to virus family Reoviridae and genus Orbivirus, it is non- enveloped, has double stranded RNA genome, and has about 10-12 segmentation. BTV disease is usually associated with ruminants, often affecting mostly sheep; cattle are affected less often.

BTV disease can go unnoticed without symptoms in animals like goats, deer, and alpacas. The virus has an of about 2-15 days. Symptoms of the disease include fever, salivation, conjunctivitis, muscle necrosis, cyanotic tongue (from which the disease gets its name), oedema of face and lips (Johnson et al., 2012). The BTV is endemic throughout the world, having a total of 24 serotypes. BTV is carried by the vector – midges (Culicoides genus). The most common of them being C. imicola, which is widely spread through Asia, the

Middle East, Africa, Southern and Eastern Europe. Other species that have been identified with

BTV disease include C. dewulfi, C. obsoletus, C. scoticus, C. pulicaris, C. chiopterus (Johnson et al., 2012).

ASFARVIRIDAE

Asfiviruses are enveloped viruses and their shapes range from spherical to pleomorphic and have diameter ranging between 175-215 nm. The viral has icosahedral symmetry with diameter 172-191 nm. The viruses have linear double-stranded DNA genomes of about 170-

190 kb. Asfiviruses are arthropod-borne DNA viruses. The natural hosts include domestic pigs, bush pigs and warthogs. Argasid ticks (Ornothodoros species) are the arthropod vectors for the

Asfarviridae family. Asfiviruses primarily infect swine, causing African swine fever; that could cause death within 15 days.

RHABDOVIRIDAE

Rhabdoviridae are enveloped and bullet-shaped, having width and length of about 75 nm and

180 nm, respectively (ViralZone, 2015). They have linear genomes, measuring between 11-15

22 kilobases in length. Rhabdoviruses contain negative-sense single-stranded RNA (ViralZone,

2015). Natural hosts of the Rhabdoviruses include vertebrates (mammals and humans inclusive), invertebrates (mosquitoes and midges) and plants. The Rhabdoviridae as a family currently has 11 genera, with about 71 species. Of these 11 genera, Ephemeroviruses,

Cytorhabdoviruses, Nucleorhabdoviruses are strictly arthropod-borne; Tiboviruses and

Vesiculoviruses are sometimes transmitted by arthropod bites (ViralZone, 2015). Some diseases associated with this virus family are fatal encephalitis from , encephalitis flu-like symptoms in humans from vesiculovirus and bovine ephemeral fever from ephemovirus (ViralZone, 2015; Johnson et al., 2012).

Bovine ephemeral fever virus (BEFV) belong to the virus family Rhabdoviridae and genus

Ephemerovirus. It is enveloped and non-segmented, having single stranded ribonucleic acid

(ssRNA) as its genome. BEFV is transmitted through flying insects (Culicoides midges/Culicine mosquitoes). The virus can cause disease commonly known as „3-day sickness‟ stiff sickness, bovine epizootic fever, lazyman‟s disease, or dengue of cattle. The disease usually affects cattle and water buffalo. Incubation period is between one to ten days, having symptoms which include sudden fever, depression, mucous discharge from the nose, reduced or ceased milk production, profuse salivation, mastitis, abortion in late pregnancy, pneumonia and temporary sterility of bulls. Mortality rate in most uncomplicated cases is usually less than 2% (Johnson et al., 2012). BEFV is currently present in all of Africa, the

Middle East, Asia, and Australia (Walker, 2005).

MESONIVIRIDAE

This family of viruses belong to the order , alongside , Roniviridae,

Coronaviridae (Warrilow et al., 2014). Mesoniviridae has one species – Mesonivirus, which

23 was first described by Junglen et al. (2009). The family comprises a group of positive-sense single stranded RNA insect viruses (Lauber et al., 2012). About six mesoniviruses have been described till date, they include Cavally (CavV), DakNong (DKNG), Hana (HanaV), Meno

(MenoV), Nam Dinh (NDiV) and Nse (NseV), which were all isolated from naturally infected mosquitoes. These viruses have been found in two continents: Côte d‟Ivoire (West Africa) and

Vietnam (Southeast Asia) (Junglen et al., 2009; Nga et al., 2011; Zirkel et al., 2013). Another mesonivirus was described by Warrilow et al. (2014) in the Northern Territory of Australia, which they named Casuarina virus (CASV). However, these mosquito-borne viruses do not appear to infect vertebrates or to cause illness in humans or livestock, they are nonetheless of interest because of the structural and genetic similarities to other members of the order

(Vasilakis et al., 2014).

Other arboviruses include the of the ; a virus family with 6-

8 segments of linear negative sense single stranded RNA genome. The virion has pleomorphic structure, and has envelope, which can be spherical or filamentous. A spherical virion has diameter of about 50-120 nm, while filamentous virions are about 20 nm wide and has length ranging between 200-300 nm. This family has 6 known genera, which are: Influenza A,

Influenza B, Influenza C, Isavirus, and Quanranjavirus (ViralZone, 2010). Of all the 6 genera, only the Thogotoviruses are arboviruses, affecting both vertebrates and invertebrates (e.g. ticks and mosquitoes) (Jones and Nuttall, 1989; Johnson et al., 2012).

Statement of Problem

Recently, there has be an increase in outbreak of emerging and reemerging diseases throughout the world, , Ebola fever, and Zika infection. A number of factors, such as, global warming, climate change, globalization, increased migration, war, poverty, flood, ease of travel, and evolution or mutation of infectious agents, may be responsible. Consequently, there is increase in the number and spread of disease vectors which include arthropods. It is therefore

24 very important to understand the role of vectors in the spread or transmission of these arboviral infections, and this understanding can be used in the development of novel strategies in controlling the spread of diseases.

Rationale

Broad objectives of study

This study was aimed at surveillance of arboviruses in mosquitoes in Kara cattle market.

Specific objectives of study

i. To identify mosquito vectors of arboviruses at Kara, Ogun State.

ii. To describe arbovirus distribution in Kara and attempt to assess the risk of human

exposure to arboviral diseases. iii. To identify and characterize existing or novel arboviruses isolated form mosquitoes

trapped.

25 CHAPTER TWO

MATERIALS AND METHODS

Study area

The study was carried out in Kara cattle market, located at Isheri, along Lagos-Ibadan expressway. This is a suburb of Lagos State, though precisely in Ogun state, Nigeria. The cattle market/abattoir is very important to the residents of both Ogun and Lagos states; as about 40-

50% of daily beef comes from this market. Kara cattle market houses cattle, sheep, goats, dogs, egrets and fowls (Plate 6). It represents a unique interaction between humans and livestock.

The market is bordered on one side by the Ogun River. The Ogun River is about 410 km long, arising from Oyo State, running through Ogun State and emptying into the Lagoon in Lagos

State.The river serves various purposes for both the human residents and the animals in Kara market. The river is used for bathing, washing, transportation and also drinking (Plate 7). It also serves as a drain for organic wastes discharge from both man and animal (Plate 8).

26

Plate 6: Cattle and sheep kept and sold at Kara market

27

Plate 7: A canoe transporting passengers on the Ogun River

28

Plate 8: An overview of the environmental conditions at Kara market/abattoir

29 The study area falls under the tropical rainforest vegetation, having high rainfall between

March and July every year, followed by a slight break in August, and then short rainy season between September and November. The dry season begins around December and lasts till

February. The study area is considered a sub-, and therefore lacks vegetation, except those along the riverbank, basically, water plants. This study involved collection of mosquitoes, which are assumed to have interactions with humans, birds and animals in the market.

Mosquito trapping

Mosquitoes were trapped between March and April, 2016; around the beginning of the rainy season. Five (5) CDC light traps baited with carbon dioxide (dry ice) were suspended at five different locations at approximately 7:00pm, and left overnight for about 12 hours (Figure 9).

Traps were retrieved every morning at about 7:00am. The trapped mosquitoes were kept in the freezer and left to die at cold temperature below 00C. The frozen mosquitoes from individual trap are then transferred into petri dishes, sealed, labelled and stored at temperature below 00C.

Weather conditions for each day was also observed and recorded.

30

Plate 9: Set-up of Carbon dioxide-baited light traps

31 Mosquito identification

Stored mosquitoes were transported on dry ice to the laboratory, Department of Biological

Sciences, Redeemer‟s University, Ede, Osun State, where identification was done. Mosquitoes were placed on an ice table, viewed under microscope and identified into genus using morphological keys. Some mosquitoes could not be identified due to missing parts such as wings, broken or missing limbs. Identified mosquitoes were pooled by species and sex into tubes; each pool containing between 1-50 mosquitoes. All laboratory processes were performed on ice.

Mosquito homogenization

About 1 ml of cooled Dulbecco‟s Modified Eagle Medium, DMEM (composed of 500 ml

DMEM high glucose, with L-Glutamine, 1 ml penicillin-streptomycin, 15 ml Fetal Bovine

Serum (FBS) and 5 ml Amphotericin B) was added into each of the tubes containing the pooled mosquitoes. About I.5 ml of Ziconia beads (Biospec; 2.0 mm) was then added to the tubes, and the mixture was vortexed twice; each time for 2 minutes or until mosquitoes were completely homogenised. The mixture was centrifuged in a centrifuge at 4,500xg for 15 minutes. The supernatant was carefully pipetted into sterile tubes and stored at -200C. All steps of the homogenization were done on ice.

EXTRACTION OF RIBONUCLEIC ACID (RNA)

For purification of viral RNA, QIAGEN QIAamp Viral RNA Mini Kit (Qiagen, Germany) was used. Lyophilised carrier RNA (310 µg) was re-suspended in 310 µl of elution buffer, AVE, giving a final concentration of 1 µg/µl. Aliquots were made from this stock solution and stored at -20oC. At the beginning of each extraction, a mixture of lysis buffer, AVL and re-suspended carrier RNA is freshly prepared.

32 One hundred and forty microliter of the mosquito homogenate was added to 560 l mixture of lysis buffer, AVL and re-suspended carrier RNA. The mixture was mixed by vortexing and spun down briefly, then allowed to incubate for 10 minutes. To the mixture was added 560 l of ethanol (99%), and mixed thoroughly by vortexing, then spun down. Six hundred and thirty of this mixture was transferred to viral RNA column and centrifuged at 8,000 rpm for 1 minute.

The collection tube and its content was discarded. The column was then transferred into a new collection tube, and the remaining 630 l sample mixture was transferred into the column, then centrifuged at 8,000 rpm for 1 minute. Again, the collection tube and its content was discarded, and the column transferred into another collection tube. 500 l of washing buffer, AW1 was then transferred into the column, centrifuged at 8,000 rpm for 1 minute. The collection tube was discarded and replaced by another. 500 l of washing buffer, AW2 was transferred into the column, centrifuged at 14,000 rpm for 3 minutes. The collection tube was discarded and column transferred into another collection tube, then centrifuged at full speed for 1 minute, so as to completely dry the column and remove residual ethanol. The column was then transferred into 1.5 ml safe-lock vial and 60 l elution buffer, AVE was transferred into the column and centrifuged at 8,000 rpm for 1 minute. The eluted solution contains the extracted RNA.

Rehydration of primers

Tubes containing lyophilised primers were centrifuged briefly. Primers were then re-suspended by adding indicated volume of sterile PCR-grade water, according to manufacturer‟s specifications (Eurofins Genomics), to make a final concentration of 100 pmol/l. The mixture was allowed to incubate for 5 minutes at room temperature, then mixed by vortexing and spun down. Working concentrations (10 pmol/l and 20 pmol/l) were made from the stock solution

(100pmol/l). Aliquots of the primers were made and stored at -20°C.

33 Table 1: Primers Sequence used for this project

S/N PRIMER PRIMER SEQUENCE NAME 5‟ – 3‟

1. mFU1 TACAACATGATGGGAAAGCGAGAGAAAAA

(29)

2. CFD2 GTGTCCCAGCCGGCGGTGTCATCAGC (26)

3. Phlebo_fwd1 TTTGCTTATCAAGGATTTGATGC (23)

4. Phlebo_fwd2 TTTGCTTATCAAGGATTTGACC (22)

5. Phlebo_Rev TCAATCAGTCCAGCAAAGCTGGGATGCATCAT

(32)

6. PVO3_1 AAT AAA TCA TAA CCA DMC BTT TTG YCK

YAR RCC TTC (36)

7. PVO4_1 AAT AAA TCA TAA RAA GGY AGR TTT TTY

KCD YTR ATG (36)

8. MESOV_F TGGHGATKCRGAATTCATGCG (21)

9. MESOV_R ATCCCAACCRCCRTATTGTGC (21)

KEY: mFU1- Pan-Flavivrus forward primer; CFD2- Pan-Flavivirus Reverse primer; Phlebo_fwd1- Pan- Phlebovirus forward primer 1; Phlebo_fwd2- Pan-Phlebovirus forward primer 2; Phlebo_Rev- Pan-Phlebovirus reverse primer; PVO3_1- Pan-Rhabdovirus forward primer; PVO4_1- Pan-Rhabdovirus reverse primer; MESOV_F- Mesonivirus forward primer; MESOV_R- Mesonivirus reverse primer

34 PCR mastermix

Various manufacturers offer one-step reverse transcription PCR kits. However, the

SuperScript® III One-Step RT-PCR Kit (Invitrogen), was used for this study.

Pan-flavivirus

The PCR mastermix for Pan-Flavivirus was prepared by mixing the following: 127.5 µl of nuclease- free, PCR-grade water; 637.5 µl of 2x reaction buffer mix; 102 µl of 10 M forward primers

(mFU1); 102 µl of 10 M reverse primers (CFD2); and 51 µl of platinum tag enzyme mix.

The total volume of the mastermix was 1020 µl, which was used for fifty (50) reactions.

Pan-phlebovirus

The PCR mastermix for Pan-Phlebovirus was prepared by mixing the following: 127.5 µl of nuclease-free, PCR-grade water; 637.5 µl of 2x reaction buffer mix; 51 µl of 20 M forward primers (Phlebo fwd 1); 51 µl of 20 M forward primers (Phlebo fwd 2); 102 µl 0f 20 M reverse primers (Phlebo Rvs); and 51 µl of platinum tag enzyme mix. The total volume of the mastermix was 1020 µl; which was used for fifty (50) reactions.

Pan-rhabdovirus

The PCR mastermix for Pan-Rhabdovirus was prepared by mixing the following: 127.5 µl of nuclease-free, PCR-grade water; 637.5 µl of 2x reaction buffer mix; 102 µl of 20 M forward primers (PVO3_1); 102 l of 20 M reverse primers (PVO4_1); and 51 µl of platinum tag enzyme mix. The total volume of the mastermix was 1020 µl; which was used for fifty (50) reactions.

35 Mesonivirus

The PCR mastermix for Mesonivirus was prepared by mixing the following: 127.5 µl of nuclease-free, PCR-grade water; 612 µl of 2x reaction buffer mix; 25.5 l of magnesium sulphate (MgSO4); 102 µl of 10 M forward primers (MESOV_F); 102 l of 10 M reverse primers (MESOV_R); and 51 µl of platinum tag enzyme mix. The total volume of the mastermix was 1020 µl; which was used for fifty (50) reactions.

The enzyme mix was left in the freezer until it was to be added. Twenty microlitre each of the

PCR mastermix was transferred into PCR tubes and 5 µl of the extracted/purified RNA sample was added, making total volume per reaction to be 25 µl.

The positive control for each virus family is as follows: Pan-Flavivirus PCR - Murray valley encephalitis virus; Pan-Phlebovirus PCR – Punto Toro virus; Pan-Rhabdovirus PCR - vesicular stomatitis virus; Mesonivirus PCR – Mesonivirus.

THERMAL PROFILE

Cycling protocol was the same for Pan-Flavivirus, Pan-Phlebovirus and Pan-Rhabdovirus

PCRs. It began with reverse transcription process: denaturation at 600C for 1 minute; synthesis of complementary DNA (cDNA) at temperature of 500C for 45 minutes. This was then followed by amplification of the cDNA: denaturation at 940C for 2 minutes, then maintaining the temperature at 940C for another 15 seconds; annealing of primers at 550C for 30 seconds; elongation at 680C for 30 seconds. The process of denaturation, annealing and elongation was repeated for forty-five (45) times; which was then followed by termination at 680C for 7 minutes. The PCR products (amplicons) were stored at 40C until needed for further analysis.

However, thermal profile for Mesonivirus PCR was different. It begins with reverse transcription process to synthesize cDNA: temperature was set at 500C for 50 minutes, the temperature was later raised to 940C for 2 minutes. The reverse transcription process was then

36 followed by the amplification cycle of the cDNA: denaturation at 940C for 20 seconds; annealing of primers at 550C for 45 seconds; and elongation at 680C for 1 minute. The amplification cycle was repeated for forty-five (45) times, which was then followed by termination at temperature of 680C for 7 minutes. The PCR products (amplicons) were stored at 40C until needed for further analysis

Agarose gel electrophoresis

Two percent (2%) standard agarose gel with 5 mm-comb was casted and placed in the electrophoresis tank. Into each well of the gel, was loaded a mixture of 5 µl PCR product and

2 µl 6x loading dye. The first well on the gel was loaded with 100 base-pair (bp) DNA ladder.

The electrophoresis machine was then programmed to run for 30 minutes at 90 V, this allowed a run distance of about 3-4 cm.

PCR products were sequenced at least twice in each direction by conventional Sanger technology (LGC, Berlin, Germany).

37 CHAPTER THREE

RESULTS

Mosquito abundance

A total of 1,650 mosquitoes were collected, of these, 1,626 were successfully identified and pooled by species and sex. Of the 1,626 successfully identified mosquitoes – 1,461 (89.85%), comprising 1,435 females; 26 males, were Culex species, 57 (3.51%) – all females, were Aedes species, 58 (3.57%) – all females, were Mansonia species, and 50 (3.08%), comprising 1 female; 49 male, were Anopheles species (Table 2). A total of 1,551 female mosquitoes and total of 75 male mosquitoes were identified. All mosquitoes were trapped with the CDC CO2 baited light traps.

38 Table 2: Identified Mosquitoes

Culex Aedes Mansonia Anopheles Total

Female 1,435 57 58 1 1,551

Male 26 0 0 49 75

Total 1,461 57 58 50 1,626

39 Sum of mosquitoes trapped by species per location

The sum of mosquito species (Anopheles, Culex, Aedes, and Mansonia) collected differed per location (Figure 1). Location A produced the highest population of Culex species – 509, while location E produced the lowest population of Culex species – 117. Location A also produced the highest population of Anopheles species – 14, while location D produced the lowest population of Anopheles species – 6. The highest population of Aedes species was trapped at locations A and B; each having 19, while location C had the lowest population of Aedes species

– 3. The highest population of Mansonia species was trapped at location B – 17, while location

C had the lowest population of Mansonia species – 2. Locations A-E were strategic places within the cattle market.

40 1600

1461

1400

1200

1000

d e Sum of Anopheles 800 pp Sum of Culex a

r T

Sum of Aedes

s e Sum of Mansoni

ito 600 qu os 509

M 443

400

236

200 156 117 50 5758 19 19 14 13 12 17 9 3 2 6 6 11 9 1015 0 A B C D E Total Mosquito species per location

Figure 1: Chart showing sum of mosquito species trapped per location

41 Sum of mosquitoes trapped by location

The sum of mosquitoes trapped by location varied significantly as shown in figure 2 below.

Point “A” had the highest catch, with a total of 555 mosquitoes. Next to this is Point “D”, with a total of 466 mosquitoes; followed by Point “B”, with total of 284 mosquitoes; then Point “C”, with a total of 170 mosquitoes. Point “E” had the lowest catch, with a total of 151 mosquitoes.

42 600

555

500 466

d 400 e

pp a

r T

s

e

ito 300 qu 284

os M

200

170 151

100

0 A B C D E Location

Figure 2: Sum of mosquitoes trapped by location

43 Sum of mosquitoes trapped by weather

During the course of this study, the weather condition varied between normal, windy, and rainy and windy. The sum of mosquitoes trapped varied with the prevailing weather conditions.

Figure 3 below shows the sum of mosquitoes trapped for the various weather conditions. The sum of mosquitoes trapped when the weather condition was normal was the highest, with a total of 1008 mosquitoes. The sum of mosquitoes trapped during the windy condition was 411.

Sum of mosquitoes trapped during the rainy and windy conditions was the least, with a total of

207 mosquitoes.

44 1200

1008 1000

800

d

e

pp

a r

T

s

e 600

ito

qu

os

M 411 400

207 200

0 Normal Rainy & Windy windy Weather Condition

Figure 3: Sum of mosquitoes trapped by weather condition

45 Sum of mosquitoes trapped by day and weather

The sum of mosquitoes trapped for each day and its prevailing weather condition is described in Figure 4 below. Day 8, having a normal weather condition, had the highest number of trapped mosquitoes – 293. Also, Day 11, with a windy weather condition, had a total number of 233 trapped mosquitoes; Day 10, with a rainy and windy weather condition, had a total of

207 trapped mosquitoes.

46 350

300 293

d

e 250

234 233 pp

a

r T

216 s

e 207

ito 200

qu

os

M

of

m 150 Su 134

110

100

68

47 50 44 36

2 2 0 DAY 6 DAY 12 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 10 DAY 1 DAY 11 DAY 9

Normal Rainy & windy Windy Day and Weather

Figure 4: Sum of mosquitoes trapped per day, with its prevailing weather condition

47 Sum of mosquitoes trapped by weather condition per location

The sum of mosquitoes trapped by weather condition for each location varied significantly

(Figure 5). During a normal weather condition, “Point A”, had the highest number of trapped mosquitoes – 351, while “Point C” had the least – 135. When the weather condition was rainy and windy, “Point D” had the highest number of trapped mosquitoes – 133, while “Point B” had the least – 21. Also, when the weather condition was windy, “Point D” had the highest number of trapped mosquitoes – 178, while “Point E” had the least – 6.

48 400

351 350

300

250 d

e

222 pp

a r

T

s

e 200

ito 178

qu

os 155 151

M 150 145 135 133

100

53 50 41 35 21 6 0 A B C D E A B D A B C D E

Normal Rainy & Windy windy Weather condition per location

Figure 5: Sum of mosquitoes trapped by weather condition per location

49 Evaluation of the RT-PCR assay analysing mosquito samples

Overall, a total of 50 mosquito pools were analysed using SuperScript® III One-Step RT-PCR

Kit (Invitrogen). Pool 1 contains Anopheles mosquitoes, pools 2-5 contain Mansonia mosquitoes, pools 6-12 contain Aedes mosquitoes, while pools 13-50 contain Culex mosquitoes. Four different arbovirus families were screened and the results for each family is discussed below.

RT-PCR for pan-flavivirus

Of the 50 mosquito pools screened, only six (6) were positive for Pan-Flavivirus. They include pools 2, 6, 9, 13, 15, and 35. This was evident in the gel electrophoresis results of the PCR products, as they showed bands around 260 bp (Figure 6). The remaining 44 pools were negative for Pan-Flavivirus.

RT-PCR for pan-phlebovirus

Of the 50 mosquito pools screened for Pan-Phlebovirus, none was positive. This was evident in the gel electrophoresis result of the PCR product, as there was no band at 370 bp (Figure 7).

RT-PCR for pan-rhabdovirus

Of the 50 mosquito pools screened for Pan-Rhabdovirus, twenty-seven (27) pools were positive. The positive pools include pools 3, 5, 6, 10, 11, 12, 17, 20, 21, 22, 23, 25, 26, 27, 28,

30, 31, 32, 34, 37, 38, 41, 43, 44, 46, 48, and 50. This was evident in the gel electrophoresis results of the PCR products, as they showed bands around 470 bp (Figure 8). The remaining

23 pools were negative for Pan-Rhabdovirus.

50

Figure 8: Gel result for Pan-Rhabdovirus showing 470 bp

51 RT-PCR for mesonivirus

Of the 50 mosquito pools screened for Mesonivirus, about five (5) pools seem to be positive.

These pools had band between 200 bp and 300 bp (Figure 9); they include pools 32, 37, 38, 46, and 47. The remaining 45 pools were negative for Mesonivirus.

52 CHAPTER FOUR

DISCUSSION, CONCLUSION AND RECOMMENDATIONS

This surveillance study was conducted to investigate the presence or absence of arboviruses in mosquitoes collected at Kara cattle market, beside Ojodu Berger bus-stop, Ogun state, southwest, Nigeria. The mosquitoes are expected to have had interactions with both man and animals (cattle, sheep, goats, dogs, cats and birds) in the market. This study screened for four mosquito-borne arbovirus families, which included Pan-Flavivirus, Pan-Phlebovirus, Pan-

Rhabdovirus and Mesonivirus.

Arboviruses are widespread in Nigeria, due to the abundance of mosquito vectors, which are responsible for their transmission (Ayukekbong, 2014). The role of arboviruses as a major source of viral infections of man in Nigeria has been established by Moore et al., 1975;

Fagbami, 1978 and Baba et al., 2013. Quite a number of researches have been done on arboviruses in Nigeria, which established that arboviruses are endemic in Nigeria. Arboviruses such as Yellow fever virus, Dengue virus, Chikungunya virus, Dugbe virus, Igbo-ora Virus,

Bwamba virus, Shuni virus, Lembombo virus, Zika virus, Tataguine virus, Thogoto virus among others. These viruses were isolated and identified using virological, immunological and seroepidemiological techniques (Moore et al., 1975; Fagbami, 1978).

Baba et al. (2013) reported evidence of arboviral co-infection in patients having malaria and typhoid. Their study adopted serological and immunological techniques to screen for Yellow fever virus, Dengue virus, West Nile virus and Chikungunya virus. Very little or no molecular technique has been used in the surveillance or detection of arboviruses in Nigeria, until very recently, by Sule and Oluwayelu (2016).

In this study, mosquitoes (Aedes, Culex, and Mansonia) were implicated as key vectors or reservoirs of the arbovirus families examined. It was observed that a greater population of

Culex mosquitoes (89.85%) than Aedes (3.51%), Mansonia (3.57%) and Anopheles (3.08%)

53 were collected. A likely cause for the very high population of Culex mosquitoes might be due to their activeness from dusk to dawn - they are considered as “night-biters”; Aedes on the other hand are mostly “day-biters” (WHO, 1997). Studies by Baba et al., 2006; Özer et al., 2007;

Vaux et al., 2015; LaBeaud et al., 2011; and Mweya et al., 2015 also reported collection of greater proportion of Culex mosquitoes (Sule and Oluwayelu, 2016).

Arboviruses are considered as one of the most important emerging/re-emerging infectious diseases in many parts of the world. There are a number of factors that favour the resurgence and spread of these arboviruses or their arthropod vectors globally. Some of these factors include , explosive growth in population, deforestation, agricultural practices such as irrigation, global warming and climate change and evolutionary changes in genomes of these viruses, among others (Baba et al., 2013). As a result of these, the need for sustainable and continuous surveillance of arboviruses is highly imperative.

In this study, the sum of mosquitoes trapped at the various location varied significantly (Figure

2). Point “A” had the highest number of trapped mosquitoes – 555, while Point “E” had the lowest number of trapped mosquitoes – 151. Point “A” was very close to the slaughter slab where there were lots of cattle, it also has form of roof, which helped to reduce the effect of wind on the collection of mosquitoes. Point “E” on the other hand was very close to the river bank, which had very little vegetation, the river flowed freely around point “E”. It also lacked any form of roof covering, hence, wind easily affected collection of mosquitoes at point “E”.

These factors – presence of animals (food) and air movement can be implicated for variation in the number of mosquitoes trapped per location.

Although the sampling in this study began at the onset of the rainy season (March and April), the weather condition during this study was classified into three; which are normal, windy, and rainy and windy. The “normal” weather condition was characterised by steady and still air movement, temperature ranging between 23-270C, lack of precipitation (in form of rain). The

54 “windy” condition was characterised by fast air movement, temperature of about 220C, slight precipitation (in form of dew). Lastly, the “rainy and windy” condition was characterised by relatively fast air movement, very cold temperature, and high amount of precipitation (rainfall).

The sum of mosquitoes trapped with regards to the three different weather conditions in this study varied (Figure 3). The sum of mosquitoes trapped when the weather condition was normal was the highest, with a total of 1008 mosquitoes. The sum of mosquitoes trapped during the windy condition was 411. Sum of mosquitoes trapped during the rainy and windy conditions was the least, with a total of 207 mosquitoes. Mosquitoes are known to be easily swept off by wind; also, they are usually in their hiding place during periods of heavy rainfall.

These two factors can be responsible for the variance in the sum of mosquitoes trapped during each weather condition, which was lowest during the “rainy and windy” condition – 207. It can be assumed that the mosquitoes were held in their hiding place when it was rainy and windy, hence, the low catch.

The Reverse-transcriptase polymerase chain reaction (RT-PCR) carried out for Pan-Flavivirus showed 12% positive samples.. Sample 2 contained RNA extracted from a pool of Mansonia mosquitoes; samples 6 and 9 were RNA extracted from pools of Aedes mosquitoes; while sample 15 and 35 were RNA extracted from pools of Culex species. Earlier studies by Moore et al. (1975); Fagbami (1978) and Baba et al. (2013) established that are considered endemic in Nigeria. In this study, it was observed that the trio of Aedes, Mansonia and Culex carried flaviviruses, and they could transmit it to any of the vertebrate hosts in the area, which include man and his economic livestock.

From the RT-PCR carried out for Pan-Phlebovirus, which belong to the Bunyaviridae family, none of the 50 samples turned out positive. This showed that Phlebovirus (e.g. Rift Valley fever virus) was not carried by any of the mosquito species in the study area. This may also

55 either mean that this virus family is totally absent in the population or that the viremia titre is low.

Results from the RT-PCR done for Pan-Rhabdovirus revealed that 54% of the samples were positive. The positive pools/samples include pools 3, 5, 6, 10, 11, 12, 17, 20, 21, 22, 23, 25,

26, 27, 28, 30, 31, 32, 34, 37, 38, 41, 43, 44, 46, 48, and 50. Samples 3 and 5 contained RNA from Mansonia mosquitoes; samples 6, 10, 11, and12 were RNA from pools of Aedes mosquitoes; while samples 17, 20, 21, 22, 23, 25, 26, 27, 28, 30, 31, 32, 34, 37, 38, 41, 43, 44,

46, 48, and 50 were RNA extracted from Culex mosquitoes. Rhabdoviruses have been poorly reported in Nigeria. However, Stremlau et al. (2015), recently discovered two novel viruses in the blood of healthy individuals from West Africa. They used next generation sequencing to discovered new rhabdoviruses which they named Ekpoma virus-1 (EKV-1) and Ekpoma virus-

2 (EKV-2). These viruses are closely related to members of the genus Tibrovirus and Bas-

Congo virus (BASV), but they are not known to be arthropod-borne. Rabies virus and vesicular stomatitis virus, to mention a few, are of public health importance, hence, more studies should be done on rhabdoviruses. From the RT-PCR results discussed above, it will be noted that the trio of Aedes, Culex, and Mansonia are culpable carriers/transmitters of rhabdoviruses.

From the RT-PCR results and gel evaluation for mesonivirus, only 1% of the samples seemed positive. The positive samples include 32, 37, 38, 46 and 47. All of these samples were RNA extracted from pools of Culex mosquitoes. However, the bands shown on the gel were somewhere between 200 and 300 bp, they were not exactly on 270 bp. This therefore calls for further analysis such as sequencing of the product, to confirm the true identities of the nucleic acids. Mesonivirus is a relatively new arbovirus family, and there has not been any report of this family in Nigeria. Mesonivirus were isolated from mosquitoes collected from Côte d‟Ivoire and Vietnam (Junglen et al., 2009).

56 Limitation

This study was however limited by finances. The cost of dry-ice which was used as CO2 was high; cost of sequencing the PCR products was high.

Conclusion and Recommendations

Further work should be carried out to sequence the positive samples and be sure of the exact viruses present. Also, surveillance of other arbovirus families such as Togaviridae, Reoviridae,

Asfarviridae and other not included in this study, should be done. From the results discussed above, it is very important to have a continuous surveillance for arboviruses as the mosquitoes

– which are vectors of these arboviruses are relatively abundant in our environment. The result also established that the prevalence of Rhabdoviruses is quite high in the study area, as 54% of the samples were positive. This therefore calls for adequate attention to monitor and control arboviral infections. Proper and regular sanitation of the market should done. Appropriate sewage disposal system should be constructed in the market. Control of the mosquito vectors can be achieved by cultivating proper hygiene practices, use of biological controls agents such as fish, Notonecta species, predatory copepods, entomopathogenic bacteria, and Lagenidium giganteum (fungus) (Lacey and Orr, 1994).

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