Dengue is becoming more common in Pakistan with its alarming spreading rate. A historical review needs to be carried out to find the root causes of dengue dynamics, the factors responsible for its spread and lastly to formulate future strategies for its control. We searched (January, 2015) all the published literature between 1980 and 2014 to determine spread/burden of dengue disease in Pakistan. A total of 81 reports were identified, showing high numbers of dengue cases in 2010, 2011, and 2013. The tendency of dengue to occur in younger than in older age groups was evident throughout the survey period and all four serotypes were recorded, with DENV1 the least common. Most dengue hemorrhagic fever (DHF) cases fell in the 20-45 years age range. High frequencies tended to be observed first in the Southern coastal region characterized by mild winters and humid warm summers and then the disease progressed towards the lowland areas of the Indus plain with cool winters, hot summers and monsoon rainfall. Based on this survey, new risk maps and infection estimates were identified reflecting public health burden imposed by dengue at the national level. Our study showed that dengue is common in the three provinces of Pakistan, i.e., Khyber Pakhtunkhwa (KP), Punjab and Sindh. Based on the literature review as well as on our study analysis the current expansion of dengue seems multifactorial and may include climate change, virus evolution, and societal factors such as rapid urbanization, population growth and development, socioeconomic factors, as well as global travel and trade. Due to inadequate remedial strategies, effective vector control measures are essential to target the dengue vector mosquito where high levels of human-vector contact occur. The known social, economic, and disease burden of dengue is alarming globally and it is evident that the wider impact of this disease is grossly underestimated. An international multi-sectoral response, outlined in the WHO Global Strategy for Dengue Prevention and Control, 2012-2020, is now essential to reduce the significant influence of this disease in Dengue endemic areas. Overall gaps were identified in knowledge around seroprevalence, dengue incidence, vector control, genotype evolution and age-stratified serotype circulation.

Aedes aegypti and Aedes albopictus play a fundamental role in transmission of dengue virus to humans. A single infected Aedes mosquito is capable to act as a reservoir/amplifier host for dengue virus and may cause epidemics via horizontal and vertical modes of dengue virus (DENV) transmission. The present and future dengue development can be clarified by understanding the elements which help the dissemination of dengue transmission. The current study deals with molecular surveillance of dengue in addition to ecological and social context of 2013 dengue epidemics in Swat, Pakistan. Herein, we reported dengue vectors surveillance in domestic and peridomistic containers in public and private places in 7 dengue epidemic-prone sites in District Swat, Pakistan from July to November 2013. Using the Flaviviruses genus-specific reverse transcriptase (RT) semi nested-PCR assay, we screened blood samples (N = 500) of dengue positive patients, 150 adult mosquito pools and 25 larval pools. The 34 adult and 7 larval mosquito pools were found positive. The adult positive pools comprised 30 pools of Ae. aegypti and 4 pools of Ae. albopictus, while among the 7 larval pools, 5 pools of Ae. aegypti and 2 pools of Ae. albopictus were positive. The detected putative genomes of dengue virus were of DENV-2 (35% in 14 mosquito pools & 39% in serum) and DENV-3 (65% in 27 mosquito pools & 61% in serum). The higher vector density and dengue transmission rate was recorded in July and August (due to favorable conditions for vector growth). About 37% of Ae. aegpti and 34% Ae. albopictus mosquitoes were collected from stagnant water in drums, followed by drinking water tanks (23% & 26%), tires (20% & 18%) and discarded containers (10% & 6%). Among the surveyed areas, Saidu was heavily affected (26%) by dengue followed by Kanju (20% and Landikas (12%). The maximum infection was observed in the age group of 45 (25%) years and was more in males (55.3%) as compare to females (44.7%). The increase in vector mosquito density and the subsequent viral transmission was determined by a complex interplay of ecological, biological and social factors. The suitable environmental conditions and discriminable role of Aedes through trans-ovarial transmission of DENV is indispensable in the recent geographic increase of dengue in Pakistan. Climate change affects the survival and dispersion of vectors as well as the transmission rates of dengue. Control of Aedes mosquitoes (vectors) and elimination of breeding sources must be emphasized and prioritized. Such actions may not only reduce the risk of dengue transmission during epidemics, but also minimize the chances of dengue viruses establishment in new (non endemic) areas of the region.

Pakistan is suffering from dengue fever since 1994. In the country, major dengue outbreaks have been documented in 2010, 2011 and 2013 in Punjab (Lahore) and Sindh (Karachi) Provinces. District Swat was hit for the first time by dengue virus in 2013, claiming 57 deaths and 8000 morbidities. The molecular and entomological aspects along with the ecological and social context of the dengue outbreaks were investigated in this study. In addition to entomological survey, the data collected from patients' files (Saidu Sharif Teaching Hospital, Swat) and by directly questioning the patients (field data) was analyzed for epidemiological trends, molecular identification (RT-PCR based serotyping of DENV), clinical profile, socioeconomic status (SES) and demographic features. The peak prevalence of dengue was documented in September (56% in 2013, 38% in 2014) and October (33% in 2013, 24% in 2014), whereas in 2015, in October (54.5%) and November (30.5%). The serotype 3 (≤60%) and serotype 2 (≤40%) were found dominant in the area. Among the reported patients (5513), 69% were males and 31% females. Majority of them were found in the age of 14–30 years (52.5%) as compared to 31–60 years (34.5%) (Chi-square: 3219.463, p-value: 0.00). About 63% cases reported in low SES. Among the different categories of government employees affected with dengue, majority (4%) were belonging to health department (Chi-square: 4541.011, p-value: 0.00). Similarly, dengue targeted the dwellers living in multiple-storey houses (65%) as compared to those in the single-storeyed houses (35%) (Chi-square: 495.630, p-value: 0.00). The overall death toll observed was of 57 persons. Dengue prevailed more (38.4%) among low qualified individuals as compared to high qualified (11.5%) (Chi-square: 884.315, p-value: 0.00). Our analysis indicated a decrease in the epidemiological trend of dengue (now) in the area, though initially it was observed affecting all types of communities on a larger scale. However, the DENV-2 and DENV-3 were dominantly circulating in the area and the prevalence (with usual peaks in post-monsoon) found high in males, illiterate (less educated) individuals and in those with low SES. Urbanization, infected human travelling, climate change, socioeconomic, sociodemographic as well as the wide range adaptation of vector mosquitoes, altogether, are the important factors playing role in the expansion of dengue. Further studies are needed to determine the association of these variables with the dengue spread in the area.

Wolbachia are intracellular bacteria that infect a wide range of invertebrates and have evolved various strategies to alter host reproduction for their own survival and dissemination. In small brown planthopper Laodelphax striatellus, Wolbachia-infected females lay more eggs than uninfected females. Our previous study has shown that Wolbachia are abundant in ovarian cells of L. striatellus and change the number of apoptotic nurse cells in a caspase-dependent manner to provide nutrition for oogenesis. The cellular and molecular bases of the Wolbachia-mediated alterations in L. striatellus oogenesis remain largely unknown. Here, we investigated whether germ cell mitosis, which has been implicated in determination of egg production rates, influences the interaction between fecundity and Wolbachia in L.striatellus. We used an anti-phospho-histone 3 (pH3) antibody to label and visualize mitotic cells. Microscopic observations indicated that the Wolbachia strain wStri increased the number of ovarioles that contained mitotic germ cells. The increased fecundity of Wolbachia-infected females was a result of mitosis of germ cells; the frequency of germ cell mitosis was much higher in infected females than in uninfected females. In addition, mitosis inhibition by Cdc20, CDK1, and CycB messenger RNA interference in Wolbachia-infected L. striatellus markedly decreased egg numbers. Live Wolbachia recolonization enhanced the egg production of uninfected L. striatellus by directly affecting mitosis regulators. Together, these data suggest that wStri might increase germ cell mitosis to enhance the fecundity of L. striatellus in a mitosis-regulating manner. Our findings establish a link between Wolbachia-induced mitosis and Wolbachia-mediated egg production effects.