Metagenomic Analysis Of Viruses In Mosquitoes

Monitoring the emergence and re-emergence of viral diseases with the goal of containing the spread of viral agents requires adequate preparedness and quick response.Identifying the causative agent of a new epidemic is one of the most key step for effective response to disease outbreaks.Previously,virus discovery required propagation of the virus in cell culture,a proven technique responsible for the identification of the vast majority of viruses known to date.However,many viruses cannot be easily grown in cell culture,thus limiting our knowledge regarding viruses.Viral metagenomic Next Generation Sequencing(mNGS)analyses of environmental samples suggest that the field of virology has explored less than 1% of the extant viral diversity.In the last decade,the culture independent and sequence-independent metagenomic approach has permitted the discovery of many novel viruses in a wide range of samples.Here we used mNGS to analyze viruses carried by mosquitoes of Shanghai and Xinjiang province of China to explore the viral burden.In first study,we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes,Anopheles,and Culex species,collected from different pig farms located in the vicinity of Shanghai,China to explore the viral community carried by mosquitoes.The resulting metagenomic data revealed that viral community in the mosquitoes was highly diverse and varied in abundance among pig farms,which comprised of more than 48 viral taxonomic families,specific to vertebrates,invertebrates,plants,fungi,bacteria,and protozoa.In addition,a considerable number of viral reads were related to viruses that are not classified by host.The read sequences related to animal viruses included parvoviruses,anelloviruses,circoviruses,flavivirus,rhabdovirus,and seadornaviruses,which might be taken up by mosquitoes from viremic animal hosts during blood feeding.Notably,sample G1 contained the most abundant sequence related to Banna virus,which is of public health interest because it causes encephalitis in humans.Furthermore,non-classified viruses also shared considerable virus sequences in all the samples,presumably belonging to unexplored virus category.Overall,the present study provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms,which is a potential source of diseases for mammals including humans and animals.These viral metagenomic data are valuable for assessment of emerging and re-emerging viral epidemics.Another investigation was conducted to determine the virome burden of mosquitoes from Xinjiang province,which is located at border of Pakistan and Central Asia and provides an important terrestrial corridor of the “One Belt one Road” connecting China with Central/Western Asia and Europe.A total of 548 mosquitoes were collected from different animal farms located near to highly populated cities in Xinjiang province,and subjected to metagenomic Next Generation Sequencing.The mNGS data demonstrated that 2 of 4 mosquito pools were containing considerable Japanese encephalitis virus(JEV)reads.The Bayesian molecular phylogenetic analyses suggest that the detected XJ1 JEV genome emerged from neighboring JEV endemic Gansu province(GSS strain).In addition,Bayesian skyline plot demonstrates that JEV demography lacks fluctuations for the last two decades,but still a plateau like pattern of population dynamics of JEV is maintained,which shows the rich genetic diversity.This indicates the potential JEV replication in reservoir host,which is helping it to maintain its genetic diversity,and to continue its dispersal into non-endemic areas.Indeed,our findings suggest that Xinjiang is probably not free from JEV and continued surveillance of JEV is required in Xinjiang.In third experiment we determine the role of Culex pipiens mosquitoes in JEV genotype shift.JEV genotype dominance has shifted from genotype III(GIII)to genotype I(GI).To date,the exact mechanism of this replacement is still not known.Culex(Cx.)mosquitoes are the most common species in China and play an essential role in maintaining JEV enzootic transmission cycle.In this study,we used Cx.pipiens mosquitoes as an in vivo mosquito model to explore if mosquitoes played a potential role in JEV genotype shift.We experimentally orally exposed female Cx.pipiens mosquitoes with either GI or GIII JEV strains.Midgut,whole mosquitoes,secondary organs,and salivary glands of JEV-infected mosquitoes were collected after 7 and 14 days of post infection(dpi)and subjected to measure the infection rate,replication kinetics,dissemination rate and transmission potential of the infected JEV strains in Cx.pipiens mosquitoes by 50% tissue culture infective dose assay.We found that Cx.pipiens mosquito was competent vector for both GI and GIII JEV infection,with similar infection rates and growth kinetics.After the establishment of infection,Cx.pipiens mosquitoes disseminated both JEV genotypes to secondary organs at similar dissemination rate levels.A few GI infected mosquito salivary glands(16.2%)were positive,whereas GIII viruses were undetectable at 7 dpi.However,29.4%(5/17)and 36.3%(8/22)were positive for GI and GIII infected mosquito salivary glands at 14 dpi,respectively,showing an increase in JEV positive rate.No significant statistical difference in the transmission rate between GI and GIII infected mosquitoes was detected.Our experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx.pipiens mosquitoes,suggesting that mosquitoes may not play an essential role in JEV genotype shift.In conclusion,our metagenomic analyses provides a comprehensive knowledge of diverse viral populations carried by mosquitoes at pig farms of Shanghai province,which is a potential source of diseases for mammals including humans and animals.In addition,our findings suggest that Xinjiang is probably not free from JEV and timely disease prevention and control strategies such as vaccination and mosquito control strategies should be implemented.Furthermore,our mosquito infectivity experiment data demonstrated that GI and GIII viruses have similar infectivity in Cx.pipiens mosquitoes,suggesting that mosquitoes may not play an essential role in JEV genotype shift.