Corrigendum: A metagenomic analysis of mosquito virome collected from different animal farms at Yunnan-Myanmar border of China.

[This corrects the article DOI: 10.3389/fmicb.2020.591478.].

Recent Population Dynamics of Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) causes acute viral encephalitis in humans and reproductive disorders in pigs. JEV emerged during the 1870s in Japan, and since that time, JEV has been transmitted exclusively throughout Asia, according to known reporting and sequencing records. A recent JEV outbreak occurred in Australia, affecting commercial piggeries across different temperate southern Australian states, and causing confirmed infections in humans. A total of 47 human cases and 7 deaths were reported. The recent evolving situation of JEV needs to be reported due to its continuous circulation in endemic regions and spread to non-endemics areas. Here, we reconstructed the phylogeny and population dynamics of JEV using recent JEV isolates for the future perception of disease spread. Phylogenetic analysis shows the most recent common ancestor occurred about 2993 years ago (YA) (95% Highest posterior density (HPD), 2433 to 3569). Our results of the Bayesian skyline plot (BSP) demonstrates that JEV demography lacks fluctuations for the last two decades, but it shows that JEV genetic diversity has increased during the last ten years. This indicates the potential JEV replication in the reservoir host, which is helping it to maintain its genetic diversity and to continue its dispersal into non-endemic areas. The continuous spread in Asia and recent detection from Australia further support these findings. Therefore, an enhanced surveillance system is needed along with precautionary measures such as regular vaccination and mosquito control to avoid future JEV outbreaks.

Bioenergy production in Pakistan: Potential, progress, and prospect.

Pakistan is a developing country with a rapidly growing population. It is currently facing serious economic and energy challenges. Pakistan's energy demand is increasing by the day, and it now stands at 84 MTOE. Currently, the use of fossil fuels dominates Pakistan's energy sector. Conversely, indigenous fossil fuel resources are rapidly depleting and will be unable to meet rising energy demands in the future. Therefore, to withstand its energy needs, the country will need to explore alternative energy production methods. Biomass is one of the alternatives that has enormous potential to help Pakistan combat its growing energy crisis. In this review, we first present an overview of bioenergy, biomass resources, and biomass conversion technologies. We then discuss in detail the current state of the energy mix of Pakistan. Subsequently, we show that annual production of about 121 MT of agricultural residues, 427 MT of animal manure, and 7.5 MT of MSW in Pakistan offer a variety of bioenergy options ranging from biofuels to bio-electricity production. Overall, these biomass resources in Pakistan have the potential to generate 20,709 MW of bio-electricity and 12,615 million m3 of biogas annually in Pakistan. Though these resources hold promising potential for bioenergy production in the country, however, there are some critical challenges that need to be considered, and some of which are extremely difficult to overcome for a developing country like Pakistan. This work is expected to provide a useful basis for biomass management and utilization in Pakistan to harvest eco-friendly and sustainable green energy locally.

Genome-Wide Analyses of Proteome and Acetylome in Zymomonas mobilis Under N2-Fixing Condition.

Zymomonas mobilis, a promising candidate for industrial biofuel production, is capable of nitrogen fixation naturally without hindering ethanol production. However, little is known about the regulation of nitrogen fixation in Z. mobilis. We herein conducted a high throughput analysis of proteome and protein acetylation in Z. mobilis under N2-fixing conditions and established its first acetylome. The upregulated proteins mainly belong to processes of nitrogen fixation, motility, chemotaxis, flagellar assembly, energy production, transportation, and oxidation-reduction. Whereas, downregulated proteins are mainly related to energy-consuming and biosynthetic processes. Our acetylome analyses revealed 197 uniquely acetylated proteins, belonging to major pathways such as nitrogen fixation, central carbon metabolism, ammonia assimilation pathway, protein biosynthesis, and amino acid metabolism. Further, we observed acetylation in glycolytic enzymes of central carbon metabolism, the nitrogenase complex, the master regulator NifA, and the enzyme in GS/GOGAT cycle. These findings suggest that protein acetylation may play an important role in regulating various aspects of N2-metabolism in Z. mobilis. This study provides new knowledge of specific proteins and their associated cellular processes and pathways that may be regulated by protein acetylation in Z. mobilis.

Bio-conversion of CO2 into biofuels and other value-added chemicals via metabolic engineering.

Carbon dioxide (CO2) occurs naturally in the atmosphere as a trace gas, which is produced naturally as well as by anthropogenic activities. CO2 is a readily available source of carbon that in principle can be used as a raw material for the synthesis of valuable products. The autotrophic organisms are naturally equipped to convert CO2 into biomass by obtaining energy from sunlight or inorganic electron donors. This autotrophic CO2 fixation has been exploited in biotechnology, and microbial cell factories have been metabolically engineered to convert CO2 into biofuels and other value-added bio-based chemicals. A variety of metabolic engineering efforts for CO2 fixation ranging from basic copy, paste, and fine-tuning approaches to engineering and testing of novel synthetic CO2 fixing pathways have been demonstrated. In this paper, we review the current advances and innovations in metabolic engineering for bio-conversion of CO2 into bio biofuels and other value-added bio-based chemicals.

Potential Role of Birds in Japanese Encephalitis Virus Zoonotic Transmission and Genotype Shift.

Japanese encephalitis (JE) is a vaccine-preventable disease caused by the Japanese encephalitis virus (JEV), which is primarily prevalent in Asia. JEV is a Flavivirus, classified into a single serotype with five genetically distinct genotypes (I, II, III, IV, and V). JEV genotype III (GIII) had been the most dominant strain and caused numerous outbreaks in the JEV endemic countries until 1990. However, recent data shows the emergence of JEV genotype I (GI) as a dominant genotype and it is gradually displacing GIII. The exact mechanism of this genotype displacement is still unclear. The virus can replicate in mosquito vectors and vertebrate hosts to maintain its zoonotic life cycle; pigs and aquatic wading birds act as an amplifying/reservoir hosts, and the humans and equines are dead-end hosts. The important role of pigs as an amplifying host for the JEV is well known. However, the influence of other domestic animals, especially birds, that live in high abundance and close proximity to the human is not well studied. Here, we strive to briefly highlight the role of birds in the JEV zoonotic transmission, discovery of birds as a natural reservoirs and amplifying host for JEV, species of birds susceptible to the JEV infection, and the proposed effect of JEV on the poultry industry in the future, a perspective that has been neglected for a long time. We also discuss the recent in vitro and in vivo studies that show that the newly emerged GI viruses replicated more efficiently in bird-derived cells and ducklings/chicks than GIII, and an important role of birds in the JEV genotype shift from GIII to GI.

Detection of Japanese encephalitis virus in mosquitoes from Xinjiang during next-generation sequencing arboviral surveillance.

A total of 548 mosquitoes were collected from different animal farms located near to highly populated cities in Xinjiang and were subjected to metagenomic next-generation sequencing (mNGS). The mNGS data demonstrated that 18,842 (XJ1 strain) and 1,077 (XJ2 strain) of Japanese encephalitis virus (JEV)-related reads were detected in XJ1 and XJ2 mosquito samples collected from Wushi and Wensu counties of Aksu area, which accounted for 0.032% and 0.006% of the total clean reads generated from XJ1 and XJ2 samples, respectively. The Bayesian molecular phylogenetic analysis suggested that XJ1 and XJ2 strains belonged to JEV genotype III and were clustered with JEV strains isolated in China. Notably, Bayesian molecular time line phylogeny revealed that XJ1 strain shared its MRCA with JEV GSS strain about 67 YA, suggesting that XJ1 strain likely originated from linages closely related to GSS strain and spread to Xinjiang later. Overall, these findings suggest that Xinjiang was probably not free from JEV, and thus, a further surveillance of JEV is required in Xinjiang.

Analyses of mitogenomic markers shed light on the divergence, population dynamics, and demographic history of Pakistani chickens.

Pakistan is one of a few sites, associated with the earliest known independent domestication event in the evolutionary history of chicken, which is socio-economically and historically the most important poultry bird in the country. However, the divergence, past population dynamics, and demographic history of Pakistani chickens have not been addressed so far. Therefore, we herein investigated the indigenous Pakistani chickens using mitogenomic markers. We first prepared individual DNA samples from the chicken feathers, and generated nucleotide sequence data, which was then subjected to various population genetics analyses. In molecular phylogenetic analysis, the Pakistani chickens were clustered under nine different clades. Among the wild fowls, the Indian red jungle fowl (IRJF) shared very close affinities to Pakistani chickens. The Bayesian skyline plot showed an increase in the effective population size of Pakistani chickens during the last 50 years. Finally, a time-calibrated phylogeny inferred molecular divergence of the Pakistani chickens. A molecular rate of 3.6 × 10-6 mutations/site/year (95% HPD interval: 2.28 × 10-8 to 9.32 × 10-6) was estimated for the data set. In a rooted tree with root-age of 12058 years (95% HPD interval: 1161-38411), the Pakistani chicken haplotypes showed divergence from IRJF haplotypes around 6987 years (95% HPD interval: 1132-20746) ago, and they shared their most recent common ancestor with Gallus gallus spadiceus, and G. g. jabouillei at the root of the tree. Overall, these results suggest that Pakistani chicken haplotypes share their ancestral gene pool with the IRJF as compared to other red jungle fowl subspecies.

Suppression of hyaluronidase reduces invasion and establishment of Haemonchus contortus larvae in sheep.

Haemonchus contortus is a hematophagous endoparasite of small ruminants, which is responsible for huge economic losses in livestock sector. Hyaluronidase produced by infective larvae of H. contortus can degrade hyaluronic acid present in the host's abomasal tissue. Thus, it facilitates larval tissue invasion and early establishment. We herein explored this ability of hyaluronidase in H. contortus, and tested whether hyaluronidase is utilized as a virulence factor by H. contortus while establishing the infection. We first successfully blocked the hyaluronidase gene in L3 larvae by RNA interference (RNAi), which was subsequently confirmed by qPCR, enzymatic activity, and immunohistochemistry assays. Using these larvae we then conducted in vivo and in vitro assays on sheep to assess the effects of hyaluronidase suppression on larval invasion and establishment of infection. The in vivo assay showed a significant drop in worm burden in siRNA treated group in comparison to control group. During in vitro assay we applied an ovine ex vivo model where siRNA treated group of larvae showed significantly reduced invasion of the abomasal tissue explants as compared to control group. These findings indicate that hyaluronidase plays a key role in host's tissue invasion and larval establishment, and it is used as a virulence factor by H. contortus while establishing the infection. As an invasive virulence molecule, its functional research is thus conducive to the prevention of haemonchosis.

A Whole Genome Re-Sequencing Based GWA Analysis Reveals Candidate Genes Associated with Ivermectin Resistance in Haemonchus contortus.

The most important and broad-spectrum drug used to control the parasitic worms to date is ivermectin (IVM). Resistance against IVM has emerged in parasites, and preserving its efficacy is now becoming a serious issue. The parasitic nematode Haemonchus contortus (Rudolphi, 1803) is economically an important parasite of small ruminants across the globe, which has a successful track record in IVM resistance. There are growing evidences regarding the multigenic nature of IVM resistance, and although some genes have been proposed as candidates of IVM resistance using lower magnification of genome, the genetic basis of IVM resistance still remains poorly resolved. Using the full magnification of genome, we herein applied a population genomics approach to characterize genome-wide signatures of selection among pooled worms from two susceptible and six ivermectin-resistant isolates of H. contortus, and revealed candidate genes under selection in relation to IVM resistance. These candidates also included a previously known IVM-resistance-associated candidate gene HCON_00148840, glc-3. Finally, an RNA-interference-based functional validation assay revealed the HCON_00143950 as IVM-tolerance-associated gene in H. contortus. The possible role of this gene in IVM resistance could be detoxification of xenobiotic in phase I of xenobiotic metabolism. The results of this study further enhance our understanding on the IVM resistance and continue to provide further evidence in favor of multigenic nature of IVM resistance.

A Metagenomic Analysis of Mosquito Virome Collected From Different Animal Farms at Yunnan-Myanmar Border of China.

Metagenomic analysis of mosquito-borne and mosquito-specific viruses is useful to understand the viral diversity and for the surveillance of pathogens of medical and veterinary importance. Yunnan province is located at the southwest of China and has rich abundance of mosquitoes. Arbovirus surveillance is not conducted regularly in this province particularly at animal farms, which have public health as well as veterinary importance. Here, we have analyzed 10 pools of mosquitoes belonging to Culex tritaeniorhyncus, Aedes aegypti, Anopheles sinensis, and Armigeres subalbatus species, collected from different animal farms located at Yunnan province of China by using metagenomic next-generation sequencing technique. The generated viral metagenomic data reveal that the viral community matched by the reads was highly diverse and varied in abundance among animal farms, which contained more than 19 viral taxonomic families, specific to vertebrates, invertebrates, fungi, plants, protozoa, and bacteria. Additionally, a large number of viral reads were related to viruses that are non-classified. The viral reads related to animal viruses included parvoviruses, anelloviruses, circoviruses, flaviviruses, rhabdoviruses, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, the presence of viral reads matched with Japanese encephalitis virus, Getah virus, and porcine parvoviruses in mosquitoes collected from different geographic sites suggested a potential circulation of these viruses in their vertebrate hosts. Overall, this study provides a comprehensive knowledge of diverse viral populations present at animal farms of Yunnan province of China, which might be a potential source of diseases for humans and domestic animals.

A viral metagenomic analysis reveals rich viral abundance and diversity in mosquitoes from pig farms.

Mosquitoes harbour a diversity of viruses and are responsible for several mosquito-borne viral diseases of humans and animals, thereby leading to major public health concerns, and significant economic losses across the globe. Viral metagenomics offers a great opportunity for bulk analysis of viral genomes retrieved directly from environmental samples. In this study, we performed a viral metagenomic analysis of five pools of mosquitoes belonging to Aedes, Anopheles and Culex species, collected from different pig farms 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.

Analysis of genome-wide SNPs based on 2b-RAD sequencing of pooled samples reveals signature of selection in different populations of Haemonchus contortus.

The parasitic nematode Haemonchus contortus is one of the world's most important parasites of small ruminants that causes significant economic losses to the livestock sector. The population structure and selection in its various strains are poorly understood. No study so far compared its different populations using genome-wide data. Here, we focused on different geographic populations of H. contours from China (Tibet, TB; Hubei, HB; Inner Mongolia, IM; Sichuan, SC), UK and Australia (AS), using genome-wide population-genomic approaches, to explore genetic diversity, population structure and selection. We first performed next-generation high-throughput 2b RAD pool sequencing using Illumina technology, and identified single-nucleotide polymorphisms (SNPs) in all the strains. We identified 75,187 SNPs for TB, 82,271 for HB, 82,420 for IM, 79,803 for SC, 83,504 for AS and 78,747 for UK strain. The SNPs revealed low-nucleotide diversity (pi= 0.0092-0.0133) within each strain, and a significant differentiation level (average Fst = 0.34264) among them. Chinese populations TB and SC, along with the UK strain, were more divergent populations. Chinese populations IM and HB showed affinities to the Australian strain. We then analysed signature of selection and detected 44 (UK) and 03 (AS) private selective sweeps containing 49 and 05 genes, respectively. Finally, we performed the functional annotation of selective sweeps and proposed biological significance to signature of selection. Our data suggest that 2b-RAD pool sequencing can be used to assess the signature of selection in H. contortus.

Microsatellite analysis reveals extensive gene flow, and lack of population structure in the farm populations of Haemonchus contortus in northern China.

The parasitic nematode Haemonchus contortus is economically an important parasite of small ruminants across the globe. China is the world's largest producer, consumer, and importer of mutton. With ubiquitous distribution across the country H. contortus is one of the potential candidates to cause huge economic losses to small ruminant farming industry in China. We herein investigated genetic diversity and population structure of six farm populations of H. contortus in northern China, and also compared them to H. contortus isolates from UK and Australia. We first prepared individual DNA samples from 240 adult worms, and generated genotyping data using eight microsatellite markers. Obtained data was then subjected to allelic frequency and population genetic analyses. The overall allelic richness (mean/locus/pop = 7.375 ±â€¯0.844-10.125 ±â€¯1.109), and expected heterozygosity (mean/locus/pop = 0.646 ±â€¯0.040-0.735 ±â€¯0.025) indicated high degree of population genetic variation across the Chinese isolates. Low level of genetic differentiation (Fst = 0.010-0.066) was observed across all the populations. AMOVA results showed high level of variation (93%) within the populations. PCA analysis revealed mixed clustering of all the populations with no visible geographical sub-structuring. Finally the population admixture analysis resulted in extensive admixing of genotypes across all the populations. With these findings we conclude that there is no obvious population genetic structure with extensive gene flow across all the farm populations of H. contortus in northern China.

Ethnogenetic analysis reveals that Kohistanis of Pakistan were genetically linked to west Eurasians by a probable ancestral genepool from Eurasian steppe in the bronze age.

Despite the unique geographic, ethnic, social and cultural features of Kohistan in Pakistan, the origin and descent of Kohistanis remain still obscure. In an effort to address questions concerning the genetic structure, origin and genetic affinities of Kohistanis, we herein applied an ethnogenetic approach consisting on mitochondrial DNA (mtDNA) analysis and dental morphology analysis. We sequenced HVS1 of mtDNA, observed 14 haplotypes and assigned a total of 9 haplogroups belonging to macrolineages M (17%) and N (83%). Genetic diversity estimates in Kohistanis (Hd = 0.910 ±â€¯0.014; Pi = 0.019 ±â€¯0.001; θw = 0.019 ±â€¯0.006) were similar to that of previous studies in other Pakistani populations. Overall, the analyses of dental morphology and mtDNA profile of Kohistanis resulted in similar findings. All the analyses indicate that Kohistanis share affinities to populations from Europe, Near East, Central Asia and South Asia. The Kohistani HVS1 haplotype 2 shares 100% identity to HVS1 haplotypes across the Europe. These results in light of recent insights into ancient genomics lead us to conclude that ancestry from Eurasian Steppe genetically linked Kohistanis to all these populations in the Bronze Age. This is consistent with linguistic evidence and also with the Indo-Aryan migration model for the peopling of South Asia.

Population structure, genetic diversity and phylogenetic analysis of different rural and commercial chickens of Pakistan using complete sequence of mtDNA D-loop.

Chicken is the most important poultry bird in Pakistan that not only provides nutrition but also contributes to country's economy. The Pakistani chicken and its germplasm resources are not genetically characterized and explored. Here, we focused at rural and commercial chickens of Pakistan and explored genetic diversity, population structure and phylogeny. We first collected feather samples from Rural and Broiler populations of Pakistani chickens, isolated DNA and sequenced complete D-loop of mtDNA. The length of complete D-loop ranged from 1231 to 1234 bp in Pakistani chickens. The GC content was 39%. Hotspots of mutations were three hypervariable sites (HVS). Most of the variations (77%) were in HVS1. In a total, 26 polymorphic sites defined 12 haplotypes and all major haplogroups (A-I) in genetic structure of Pakistani chickens. Genetic diversity remained relatively very low in Broiler (Pi = 0.00212 ± 0.00136). There was a low sharing of matrilineages between the two populations (Fst = 0.170). With high Hd value (0.825 ± 0.051) and presence of all nine major haplogroups the rural chicken population showed relatively rich genepool. Finally we did molecular phylogenetic analysis and inferred phylogeny. Presence of subcontinent specific haplogroups E3 and I and clustering of Indian red junglefowl closely with Pakistani chickens in Bayesian inference tree, provide further evidence for an independent domestication event of chicken in subcontinent.