Wolbachia pipientis： A potential candidate for combating and eradicating dengue epidemics in Pakistan

Uruj Tahir， Umair Hassan Khan， Muhammad Saad Zubair， Bahar-e-Mustafa

1Department of Environmental Sciences， Fatima Jinnah Women University， Rawalpindi， Pakistan

2Department of Microbiology， University of Agriculture Faisalabad， Sub-Campus Toba Tek Singh， Pakistan3Department of Microbiology， Government College University Faisalabad， Pakistan

2Department of Microbiology， University of Agriculture Faisalabad， Sub-Campus Toba Tek Singh， Pakistan

Wolbachia pipientis： A potential candidate for combating and eradicating dengue epidemics in Pakistan

Uruj Tahir1*， Umair Hassan Khan2， Muhammad Saad Zubair3， Bahar-e-Mustafa2

1Department of Environmental Sciences， Fatima Jinnah Women University， Rawalpindi， Pakistan

2Department of Microbiology， University of Agriculture Faisalabad， Sub-Campus Toba Tek Singh， Pakistan3Department of Microbiology， Government College University Faisalabad， Pakistan

2Department of Microbiology， University of Agriculture Faisalabad， Sub-Campus Toba Tek Singh， Pakistan

ARTICLE INFO

Article history：

in revised form 3 October 2015

Accepted 3 November 2015

Available online 20 December 2015

Dengue

Pakistan

Antiviral agents

Chemical methods

Biological control

Wolbachia pipientis

Dengue virus syndrome is an emerging global health challenge which is endemic in tropical countries like Pakistan. In recent years dengue incidences have increased considerably in different areas of Pakistan with more sever impacts on urban and peri-urban populations. This review is an effort to highlight the changing epidemiology of dengue fever， role of Government of Pakistan in disease management and control using preventive and community based approaches in the region. Moreover， there is an emphasis on application of Wolbachia as novel， inexpensive and environmentally benign candidate for control and eradication of dengue transmitting vectors.

Document heading doi：10.1016/j.apjtm.2015.11.012

1. Introduction

Infectious diseases continue to plague the global community with high rates （26%） of morbidity and mortality. The main causes for transmittance of such emerging and reemerging disease outbreaks are unprecedented shifts in （a） environmental or climatic conditions either due to anthropogenic activities， （b） overlapping of geographical ranges， （c） change in land-use patterns or （d）emergence of drug and insecticidal resistances among vector populations［1，2］. Vector-borne diseases such as Congo hemorrhagic fever， Malaria， Dengue， Yellow fever， Chikungunya etc. account for a significant threat to human population in terms of health and economic losses all over the world. Such outbreaks lead to widespread epidemics which may result in deaths of many hundreds and millions of people annually［3，4］. These diseases have been reported to exert more devastating effects on developing orunder transition countries. Because different variables eg. water and sanitation facilities， population density， literacy rate etc. are the major players for spread of epidemics in such countries as compared to developed ones， where main contributors include ambient temperature， moisture or humidity along with rainfall patterns［1，2，5-7］. Most of these transmitted ailments are concentrated and prevalent with higher impacts in regions of South-East Asia，Southern Europe， Western Pacific and Eastern Mediterranean countries， Latin America， Australia and Sub-Saharan Africa［1，2，6，7］. As for instance， approximately 1.1 million deaths at global scale have been attributed to malaria alone［8］. Around 91% deaths involving 86% of children （＜5 years of age） were recorded in Africa because of malaria［9］.

Recently dengue fever （DF） has been recognized as one of emerging infectious diseases worldwide. It represents a considerable havoc to people living in urban and peri-urban localities of tropics and subtropics［10］. The viral epidemic has led to hospitalization of 20 million people with around 24 000 deaths as documented by Sulehri et al［7］， which now has raised up to 50-100 million infected people with 2.5 billion at risk of contracting dengue［11,12］.Pakistan has experienced a number of dengue outbreaks with more server impacts in recent years. These outbreaks are affecting larger proportion of urban population and putting significant stress on health care facilities. Unfortunately the magnitude and severity index of the disease remains unreported due to deficiency and difficulty in data collection［6,11,13］. This review attempts to describe the prevalence of dengue virus infection and its associated serotypes in Pakistan with importance of potential biological control agents in eradication of the transmitting vector/s.

2. Dengue fever， signs and symptoms

Dengue globally affects 40% of world's population and occurs in repetitive cycles （3-5 years interval）. The ailment presents high fever accompanied by frequent headaches， nausea， skin rashes，enlargement of lymph nodes， pain behind eyes， severe muscular，bone and joint pains， epitaxis along with leukocytopenia ie. reduction in white blood cells count. Thus render individuals more prone to viral infection. DF is also characterized as bone break fever or bone crush fever due to severe pain in joints and bones［6,7,12,14-18］.

3. Virology and serotypes

Being a member of a medically important viral family， Flaviviridae，the virus possesses single stranded positive sense RNA （+ve ssRNA）. Albert Sabin in 1944 classified dengue virus into four antigenically related but immunologically distinct serotypes namely DENV-1，DENV-2， DENV-3 and DENV-4［19-25］. All DENVs comprise of three structural ［Capsid （C）， membrane （prM/M） and envelope （E）］and seven nonstructural proteins （NS1， NS2a， NS2b， NS3， NS4a，NS4b and NS5）. Each of these serotypes has been reported to exhibit extensive genetic variability due to distinct genotypes. Each of these genotypes is associated with severe dengue epidemics［17,22,23,26］.

4. Principal transmission vector， breeding habitats and virus acquisition

The infection is usually transmitted through bite of infected Aedes aegypti （Ae. aegypti） （female mosquito）. The mosquito is adapted to feed exclusively on human blood and is a day time feeder［6,11,15,17］. Ae. aegypti is well-known for being susceptible to all dengue viral serotypes and as an efficient vector with capability of transmitting dengue virus to many individuals in shorter time span［20,26,27］. This species of mosquito has spread extensively in tropical and subtropical regions of the world as a result of increased national and international trade， shipping and tourism［6,11,15,17,28］. Aedes albopictus （Ae. albopictus） has also been reported to transmit dengue viruses however to much lesser extent［29］.

Ae. aegypti usually breeds in dark places， stagnant water kept in containers for （indoor and outdoor） storage， water coolers， drums，barrels， plant saucers， open buckets， in used tyres and places where rainwater collects. The mosquito unlike rural settings is specifically adapted to urban and peri-urban settings （ie. cleaner environment）. In such localities mosquito population density has been reported to be proportional to human population density. Moreover， breeding habitats of mosquitoes have strongly been associated with elevated temperatures， water supplies， sewage and sanitation facilities［6,15］.

Virus is acquired by mosquito during blood meals of viraemic（infected） patients. The acquisition is followed by incubation period（8-10 d） where viral genome is replicated within the mosquito，transferred to salivary glands via intestinal tract of mosquito. Mosquito then transmits virus to healthy persons upon subsequent biting［17,21,30,31］ and also to off springs via trans-ovarian transmission during reproductive cycles［7,21］. Once inside the human host virus starts replicating within the target organs like white blood cells， lymphatic tissues etc. and gets released into blood following circulation［32］.

5. Dengue viral syndromes

Depending upon age and immunological status dengue virus results in clinically different syndromes ranging from （a） classic DF characterized as a milder a typical flu-like form with high grade fever，head and body aches. It affects infants， young children and adults but is not fatal. Nonspecific febrile sickness with rashes has been reported in infants and young children. While mild febrile syndrome with high fever， head and joint-muscular pains along with skin rashes has been observed in older children and adults （b） dengue hemorrhagic fever（dhf） characterizes acute and continuous fever （40-41 ℃） lasting for 2-7 d， accompanying hemorrhagic manifestations （ecchymosis and petechiae） with reduction in platelets. It may lead to liver enlargement and circulatory dysfunction in severe cases， and （c） dengue shock syndrome， a complicated and prolonged illness， in which DENV-1 viral infection is followed by DENV-2 and DENV-3. Thus exhibits symptoms of both DF or DHF. Such infections lead to hypervolemic shock （i.e. increased plasma leakage due to vascular permeability） and are associated with higher morbidity and mortality rates［6，7，14，31，33-37］. Furthermore， dengue infections have been reported to be life threatening upon coexistence in asthmatic and diabetic patients or in individuals with other chronic diseases［31］. According to Raza et al［12］and Mukhtar et al［38］ approximately 250 000-500 000 people suffer from DHF and/or dengue shock syndrome and about 20 000-25 000 are succumbed to death every year.

6. Dengue in Pakistan

Pakistan being a developing urban and agricultural economy is more likely to be at risk of vector-borne epidemics. Mosquito-bornediseases such as dengue have now become a public health concern. This is due to exertion of considerable burdens of morbidity，mortality and economic distresses in many parts of the region. Over populated cities， lack of access to proper sanitation facilities，unavailability and shortage of clean drinking water， population influxes at massive scale and lack of awareness about possible health effects posed by vector transmitted pathogens are some of the leading causes of spread of mosquito-borne diseases［1，2，39］. Moreover， raised ambient temperature， huge population， overcrowded cities with poor resource settings and lack of health facilities （in terms of vaccination access and coverage） also serve as important prerequisites for reproduction of mosquitoes. This situation further exacerbates transmission of pathogens dwelling within these host vectors causing long-term illnesses［31，40］.

Dengue viral infection killing 365 individuals with 21 597 positive cases is becoming endemic in Pakistan. The viral epidemic persists all the year round with greater intensity in months of October-December （post-monsoon period）. Additionally， recent floods have enhanced the prevalence of dengue in entire country through provision of large number of breeding sites for viral vectors， thereby made the situation more worst［13，15，17，24，41］.

7. Prevalence of dengue viral serotypes in Pakistan

Outbreaks of DF recorded during 1994-2004 showed prevalence of DENV-1 and DENV-2 serotypes in different regions of Pakistan. Whereas DENV-3 was the commonest serotype in 2005 and led to severe dengue hemorrhagic infections in previously DENV-1 and DENV-2 sensitized populations. In recent years Pakistan is experiencing dengue outbreaks either major or minor mainly by DENV-2 and DENV-3［42］. For instance， the predominance and cocirculation of DENV-2 and DENV-3 serotypes have been reported during 2006， 2007 and 2009 in Pakistan. Research studies revealed that DENV-2 （subtype IV）， DENV-3 （subtype III） and DENV-4 prevailed in 2008. Likewise， serotype DENV-2 was reported to follow DENV-1 during dengue epidemics in 2010. However DENV-2 and DENV-3 serotypes caused most critical outbreaks in 2011［12，18，21，41］ suggesting that magnitude of dengue outbreaks is entirely dependent upon serotype， strain of virus， density of infected vector， number of susceptible individuals［25］.

8. Demographical facts about DF in Pakistan

History of dengue outbreaks in Pakistan dates back to early nineties（1990） usually after the commencement of rainy season. The first ever case of dengue was reported in Karachi in 1994， followed by second dengue outbreak in Baluchistan in 1995［6，7，18，43］. But now the infection is appearing on regular basis with greater impacts in densely populated urban areas including upper and central Punjab，Sindh （Hyderabad， Karachi） etc. during and soon after the rainy season［43］. In 2004， 25 cases of dengue were reported from Karachi and Islamabad. Around 500 positive cases （including 13 deaths）were recorded in Karachi by the year 2005， followed by 5 400 cases （55 deaths） in Nawabshah， Karachi， Sukhar， Islamabad and Rawalpindi and a larger proportion of dengue cases in Azad jammu and Kashmir in 2006［6，44，45］. Hyderabad， Karachi， Mirpurkhas，Haripur， Lahore， Islamabad and Rawalpindi were the more affected areas due to dengue outbreak with 2 700 positive cases and 24 deaths in 2007. Likewise 1 800 cases were reported in Lahore in 2008， followed by 320 cases reported in Gujranwala， Lahore and Sheikhpura in 2010［46，47］. The worst strike of dengue （DF and DHF）was witnessed in 2011， with ＞ 20 000 cases （including 300 deaths），with major havoc in Lahore followed by Faisalabad， Rawalpindi and Sargodha［21，43］.

9. Strategies for management and control of dengue viral outbreaks

Since the epidemiology of DF with reference to Pakistan has changed during the recent era with increased frequency of occurrence and more drastic effects. Therefore immediate actions need to be taken for controlling dengue transmitting vector in order to prevent wide spread of epidemics. Government of Pakistan has taken some crucial and important steps in managing the disease by launching various health line projects and awareness campaigns etc.

10. Role of Government of Pakistan in reducing viral epidemics at administrative level.

Government of Pakistan as well as government of Punjab has taken many initiatives at administrative level for controlling the spread of disease. For instance， Punjab Health Line Project for Dengue has been launched by government of Punjab for sharing information on disease recognition （signs and symptoms）， identification of epidemically affected areas along with provision of help to suspected individuals. The government has also established 24/7 operating mobile teams for providing free treatment to affectees with particular emphasis in rural areas［6］.

11. Environmental management and community based approaches for reduction of mosquito population

Government of Pakistan has also taken environmental management initiatives in an effort to control dengue transmitting vector. Specialized community based programs have been started for proper solid waste collection and disposal in addition to improved water storage practices. The local community has been actively engaged along with concerned （municipal and public health） authorities to ensure street cleaning， proper drainage systems and elimination ofpools of stagnant water. Proper coverage of water storage containers is made mandatory for preventing access of female mosquitoes for egg laying. The water air coolers must be emptied or drained out when not in use. Water containing objects like plant saucers should be removed from house-gardens， old tyres must be recycled or disposed off properly［6，7，15，17，48］. Moreover latest GIS based systems and tools are now being used for identification and mapping of hotspots for dengue infection in order to develop and implement national preparedness plans such as disease early warning and response systems［6，12，18，21］.

12. Educational initiatives

Numerous awareness campaigns have been launched in association with electronic and print media. Additionally， dengue related information has also been included in the syllabi of various educational institutes especially at school level［17］. The Health Services Academy has also launched （12 months） diploma courses in medical entomology and disease vector control in Pakistan at postgraduate level for training the manpower to effectively and efficiently deal with vector-borne diseases［49］. Such programs will be more effective in producing more specialists to address the challenges posed by vector-borne diseases like dengue.

13. Reducing contact with dengue mosquito through personal actions

One of the most important measures for reduction in dengue virus transmission is the interruption of host-mosquito contact. Individuals should wear full length pants， trousers and full sleeved clothes to cover the limbs. The conventional measures such as mosquito repellents or lotions must be used for protection and to avoid mosquito biting during day time hours. Moreover coils，electric vapor mats， trap lights， insecticide treated nets and curtains etc. should be used to prevent direct contact with mosquitoes［7，17，21］. Windows and doors should be kept closed. Moreover， use of indoor air conditioning systems for cooling can also be helpful in avoiding direct contact of humans with transmitting vector［17］.

14. Chemical methods for controlling mosquito population

Spray teams have been assembled by governmental authorities. These teams are responsible for time to time spraying， fumigation or fogging in residential areas and streets of various cities［6］. Suitable and long-lasting insecticides such as larvicides or adulticides are periodically applied to breeding habitats for reducing and controlling vector population［7，17］.

15. Discovering effective antiviral vaccinations

Since no effective treatment or medication is available for dengue virus and/or fever to date. Therefore discovery of effective antiviral drugs and/or vaccines capable of targeting multiple serotypes of dengue virus is exigently needed. The vaccination needs to be effective， must provide life time immunity against viral pathogens as well as lessen the risks of formation of cross reacting antibodies［18，50］. Live attenuated and inactive dengue viruses， cloned and chimeric viruses， subunit and DNA vaccines are some of the approaches that are recently being used for developing effective vaccination for treating dengue viral infections［31］.

Phytochemicals （plants and plant-derived compounds） have always served as important source in discovery of many antiviral drugs. Therefore activity of various plant-derived phenolic compounds has been evaluated by the researchers. For instance， Zandi et al［51］assessed antiviral activity of bioflavonoids against DENV-2 serotype at different infectious stages and virus replication. They concluded that quercetin was most effective viral inhibitor amongst all the tested compounds. Similarly， Carica papaya leaf extracts exhibited potential activity against dengue on administration to dengue infected patient［52］. Moreover， significance of various phytochemicals against DF has been shared in details by Sohail et al［53］.

Most recent advancements related to dengue specific antiviral agents have been reviewed and explained explicitly by Julander et al［50］. Idrees and Ashfaq［54］ have also reviewed the potential of siRNA as next generation therapeutic agents against dengue viral infection. Reports have revealed that vaccination （comprised of proteins from all the 4 viral strains） against dengue virus is under trial and testing processes （on monkeys）. It is likely to be proved effective in treating the disease caused by all serotypes［18］. Moreover，the trials of tetravalent vaccines exhibited 89% sero-conversion against four serotypes after administration of third dose. While the administration of this vaccine in two doses provided 80%-90% protection in children. Similar results for sero-conversion rates have been recorded in adult volunteers for other vaccines prepared by Walter Reed Army Institute of Research［7］. Furthermore， numerous academic and academy associated research centers are actively engaged in discovering and developing anti-dengue viral drugs with hopeful results［17］. Researches and fully funded projects on various aspects of dengue virus and transmitting vectors （mosquito）with reference to serotypic and genotypic analysis also need to be launched by government of Pakistan on large scale at national level for understanding disease development and complications. These researches will hopefully be leading to breakthroughs in discovering and developing ideal dengue vaccines［55，56］.

16. Biological methods for controlling mosquito population

Due to absence of effective vaccination and/or antiviral agents，current dengue vector control methods heavily rely on usage of chemically based insecticides. Though seem to be useful in reducing larval or adult populations of mosquitoes during dengue epidemics，these insecticides are effective against Ae. aegypti population only over a small geographical scale. Conversely， non-targeted populations of other insects are also at risk of being affected by insecticides. Moreover accumulation of these insecticides in various environmental compartments leads to health and pollution problems along with evolution of insecticidal resistance among mosquito populations over passage of time. Hence， failure of chemical insecticide-based programs in preventing disease incidences and their escalating costs， especially in developing countries， has spurred the exploration of novel， cost effective and environment friendly control measures. These strategies should be capable of efficiently eliminating reliance upon such chemical toxins［16，57，58］.

Biological control usually refers to introduction and/or release of biological agents persisting in nature into particular environment for controlling pests either through predation， disease or parasitism. These techniques have been widely utilized for protection of agriculturally important crops and livestock against pest outbreaks，plants or animals causing damage to environment. However，application of such biological control agents in public health sector is relatively recent approach that is still at infancy. This strategy for combating and controlling mosquito-borne epidemics has attracted the attention of many researchers. Presently there exists numerous biological control methods and many are under development process for controlling dengue vector for instance copepods， pathogenic bacteria and fungi etc.［58-60］.

17. Fish and copepods as biological control agents

Introduction of mosquito predators such as fish， spiders， geckos and copepods （small crustaceans） is somewhat beneficial in combating mosquito-borne diseases. They prey or feed on mosquito larvae thus minimizing the social distress［58］. Such copepods trails for eradication of dengue have been conducted in Honduras， Vietnam［60-63］， where field trails proved to be successful together with government and community participation. However being expensive and requiring continuous intercessions these strategies are unlikely to be implemented in peri-urban and urban communities［16，58］.

18. Bioinsecticides as biological control agents

Constant monitoring and community interventions for applications of natural predators along with adverse environmental impacts（including humans and nature） posed by chemically-based insecticide vector control approaches. Additionally the loss of sensitivity of these vectors against insecticides has necessitated the discovery of environment friendly alternatives ie. biopesticides［64，65］. Microbial insecticides comprised of microorganisms and their byproducts seem to be more effective and valuable alternatives for vector reduction in terms of target specificity and lower toxicity to pesticide user［66］. The entomopathogenic bacterium Bacillus therungenesis subsp. israelensis （BTi） was found to be a safer alternative for controlling mosquitoes. Larvicidal activities of BTi described explicitly by Boyce et al［67］ and Ben-Dov［68］ have proven to be very effective in eradication of mosquito larvae［69］. However， short residual activity of BTi formulations limits its applicability in controlling mosquitoborne diseases［70］.

Use of fungi as biocontrol agents has also been proposed because of their natural tendency to produce infective spores （conidia）. These spores can adhere and penetrate within insect's cuticle where they flourish on nutritional reserves of host along with release of toxins that eventually kill the host［71］. Beauveria bassiana has emerged as potential candidate for control of Ae. aegypti causing significant mortality at larval as well as adult phases［58］. Metarhizium anisopliae has also been reported to cause higher mortality in adult Ae. aegypti［72］. Luz et al［73］ and Garcia-Munguia et al［74］ documented that fungal infection not only kills Ae. aegypti larvae and adults but also reduces fertility and slay the eggs. Such applications offer effective control of vector population with slow evolution of fungal resistance in contrast to insecticides. However the application and implementation of fungal formulations requires lots of research about the effects exerted by fungi on mosquito vectors， as these evidences revealed success stories from laboratories instead of field settings［75］.

19. Wolbachia as biological control agent

Microbes associated with numerous arthropod vectors eg. symbiotic bacteria as potential biocontrol agents have now gained much of attraction due to their ability to interfere with transmission of pathogens. Use of endosymbionts such as Wolbachia pipientis （W. pipientis） for control of pathogen transmission through mosquito vectors has been reported by Bordenstein and Rosengaus［59］ and Thomas et al［76］. W. pipientis has recently emerged as biological weapon for control of mosquito-borne diseases because of its natural capability of interfering with viruses persisting within mosquitoes，environmental benignity and cost effectiveness in contrast to chemical control methods［16，76，77］.

W. pipientis is a Gram negative bacterium having intracellular lifestyle and infects somatic and germ line tissues of hosts［78］. The bacterium is known to infect up to 70%-76% insect species existing on earth［16］. The bacterium usually resides in insects， filarial nematodes， arachnids， crustaceans and different arthropod species［79-82］. Wolbachia is known to have an essential role in embryogenesis，induction of alterations in reproductive cycles of hosts， as antibiotic treatment of infected individuals and in animals for inhibition of microfilariae［81，83］.

Utilization of W. pipientis for controlling mosquito-borne diseases has been pursued as a recent strategy with more promising results toalter Ae. aegypti populations and reduce their capability to transmit dengue virus pathogens in humans［84，85］. A number of different Wolbachia strains including wMel and wMelPop from Drosophila and aAlbB from Ae. albopictus have been reported to be introduced into Ae. aegypti［86，87］. Wolbachia invades and sustains itself inside mosquitoes where it induces series of reproductive abnormalities within the host for increasing reproduction of female mosquitoes. Thereby enhancing their own reproduction and transmission within host populations［16，79，88，89］. These reproductive abnormalities include feminization （transformation of genotypic males into phenotypic females）， modifications within male sperm and parthenogenic reproduction of females along with other behavioral effects plus altering the biting position etc.［16，89］. The bacterium has also been reported to affect nutritional status， development of their hosts，reduction of host's lifespan due to over-replication and occupation of brain tissues of the infected hosts and interference with pathogen replication［16，90-92］.

Before migration into salivary glands of mosquito and transmission into humans， dengue virus requires sufficient incubation period. Hence lifespan of mosquitoes is very critical in disease transmission. Consequently wMelPop strain has been proposed to alter and shorten the lifespan of Ae. aegypti and favors elimination of older mosquitoes to lessen the risks of dengue transmission［90，93］. Similarly， wMel strain has also been reported to reduce （adult） mosquito's lifespan around 10%［94］.

Wolbachia transfections can also induce cytoplasmic incompatibilities a type of embryonic lethality that occurs during mating of Wolbachia infected males with uninfected females. Thereby causes reduction in egg hatching and producing unviable offsprings［58，95］. Cook et al［96］ and Helinski et al［97］ reported transfection of Ae. aegypti with two Wolbachia strains namely wMel （benign） and wMelPop （virulent） and observed induction of cytoplasmic incompatibilities within their hosts. Such transfections offer a reproductive advantage to Wolbachia infected females over uninfected populations due to reduction in fecundity of uninfected females and impacting dengue virus transmission［98］. The cytoplasmic incapability is basically meant for Wolbachia for enhancing number of infected individuals within host population in order to boost their own maternal transmission［85］.

To date many stable Wolbachia transfected cell lines have been established and maintained for controlling disease transmitting vector （Ae. aegypti）. McMeniman et al［92］ transferred wMelPop into Ae. aegypti after 4 years of maintenance of Wolbachia within Ae. albopictus and generated stable wMelPop-CLA （cell-line-adapted）lines with 100% maternal transmission rates. These transfected mosquitoes have exhibited 50% reduction in adult lifespan in contrast to uninfected counterparts［92，99，100］. Similarly wAlbB strain of Wolbachia （from Ae. albopictus） has also been transfected successfully into Ae. aegypti［101］.

Additionally， Wolbachia transinfections have also been reported to reduce the susceptibility of Ae. aegypti to dengue virus［102］. As depicted by Pan et al［103］ that wAlbB transfected Ae. aegypti exhibited inhibition of dengue virus due to activation of ROS of Toll pathway. Such Wolbachia mediated pathogen interferences have caused significant reduction in competency of vector for dengue virus. These transfections also offer some sort of protection against DENV within mosquito cell lines despite lack of whole organism or tissue specific immunity within these cells［94，101，104，105］.

Moreover， considerably higher bacterium transmission frequencies to next progeny of Wolbachia transfected mosquitoes （females）have been documented by McMeniman et al［92］， where the bacterium invades uninfected populations by inducing reproductive abnormalities. Thus favors propagation of infected females［92，106］. For example， 100% transmission efficiency of wMelPop-CLA strains have been reported in offsprings of transinfected Ae. aegypti［92］. wMelPop-CLA transfection in Ae. aegypti are known to induce higher metabolic rates in mosquitoes［107］， feeding impairments in older mosquitoes［108］， reduced hatching and egg survival during phases of dormancy［109］. Such transfections suggest Wolbachia as best control strategies against dengue vector by suppressing egg hatching in wet seasons［16］. Furthermore， modifications in larval nutritional levels， duration of larval development as well as wing size of male Ae. aegypti has also been observed by Barrett et al［110］and Yeap et al［111］. wMelPop-CLA infected mosquitoes also showed significant reduction in replication of DENV-2 along with 56% reduction in fertility of infected females as compared to uninfected ones［94,104］. Additionally， reduction in blood meal ingestion by wMelPop-CLA transfected mosquitoes has been observed by Turley et al［108］ and Moreira et al［112］. All the three types of Wolbachia ie. wMel， wMelPop and wAlbB have been reported to inhibit dengue virus replication at significant levels along with dissemination within Ae. aegytpi population， thereby causing either partial or complete blockage of virus transfer in humans［94，101，104］. Analysis of pooled saliva extracts revealed complete blockage of viral transmission by wAlbB and wMelPop-CLA infected mosquitoes［94］.

Wolbachia transfections within host tissues have been reported to interfere with DENV replication where the bacterium competes for cellular resources and sequesters fatty acids of host cells that are required for DENV replication［104，113，114］. The immunofluorescence studies of Wolbachia transfected Ae. aegypti revealed no colocalization of bacterium with dengue virus and suggested metabolic competition between the two［104］. This phenomenon has been illustrated in detail by Bourtzis et al（2014）. This metabolic competition is further supported by the fact that significant differences were observed in density as well as tissue distribution between wMel and wMelPop strains （though closely related to each other） in infected Ae. aegypti. This wMelPop strain is known for boosting phenotypic interferences in mosquitoes against dengue virus due to higher concentration［94，104］. Studies have also revealed a strong negative correlation between genome copy of dengue virus and Wolbachia， where dengue infection has been inhibited completely at Wolbachia density of up to 961.8wsp/actin［115］. This has suggested dependency of Wolbachia mediated viral interferences on bacterial density within the transfected hosts［16］.

Many researchers have reported that Wolbachia strains utilize host microRNAs （miRNAs） for altering and manipulating gene expression of host and displaying antiviral response by establishing infections in Ae. aegypti［116-120］. The upregulation of host aaemiR-2940 in cell lines of Wolbachia transfected mosquitoes has been reported by Hussain et al［116］ where it targets metalloprotease genes and facilitates colonization of bacterium within the host. Similarly Wolbachia exploits this MiRNA of Ae. aegypti for targeting methyltransferase gene DnmmtA2 and alters it expression which eventually interferes with dengue virus replication. This interference inhibits virus establishment within the host thus providing a pathway for controlling disease transmission. Moreover， induction of aaemiR12 by Wolbachia down regulates DNA replication （MCM6） and monocarboxylate transporter （MCT1） genes. Therefore， plays an important role in maintaining Wolbachia infection within cell lines of host［117］.

Field trials for release of Wolbachia infected mosquitoes have been performed in different countries. For instance， wMel infected Ae. aegypti were introduced in two towns in North Queensland， Australia during wet season where wMel strains successfully invaded and transformed the local populations of Ae. aegypti within few months of being released［118-122］. While persistence of virus blocking property of Wolbachia in field released infected mosquitoes was confirmed by Frentiu et al［123］. These trials suggested that Wolbachia strains can efficiently be deployed as biocontrol agents against dengue vectors. No further evidences of transfer of bacteria into humans via mosquito bites， or the lateral transfer to nontarget species as well as to mosquito predators has been observed［16］. But the likelihood for horizontal transfer of Wolbachia DNA within mosquito genomes has been reported by McNaughton et al［124］ and Popovici et al［125］.

20. Wolbachia based biocontrol research being carried out in Pakistan

The Wolbachia strains that have already been explored and successfully exploited in other parts of the world can also be evaluated for stopping dengue virus transmission through eradication of vector populations in Pakistan. However implementation and application of such control strategies is largely dependent upon geographical and climatic conditions of particular settings， scientific evidences as well as region specific appropriateness［126］. Therefore，scientific community needs to explore the potentials of native or indigenous endosymobiotic strains and their transfer within Ae. aegypti. Many research projects in collaboration with international institutes are currently being conducted in Pakistan for reduction in transmission of dengue virus. For example， a project aimed at making Ae. aegypti incapable of carrying and transmitting dengue virus in humans through genetic modifications using Wolbachia has been launched in collaboration with Michigan State University，USA at Government College University， Lahore［127］. Moreover，interagency partnerships and collaborations of research organizations for Wolbachia based control technologies in Pakistan can be helpful in providing new means and tools for precise assessment of Wolbachia transfections in mosquitoes， their impact on vector population as well as role in blocking virus transmission etc. These collaborations among research organizations at international level may facilitate and strengthen the research networks by encouraging capacity building. They can also play crucial role in filling the funding gaps that hinder focused research efforts［128，129］. Resource mobilization， political participation and support are also needed to promote the efforts being made to prevent and control dengue using biological control agents in the region. Government of Pakistan and higher education commission needs to setup and release dengue virus research funds for reproducing researches going on in other parts of the world like Australia， USA and British biotechnology company Oxitec etc.［129，130］. Such initiatives will encourage the researchers to explore and evaluate the capability of indigenous Wolbachia species to eliminate dengue epidemics through hightech facilities and equipment. Various institutes of Pakistan intend to collaborate with international institutions to figure out the potentials and implementation of W. pipientis for minimizing and eradicating dengue vector populations form the region.

Though W. pipientis seems to be very efficient in reduction and eradication of dengue vector， still the interactions and molecular mechanisms which affect the performance of mosquitoes are largely unknown. Moreover， effects exerted by Wolbachia on dengue virus transmission and epidemiology in endemic areas are yet ambiguous and require large scale clinical trials before being released into field［131］. These untold stories have opened new portals for research activities to understand biological and evolutionary relations between Wolbachia and their symbionts. The knowledge obtained can potentially be applied for control of mosquito-borne diseases.

21. Conclusion

Prevalence of dengue epidemics throughout Pakistan is a leading cause of morbidity and mortality of thousands of individuals every year. This situation has spurred the development of effective control measures for controlling and eradicating the transmission vectors. Though many efforts at governmental and community levels have been made to effectively deal with Ae. aegypti. Unfortunately the failure of chemical based strategies， development of resistance against insecticides with subsequent threats to humans and environment， hurdles in implementation of other biological control measures due to effectiveness over small geographical scales has forced the scientific community to discover economically feasible and environment friendly alternatives. Wolbachia mediated vector control strategies as described above offer promising results for control of dengue virus. Though still at infancy this strategy seems to be very effective without posing any threat to human health as well as the environment. So there is a dire need to explore the potentials of native and/or indigenous endosymobiotic Wolbachia strains andtheir transfer within Ae. aegypti for ensuring our region to be free of dengue virus. All this seems to be possible only with support of government and private funding agencies.

Conflict of interest statement

We declare that we have no conflict of interest.

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15 September 2015

Uruj Tahir， Department of Environmental Sciences， Fatima Jinnah Women University， Rawalpindi， Pakistan.

E-mail： urujtahirjavaid@gmail.com