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Among the various species of livestock, the domestic yak (Bos grunniens) belongs to the class Bovidae and is specially adapted for survival at high altitudes under extreme climatic conditions. Yak is a very useful species due to the multiple products it provides, such as meat, hide, milk and other dairy products. Like other livestock species, domestic yak is also in danger of infection by microbial infections. However, multiple immunity genes encode special protein products to fight infection. One of these immunity genes is interferon-α-A (IFN-α-A), which encodes proteins that belongs to cytokines and fights viral infections. The current study analysed the genetic variation and phylogenetic analysis of the IFN-α-A gene in domestic yak, with comparisons to other mammalian species to investigate immune diversity level, with the aim of designing molecular selection strategies for better disease resistant animals.

The genetic diversity of Pakistani dromedary camels is poorly documented. The present study evaluated the genetic variations of two well-known Pakistani camel breeds, Mareecha and Barela, that are well-adapted to the Cholistan desert climate. Camel can serve as a beneficial participant in the food supply chain by providing milk, meat and other food products for the livelihood of pastoral peoples. To explore a new world of resources, greater attention to needed to create standard procedures to genetically characterize, classify and identify camel breeds in the country. For this purpose, 66 unrelated animals of the Mareecha (n=35) and Barela (n=31) breeds were genotyped using a set of 12 labelled microsatellite loci. DNA fragment sizes were determined in an ABI 3130 Genetic Analyzer. All microsatellite markers were successfully amplified and exhibited a polymorphic nature, with an average Polymorphic Information Content (PIC) of 0.72 and 0.70 in Mareecha and Barela, respectively. A total of 107 alleles with an average of 8.91 alleles per locus were identified by these markers in both breeds. CMS15 was highly polymorphic with 13 alleles, while VOLP-032 was the lowest with two loci. The Fit and Fis values were low but high population differentiation (17%) was observed in both breeds, due to the migrations of pastoral people to different remote areas during climate fluctuations., Genetska raznolikost pakistanskih jednogrbih deva slabo je dokumentirana. Ova studija procijenila je genetske varijacije dviju dobro poznatih pasmina pakistanskih deva - mareecha i barela - koje su vrlo dobro prilagođene klimi pustinje Cholistan. Deva može poslužiti kao koristan sudionik u lancu opskrbe hranom, osiguravajući mlijeko, meso i druge prehrambene proizvode za život pastira. Da bi se istražio novi svijet resursa, potrebno je više pozornosti za obavljanje standardnih postupaka za genetsku karakterizaciju, razvrstavanje i identifikaciju pasmina deva u zemlji. U tu svrhu, ukupno 66 životinja koje nisu u srodstvu pasmine mareecha (n=35) i barela (n=31) genotipizirane su uporabom 12 označenih mikrosatelitskih lokusa. Veličine fragmenata DNK određene su u ABI 3130 genetskom analizatoru. Svi mikrosatelitski markeri uspješno su pojačani i pokazali su polimorfnu narav s prosječnim sadržajem polimorfnih informacija (PIC) od 0,72 i 0,70 za mareecha, odnosno barela pasminu. Ukupno 107 alela s prosječno 8,91 alelom po lokusu u obje pasmine identificirano je tim markerima. CMS15 bio je vrlo polimorfan s 13 alela dok je VOLP-032 bio najniži s 2 lokusa. Fit i Fis vrijednosti bile su niske, ali je uočena velika diferencijacija populacije (17 %) za obje pasmine koje su posljedica migracije pastira u različite udaljene krajeve tijekom promjena klime.

Host defense against infectious disease is a tormenting task considering not only the enormous pathogens diversity, but also their rapid genome replication and mutation rates. Front line and anti-microbial defense is accomplished by the innate immune system with the help of pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) in prompt pathogens detection. In this study, distribution of single nucleotide polymorphisms (SNPs) in TLR2 gene was studied in two cattle breeds Dhanni and locally adapted exotic Jersey. The complete sequences of TLR2 gene composed of 5'UTR 136 bp, coding sequence 2355 bp and 3'UTR 1316 bp. Phylogenetic analysis revealed clustering of Dhanni with Bos indicus and Jersey with Bos taurus as the nearest neighbor. Sequencing of TLR2 in Dhanni cattle showed the occurrence of 35 variable sites within coding region including 37% non-synonymous and 63% synonymous, while in Jersey cattle, 21 SNPs were identified within coding region including 48% non-synonymous and 52% synonymous. Mostly SNPs fell within the leucine-rich repeats (LRR) of extracellular domain, regions responsible for ligand recognition. The ratio of dS/dN substitutions was

Chlorpyrifos (ChF) is an organophosphate pesticide that is widely used in agricultural fields and indoor for controlling pests. Aquatic ecosystems are the recipients of various pesticide residues due to leaching spray drift and agricultural runoff and pose toxicity for aquatic organisms. Therefore, the current study was designed to investigate the oxidative stress enzymes and histological alterations in the vital organs of tilapia due to ChF exposure. LC50 (24 h) was calculated as 52.78 μg/l by exposing tilapia with different acute concentrations of ChF. For assessment of sub-lethal toxicity of ChF, the fish were divided into four groups (ChF1, ChF2, ChF3, and control group). ChF1 group was treated with 1/15th of LC50, whereas ChF2 and ChF3 groups were treated with 1/10th and 1/5th of LC50, respectively for 14 days. After that, ChF induced changes in oxidative stress enzymes and histological alterations were evaluated. It was found that the level of glutathione S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) increased significantly in the liver of ChF-treated tilapia. Histological study of liver tissues showed an increased number of Kupffer cells, hydropic degeneration, necrosis, and hemorrhage. In the spleen of treated fish, increased melanomacrophage centers, necrosis, and congestion were detected. Disorganized muscle fibers, cardiac muscle fiber degeneration, and coagulative necrosis were observed in the heart of ChF-treated fish. It is concluded that sub-lethal concentrations of ChF can induce oxidative stress and histological alterations in the tissues of tilapia.

Alongshan virus (ALSV) is an emerging tick-borne pathogen that infects humans, causing febrile disease. ALSV uses Ixodes Persulcatus ticks to infect humans with a wide range of signs, from asymptomatic to encephalitis-like syndrome. There is an increasing public health concern about the ALSV infection. To get insight into the impacts of viral relations with their hosts on viral ability, survival, and evasion from hosts immune systems remain unknown. The codon usage is a driving force in viral genome evolution; therefore, we enrolled 41 ALSV strains in codon usage analysis to elucidate the molecular evolutionary dynamics of ALSV. The results indicate that the overall codon usage among ALSV isolates is relatively similar and slightly biased. Base compositions for the cds were in order of G >A >C >U and in the third position of codons G3 >A3 >C3 >T3. The RSCU values revealed that the more frequently used codons were mostly GC ended. Different codon preferences in ALSV genes in relation to codon usage of H. sapiens and Ixodes Persulcatus genes were found. Neutrality plot was determined to reveal the superiority of natural selection over directional mutation pressure in causing CUB based on GC12 versus GC3 contents. The results of these studies suggest that the emergence of ALSV in China, Russia and Finland may also be reflected in ALSV codon usage. Altogether, the presence of both mutation pressure and natural selection effect in shaping the codon usage patterns of ALSV.

Highly virulent Newcastle disease virus (NDV) causes Newcastle disease (ND), which is a threat to poultry production worldwide. Effective disease management requires approaches to accurately determine sources of infection, which involves tracking of closely related viruses. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. Previously formalin-fixed paraffin-embedded (FFPE) tissues have been used to conduct retrospective epidemiological studies of related but genetically distinct viruses. However, this study extends the applicability of NGS for complete genome analysis of viruses from FFPE tissues to track the evolution of closely related viruses. Total RNA was obtained from FFPE spleens, lungs, brains, and small intestines of chickens in 11 poultry flocks during disease outbreaks in Pakistan. The RNA was randomly sequenced on an Illumina MiSeq instrument and the raw data were analyzed using a custom data analysis pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (> 95% coverage of concatenated coding sequence) and two partial genomes. Phylogeny of the NDV complete genome coding sequences was compared to current methods of analysis based on the full and partial fusion genes and determined that the approach provided a better phylogenetic resolution. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct epidemiological and evolutionary studies to facilitate management of ND in Pakistan.

Picrotoxinin (1), a stable natural neurotoxin containing a highly oxygenated polycyclic rigid compound, was first isolated from berries of Anamirta cocculus (L.) Wight & Arn. (formerly known as Menispermum cocculus). The present work was aimed to study the modification of the complex polycyclic structure of picrotoxinin (1) by using microbial transformation. Initially various fungi, such as Macrophomina phaseolina, Cunninghamella blakesleeana, Aspergillus flavus, Aspergillus niger, Cephalosporium aphidicola, and Fusarium lini were screened for their ability to modify the structure of 1. Only one fungus Macrophomina phaseolina was able to catalyze the structural modification of picrotoxinin (1). The subsequent whole cell catalysis produced an intramolecular trans-esterified product 2. The structure of the transformed product was deduced by spectroscopic, and single-crystal X-ray diffraction analyses. A comparative study on convulsant activity of picrotoxinin (1) and its metabolite 2 was conducted in mice. Interestingly, metabolite 2 showed a delayed convulsant activity, as compared to the compound 1.

Spring maize is highly sensitive to low temperatures during the early development of seedlings and to high temperatures during its reproductive stage. Different strategies are being used to minimize the adverse effects of temperature extremes. Therefore, a field experiment was conducted to enhance the performance of spring hybrid maize by seed priming (3% MLE) and transplanting 20 and 30-day-old seedlings. Seed priming with moringa leaf extract (MLE) significantly enhanced stand establishment in both direct sowing and in transplanting, as indicated by the higher emergence percentage, emergence index, and lower time taken to start of emergence and mean emergence time. Minimum days from sowing to tasseling and silking were found in MLE primed 20-day-old seedlings grown in a nursery. However, all the agronomic parameters increased considerably with MLE priming of 20-day-old seedlings. Thus, MLE priming reduced chilling damage by improving stand establishment, whereas transplanting 20-day-old seedlings further enhanced the agronomic traits, yield, and quality of maize. However, the perfor-mance of maize plants from 30-day-old transplanted seedling and direct sowing was substandard.

Circulation of the dominant sub-genotype VII.2 of Avian Orthoavulavirus-1 (AOAV-1) is affecting multiple poultry and non-poultry avian species and causing significant economic losses to the poultry industry worldwide. In countries where ND is endemic, continuous monitoring and characterization of field strains are necessary. In this study, genetic characteristics of eleven AOAV-1 strains were analyzed isolated from wild birds including parakeets (n = 3), lovebird parrot (n = 1), pheasant (n = 1), peacock (n = 1), and backyard chickens (n = 5) during 2015–2016. Genetic characterization (genome size [15,192 nucleotides], the presence of typical cleavage site [112−RRQKRF−117]) and biological assessment (HA log 27 to 29 and intracerebral pathogenicity index [ICPI] value ranging from 1.50 to 1.86) showed virulent AOAV-1. Phylogenetic analysis showed that the studied isolates belonged to sub-genotype VII.2 and genetically very closely related (> 98.9%) to viruses repeatedly isolated (2011–2018) from commercial poultry. These findings provide evidence for the existence of epidemiological links between poultry and wild bird species in the region where the disease is prevalent. The deduced amino acid analysis revealed several substitutions in critical domains of fusion and hemagglutinin-neuraminidase genes. The pathogenesis and transmission potential of wild bird-origin AOAV-1 strain (AW-Pht/2015) was evaluated in 21-day-old chickens that showed the strain was highly virulent causing clinical signs and killed all chickens. High viral loads were detected in different organs of the infected chickens correlating with the severity of lesions developed. The continuous monitoring of AOAV-1 isolates in different species of birds will improve our knowledge of the evolution of these viruses, thereby preventing possible panzootic.

Drought stress, being the inevitable factor that exists in various environments without recognizing borders and no clear warning thereby hampering plant biomass production, quality, and energy. It is the key important environmental stress that occurs due to temperature dynamics, light intensity, and low rainfall. Despite this, its cumulative, not obvious impact and multidimensional nature severely affects the plant morphological, physiological, biochemical and molecular attributes with adverse impact on photosynthetic capacity. Coping with water scarcity, plants evolve various complex resistance and adaptation mechanisms including physiological and biochemical responses, which differ with species level. The sophisticated adaptation mechanisms and regularity network that improves the water stress tolerance and adaptation in plants are briefly discussed. Growth pattern and structural dynamics, reduction in transpiration loss through altering stomatal conductance and distribution, leaf rolling, root to shoot ratio dynamics, root length increment, accumulation of compatible solutes, enhancement in transpiration efficiency, osmotic and hormonal regulation, and delayed senescence are the strategies that are adopted by plants under water deficit. Approaches for drought stress alleviations are breeding strategies, molecular and genomics perspectives with special emphasis on the omics technology alteration i.e., metabolomics, proteomics, genomics, transcriptomics, glyomics and phenomics that improve the stress tolerance in plants. For drought stress induction, seed priming, growth hormones, osmoprotectants, silicon (Si), selenium (Se) and potassium application are worth using under drought stress conditions in plants. In addition, drought adaptation through microbes, hydrogel, nanoparticles applications and metabolic engineering techniques that regulate the antioxidant enzymes activity for adaptation to drought stress in plants, enhancing plant tolerance through maintenance in cell homeostasis and ameliorates the adverse effects of water stress are of great potential in agriculture.

The mitochondrial DNA sequences of DNA-D loops (mtDNA) from 105 individuals (11 Beetal goat Rajanpuri strains and 94 sequences extracted from GenBank) from 19 geographically and phenotypically defined domestic goat breeds in Pakistan were analysed in this experiment. In this study, we examined variability and molecular phylogeny of breeds. A total of 81 haplotypes were observed in 105 individuals, with a haplotype diversity of 0.984±0.006 and nucleotide diversity of 0.03953±0.00843. Phylogenetic analysis based on the mtDNA hyper variable segment (HVI) of the control region (481 bp), showed four mtDNA haplogroups (A, B1, C, and D) identified in Pakistani domestic goats, in which haplogroup A (84.11%) was dominant and widely distributed among all investigated breeds. The study revealed that all Rajanpuri strain haplotypes belonged to haplogroup A. The Rajanpuri is a rare local strain of Beetal goat breed located in western Punjab province of Pakistan. The results of genetic diversity based on 11 microsatellite loci revealed allelic diversity (3.6363) and high genetic diversity (0.8342) in the examined Rajanpuri goat breeds. The analysis for signature bottleneck events, using three models, revealed significant deviation of Rajanpuri goats from mutation drift equilibrium. The qualitative test of mode shift analysis also supported the results obtained under three models, indicating the presence of a recent genetic bottleneck in the Rajanpuri strain. This study provides the first information on the mtDNA architecture, genetic diversity and bottleneck analysis, which will be useful in the conservation and management of the highly valued Rajanpuri goat., Mitohondrijske DNK sekvence DNK-D petlji (mtDNK) 105 jedinki (11 ratalpuri sojeva beetal koze i 94 sekvence iz GenBank) od 19 geografskih i fenotipičnih domaćih pasmina koza u Pakistanu rabljeno je u ovom eksperimentu. U ovoj smo studiji istražili varijabilnost i njezinu molekularnu filogeniju. Zamijećen je ukupno 81 različit haplotip u 105 jedinki s raznolikošću haplotipova 0,984±0,006 i nukleotidnom raznolikošću 0,03953±0,00843. Filogenetskom analizom ustvrđen iz mtDNK hipervarijabilni segment (HVI) kontrolnog područja (481 bp) i pokazala je četiri haploskupine mtDNK (A, B1, C i D) identificirane u pakistanskih domaćih koza, u kojima se haploskupina A (84,11 %) pokazala dominantnom i široko rasprostranjenom među svim istraživanim pasminama. Studija je otkrila da svi haplotipovi rajanpuri soja pripadaju haploskupini A. Rajanpuri je rijedak lokalni soj beetal pasmine koza smješten u zapadnoj regiji provincije Punjab u Pakistanu. Rezultati genetske raznolikosti na temelju 11 mikrosatelitnih lokusa otkrili su alelnu raznolikost (3,6363) i visoku genetsku raznolikost (0,8342) istraživane rajanpuri pasmine koza. Rezultati analize za potpis za događaje uskog grla uporabom tri modela otkrili su značajno odstupanje rajanpuri koza od ravnoteže mutacija i pomaka. Kvalitativni test analize modalnog pomaka podržao je i rezultate dobivene trima modelima, ukazujući na prisutnost genetskog uskog grla u rajanpuri soja u nedavnoj prošlosti. Ova studija po prvi put pruža informacije o arhitekturi mtDNK, genetskoj raznolikosti, analizi uskog grla koje će biti korisne u donošenju odluke o očuvanju i upravljanju cijenjenom i skupocjenom rajanpuri kozom.

Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis.

The transporters belonging to the MATE family are involved in the transportation of diverse ligands, including metal ions and small organic molecules, and, therefore, play an important role in plant biology. Our genome-wide analysis led to the identification of 138 MATE genes in N. tabacum, which were grouped into four major phylogenetic clades. The expression of several NtMATE genes was reported to be differential in different tissues, namely young leaf, mature leaf, stem, root, and mature flower. The upstream regions of the NtMATE genes were predicted to contain several cis-acting elements associated with hormonal, developmental, and stress responses. Some of the genes were found to display induced expression following methyl jasmonate treatment. The co-expression analysis revealed 126 candidate transcription factor genes that might be involved in the transcriptional regulation of 21 NtMATE genes. Certain MATE genes (NtMATE81, NtMATE82, NtMATE88, and NtMATE89) were predicted to be targeted by micro RNAs (nta-miR167a, nta-miR167b, nta-miR167c, nta-miR167d and nta-miR167e). The computational analysis of MATE transporters provided insights into the key amino acid residues involved in the binding of the alkaloids. Further, the putative function of some of the NtMATE transporters was also revealed. The present study develops a solid foundation for the functional characterization of MATE transporter genes in N. tabacum.

Twenty-nine avian avulavirus-1 viruses (AAvV-1s) from healthy domestic and wild ducks, geese and black swans collected in Pakistan between 2014-2017 have been pathotyped and genetically characterized. A phylogenetic analysis revealed that 21 of the isolates belonged to sub-genotype VIIi, whereas eight isolates were highly similar to vaccine-like viruses of genotype II. In addition to confirming the continued presence of sub-genotype VIIi AAvV-1s in Pakistan, this study identifies the probable spill-over of vaccine-like viruses from vaccinated poultry to wild and domestic waterfowl and, as such, has important implications for the control and management of Newcastle disease in Pakistan.

The present research was conducted under laboratory conditions. The purpose of research was to investigate the potential of priming with press mud, peat moss, sand, gunny bags, compost, farm yard manure and moringa leaf extract (MLE) on seedling growth and germination capacity of maize seed. Untreated or non-primed seeds were used as a control treatment. Priming treatments improved germination capacity, stand establishment and seedling vigor, compared with control. Priming with moringa leaf extract enhance germination and seedling vigor of maize seed, compared with the control and other seed primed treatments. In moringa leaf extract primed seeds, root and shoot growth was improved. Overall, moringa leaf extract primed maize seeds performed better than all other treatments and it could be related by seedling vigor enhancement and lowering the mean germination time, due to imbibition of higher quantity of water and earlier enzymatic activity. The results propose that moringa leaf extract priming treatment had the potential to enhance germination, stand establishment and early growth of maize seeds.

Virulent viruses of the panzootic Avian avulavirus 1 (AAvV-1) of sub-genotype VIIi were repeatedly isolated (2011–2016) from commercial chickens and from multiple non-poultry avian species in Pakistan. These findings provide evidence for the existence of epidemiological links between Newcastle disease outbreaks in commercial poultry and infections with virulent AAvV-1 strains in other avian species kept in proximity to poultry. Our results suggest that the endemicity of Newcastle disease in Pakistan involves multiple hosts and environments.

Avian orthoavulavirus 13 (AOAV-13), also named avian paramyxovirus 13 (APMV-13), has been found sporadically in wild birds around the world ever since the discovery of AOAV-13 (AOAV-13/wild goose/Shimane/67/2000) in a wild goose from Japan in 2000. However, there are no reports of AOAV-13 in China. In the present study, a novel AOAV-13 virus (AOAV-13/wild goose/China/Hubei/V93-1/2015), isolated from a wild migratory waterfowl in a wetland of Hubei province of China, during active surveillance from 2013 to 2018, was biologically and genetically characterized. Phylogenetic analyses demonstrated a very close genetic relationship among all AOAV-13 strains, as revealed by very few genetic variations. Moreover, pathogenicity tests indicated that the V93-1 strain is a low virulent virus for chickens. However, the genome of the V93-1 virus was found to be 16,158 nucleotides (nt) in length, which is 12 nt or 162 nt longer than the other AOAV-13 strains that have been reported to date. The length difference of 12 nt in strain V93-1 is due to the existence of three repeats of the conserved sequence, “AAAAAT”, in the 5′-end trailer of the genome. Moreover, the HN gene of the V93-1 virus is 2070 nt in size, encoding 610 aa, which is the same size as the AOAV-13 strain from Japan, whereas that of two strains from Ukraine and Kazakhstan are 2080 nt in length, encoding 579 aa. We describe a novel AOAV-13 in migratory waterfowl in China, which suggests that diversified trailer region sequences and HN gene lengths exist within serotype AOAV-13, and highlight the need for its constant surveillance in poultry from live animal markets, and especially migratory birds.

Despite intensive vaccination, endemicity of Avian paramyxoviruses-1 (APMV-1) is a significant problem in developing countries in Africa, Middle East, and Asia. Given the importance of APMV-1 in poultry and multiple non-poultry avian species, it is important to continue surveillance programs, routine monitoring and characterization of field isolates in the region where viruses are endemic. The purpose of this study was to pathotyped and genetically characterized 21 APMV-1s isolated from multiple avian species reared in different regions of Azad Jammu and Kashmir (AJK). Phylogenetic analysis based on complete fusion (F) gene sequences showed that 17 APMV-1 isolates obtained from commercial poultry and backyard birds belonged to sub-genotype VIIi. Though, one pigeon-origin APMV-1 isolate was clustered in sub-genotype VIg and three in recently designated new sub-genotype VIm of genotype VI. The pigeon-origin isolates had the following two motifs (113)-RKKR↓F-(117) and (113)-RQRR↓F-(117), while all other isolates had the polybasic amino acid sequence (113)-RQKR↓F-(117) at the F-cleavage site, which is characteristic of virulent APMV-1 strains. These results are consistent with the five viruses that had intracerebral pathogenicity indices (ICPIs) of between 1.50 and 1.73, corresponding to a velogenic pathotype. The APMV-1s isolated from commercial poultry and backyard birds in this study showed low nucleotide distance (0.3–0.9%) and genetically closely related (> 97%) to viruses repeatedly isolated (2011–2017) from multiple avian species in other states of Pakistan. Strengthened surveillance programs in both commercial poultry and backyard flocks are needed to better assess the commercial–backyard bird interface and form a basis for evidence-based measures to limit and prevent APMV-1 transmission.

Avian influenza (AI) virus (AIV) subtype H9N2 continues to cause significant outbreaks among commercial and backyard poultry in Pakistan. Despite this, the characterization of H9N2 viruses in avian hosts other than chickens in Pakistan has not been thoroughly investigated. In this study, 12 low pathogenicity avian influenza viruses subtype H9N2 were isolated from peacocks (n = 4), ducks (n = 4), pheasants (n = 2), geese (n = 1), and black swans (n = 1) in Pakistan during 2016 and were characterized on the basis of the hemagglutinin (HA) and neuraminidase genes. All of the viruses possessed an amino acid substitution Q226L in the receptor-binding site of the HA protein, which is known to contribute to increased viral replication and virulence in mammals. In addition, phylogenetic studies showed that these H9N2 AIVs belonged to the Middle East B genetic group of sublineage G1 and were very similar to viruses isolated from an outbreak in chickens in Pakistan in 2017. This demonstrates an epidemiologic link between poultry and other avian species, which is a fact to consider in future H9N2 disease management programs.

Neural crest cells migrate to the embryonic heart and transform into a small number of cardiomyocytes, but their functions in the developing and adult heart are unknown. Here, we show that neural crest derived cardiomyocytes (NC-Cms) in the zebrafish ventricle express Notch ligand jag2b, are adjacent to Notch responding cells, and persist throughout life. Genetic ablation of NC-Cms during embryogenesis results in diminished jag2b, altered Notch signaling and aberrant trabeculation patterns, but is not detrimental to early heart function or survival to adulthood. However, embryonic NC-Cm ablation results in adult fish that show severe hypertrophic cardiomyopathy (HCM), altered cardiomyocyte size, diminished adult heart capacity and heart failure in cardiac stress tests. Adult jag2b mutants have similar cardiomyopathy. Thus, we identify a cardiomyocyte population and genetic pathway that are required to prevent adult onset HCM and provide a zebrafish model of adult-onset HCM and heart failure., A small percentage of cardiomyocytes (CM) are of neural crest origin but the function of such cells in the adult zebrafish is unclear. Here, the authors identify this CM subpopulation as expressing the Notch ligand jag2b and if deleted in the embryo, cause severe hypertrophic cardiomyopathy in adulthood.

The first objective of the present study was to evaluate if the antibodies induced by the live LaSota and killed Newcastle disease (sub-genotype VIIi) vaccines protect the chickens against exposure with pathogenic avian avulavirus-1 (AAvV-1) of chicken and/or pigeon origins. The second objective was to study the effect of vaccines on stressed birds (dexamethasone, aflatoxin, and heat stressed) with respect to antibody production and protection against pathogenic AAvV-1 challenge. Sixty-one-day-old Hubbard chickens were divided into six groups (gA–gF) with ten animals each. All the groups received LaSota (105 EID50, 0.1 ml per chick) on days 7 and 27 via eye drop and one intramuscular injection of a killed vaccine (sub-genotype VIIi) (107.5 EID50, 1 ml) on day 18, except the control birds received the PBS only. Moreover, group gC-DEX received dexamethasone intramuscularly at a dose rate of 1-mg/kg body weight daily; gD-AFLA had received aflatoxin as oral gavage at a dose rate of 30 ppb daily, and gE-HEAT was kept under heat stressed (38 °C) till challenged. All the groups were challenged with AAvV-1 strain of chicken origin of sub-genotype VIIi, except the group gA-pigeon was challenged with pigeon-origin strain (sub-genotype VIm). The result showed that the gA-pigeon and gB-chicken vaccinate showed 100% and 80% protection. The immunosuppressive birds produced low pre-challenge HI titer, and protection was observed at 40%, 50%, and 70% in gC-DEX, gD-AFLA, and gE-HEAT, respectively. Our findings suggest the stress factors such as aflatoxin in the feed and dexamethasone are immunosuppressive in nature and suppress the immune response and its associated protective role during infection.

Biotransformation of a synthetic progestonic hormone dydrogesterone (1), C21H28O2, with a plant pathogenic fungus Macrophomina phaseolina yielded two new 2 and 3, and a known 4 metabolites. These analogues were identified as, 3β,11α-dihydroxy-5β,9β,10α-pregna-7-ene-6,20-dione (2), 15β-hydroxy-9β,10α-pregna-4,6-diene-3,20-dione (3), and 8α-hydroxy-9β,10α-pregna-4,6-diene-3,20-dione (4). Major structural changes were observed in metabolite 2. New metabolite 3 showed anti-inflammatory potential, and was found to be the potent inhibitor of intracellular reactive oxygen species (ROS) from whole blood phagocytes (IC50 = 4.2 ± 0.3 μg/mL), as compared to standard drug Ibuprofen (IC50 = 11.2 ± 1.9 μg/mL). The metabolites 2, 3, and 4 were found to be non-toxic to NIH-3T3 (CRL-1658) normal cell line. This indicated anti-inflammatory potential of resulting metabolites.

Here, we present the draft genome sequences of three Ochrobactrum sp. strains with multidrug-resistant properties, isolated in 2015 from a pigeon and two chickens in Pakistan.

Newcastle disease (ND) is a devastating disease and cause high t mortality and morbidity in poultry and nonpoultry avian species worldwide. An intensive vaccination program against ND is a routine practice in Pakistan like other developing countries, but still frequent outbreaks have been recorded in the field. In this study, vaccines prepared from ND viruses corresponding to four different genotypes were compared, to determine if the phylogenetic distance between vaccine and challenge strain influences the protection induced the amount of challenge virus shed. In the experiment, 1-day-old pathogen-free Hubbard chicks were divided into five groups and all groups except control were received live LaSota vaccine. The chicks were re-vaccinated at day 5 and were given oil-adjuvanted inactivated vaccines prepared from one of four different inactivated NDV strains including SFR-55 (genotype-VIIi), Chicken-12 (XIIIb), Mukteswar (III), and LaSota (II), and control group was treated with PBS only. Pre- and post-challenge serum was collected from all groups and tested for antibody against NDV using hemagglutination inhibition (HI) assay. After challenged with virulent SFR-55, the birds were examined daily for morbidity and mortality and were monitored at selected intervals for viral shedding. All the vaccines induced high immune response, and all the groups except the control induced > 84% protection against vNDV challenge. The vaccine genetically and antigeneically similar with challenge NDV strain reduced oral shedding significantly as compared to mismatched strains. From the present study, it was concluded that genotype-matched vaccine has potential to result in better protection by limiting the viral shedding.

Neural crest cells migrate to the embryonic heart and transform into a small number of cardiomyocytes, but their functions in the developing and adult heart are unknown. Here, we map the fates of neural crest derived cardiomyocytes (NC-Cms) and genetically ablate them in embryogenesis in zebrafish. Specific NC-Cm ablation results in aberrant trabeculation patterns and altered Notch signaling, but is not detrimental to the development of the fish or early heart function. Strikingly, embryonic NC-Cm ablation results in adult fish that show severely hypertrabeculated hearts, altered cardiomyocyte size, diminished adult heart capacity and consequently poor physiological response to cardiac stress tests. Thus, we identify a novel developmental mechanism and genetic pathway that predisposes adults to hypertrophic cardiomyopathy and provides the first zebrafish model of adult-onset heart failure.

A total of 17 Y-linked short tandems repeat (Y-STRs) loci (DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATA H4) were genotyped in the Pashtun (Pathans) male population residing in the northern region of Pakistan. The Pashtun population, in spite of its decisive geographic location for repopulation movements between Pakistan, Afghanistan and India over time, has been insufficiently characterised at the genetic level, especially for Y chromosomal DNA variation. To understand the genetic structure characterisation of the Pashtun resident population of the northern region, we have utilised the Y chromosome STRs diversity, haplotype diversity and discrimination capacity. In this work, a total of 67 distinct haplotypes were observed in the resident population of Pashtun living in the northern region of the country, and they showed a high haplotype diversity (0.9756) and discrimination capacity (89.3%); five individuals s...

SummaryA major class of human birth defects arise from aberrations during neural tube closure (NTC). We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs) that is conserved between primitive chordates (Ciona) and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal.

Virulent Newcastle disease virus (NDV) isolates from new sub-genotypes within genotype VII are rapidly spreading through Asia and the Middle East causing outbreaks of Newcastle disease (ND) characterized by significant illness and mortality in poultry, suggesting the existence of a fifth panzootic. These viruses, which belong to the new sub-genotypes VIIh and VIIi, have epizootic characteristics and do not appear to have originated directly from other genotype VII NDV isolates that are currently circulating elsewhere, but are related to the present and past Indonesian NDV viruses isolated from wild birds since the 80s. Viruses from sub-genotype VIIh were isolated in Indonesia (2009–2010), Malaysia (2011), China (2011), and Cambodia (2011–2012) and are closely related to the Indonesian NDV isolated in 2007, APMV1/Chicken/Karangasem, Indonesia (Bali-01)/2007. Since 2011 and during 2012 highly related NDV isolates from sub-genotype VIIi have been isolated from poultry production facilities and occasionally from pet birds, throughout Indonesia, Pakistan and Israel. In Pakistan, the viruses of sub-genotype VIIi have replaced NDV isolates of genotype XIII, which were commonly isolated in 2009–2011, and they have become the predominant sub-genotype causing ND outbreaks since 2012. In a similar fashion, the numbers of viruses of sub-genotype VIIi isolated in Israel increased in 2012, and isolates from this sub-genotype are now found more frequently than viruses from the previously predominant sub-genotypes VIId and VIIb, from 2009 to 2012. All NDV isolates of sub-genotype VIIi are approximately 99% identical to each other and are more closely related to Indonesian viruses isolated from 1983 through 1990 than to those of genotype VII, still circulating in the region. Similarly, in addition to the Pakistani NDV isolates of the original genotype XIII (now called sub-genotype XIIIa), there is an additional sub-genotype (XIIIb) that was initially detected in India and Iran. This sub-genotype also appears to have as an ancestor a NDV strain from an Indian cockatoo isolated in1982. These data suggest the existence of a new panzootic composed of viruses of subgenotype VIIi and support our previous findings of co-evolution of multiple virulent NDV genotypes in unknown reservoirs, e.g. as recorded with the virulent NDV identified in Dominican Republic in 2008. The co-evolution of at least three different sub-genotypes reported here and the apparent close relationship of some of those genotypes from ND viruses isolated from wild birds, suggests that identifying wild life reservoirs may help predict new panzootics.

Boerhavia procumbens is a medicinal herb used in the treatment of various ailments including asthma, cough and jaundice. The concentration of calcium (280.7 ± 0.0 mg/100 g, 102.3 ± 0.0 mg/100 g), potassium (240.4 ± 0.0 mg/100 g, 92.1 ± 0.0 mg/100 g), aspartic acid (30.9 ± 0.0 g/100 g, 26.1 ± 0.0 g/100 g), glutamicacid (25.2 ± 0.0 g/100 g, 17.7 ± 0.0 g/100 g) and alanine were the abundant in the stem and root than leaf while carbohydrate (79.9 ± 0.0%) was found higher in the leaf of B. procumbens. The leaf and root extracts revealed the presence of alkaloids, flavonoids, tannins and phenol. Leaf extract was found to be more active against S. aureus (11.5 ± 0.3 mm), C. michiganensis (12.1 ± 0.2 mm) and F. oxysporum (23.6 ± 0.2 mm) than the root extract of B. procumbens. It is concluded that stem and root of B. procumbens contain essential nutrients and the antimicrobial study encourages the use of leaf extract for the treatment of most common microbial diseases.Video Clip of Methodology:4 min 54 sec Full Screen Alternate

Background The remarkable diversity and mobility of Newcastle disease viruses (NDV) includes virulent viruses of genotype VI. These viruses are often referred to as pigeon paramyxoviruses 1 because they are normally isolated and cause clinical disease in birds from the Columbidae family. Genotype VI viruses occasionally infect, and may also cause clinical disease in poultry. Thus, the evolution, current spread and detection of these viruses are relevant to avian health. Results Here, we describe the isolation and genomic characterization of six Egyptian (2015), four Pakistani (2015), and two Ukrainian (2007, 2013) recent pigeon-derived NDV isolates of sub-genotype VIg. These viruses are closely related to isolates from Kazakhstan, Nigeria and Russia. In addition, eight genetically related NDV isolates from Pakistan (2014–2016) that define a new sub-genotype (VIm) are described. All of these viruses, and the ancestral Bulgarian (n = 2) and South Korean (n = 2) viruses described here, have predicted virulent cleavage sites of the fusion protein, and those selected for further characterization have intracerebral pathogenicity index assay values characteristic of NDV of genotype VI (1.31 to 1.48). A validated matrix gene real-time RT-PCR (rRT-PCR) NDV test detect all tested isolates. However, the validated rRT-PCR test that is normally used to identify the virulent fusion gene fails to detect the Egyptian and Ukrainian viruses due to mismatches in primers and probe. A new rapid rRT-PCR test to determine the presence of virulent cleavage sites for viruses from sub-genotypes VIg was developed and evaluated on these and other viruses. Conclusions We describe the almost simultaneous circulation and continuous evolution of genotype VI Newcastle disease viruses in distant locations, suggesting epidemiological connections among three continents. As pigeons are not migratory, this study suggests the need to understand the possible role of human activity in the dispersal of these viruses. Complete genomic characterization identified previously unrecognized genetic diversity that contributes to diagnostic failure and will facilitate future evolutionary studies. These results highlight the importance of conducting active surveillance on pigeons worldwide and the need to update existent rapid diagnostic protocols to detect emerging viral variants and help manage the disease in affected regions. Electronic supplementary material The online version of this article (10.1186/s12917-017-1211-4) contains supplementary material, which is available to authorized users.

Objective To assess activation of immune system in rheumatic heart disease (RHD) patients in the form of AECA, ACL and anti GBM antibodies. Study design Descriptive, observational study. Place and duration of study Department of Immunology, University of Health Sciences (UHS), Lahore, and Outpatient Department, Punjab Institute of Cardiology, from February 2015 to January 2016. Methodology Clinically suspected patients of RHD and confirmed by echocardiography were included. AECA, ACL and anti GBM antibodies were investigated in the sera of RHD patients. Results Eighty-six RHD patients were included in the study; the mean age of the patients was 30 ±9.3 years. Among these patients, 59 (68.6%) were females and 27 (31.4%) were males. AECA was most commonly detected autoantibody i.e. in 17 (19.8%) patients; whereas, ACL was detected in only 2 (2.3%) subjects. Another 2 (2.3%) patients had both AECA and ACL antibodies. AGBM was not detected in any of the patients. ACL was seen in females with isolated MR. AECA were seen in mixed valvular heart disease patients. Conclusion Immune system gets activated in RHD patients leading to formation of different antibodies, and they are also related to the type of lesion. ACL antibodies are present in females with isolated mitral regurgitation, while AECA are present in both the genders with mixed valvular heart disease. Anti GBM antibodies are not seen in RHD patients.

Background. The vitamin D receptor (VDR) gene regulates insulin secretion from the pancreas and acts as a mediator of the immune response through vitamin D. Polymorphism in VDR causes alterations in the functioning of vitamin D, leading to type 1 diabetes (T1D) predisposition. The aim of the present study was to determine VDR gene polymorphism in association with T1D in Pakistanis. Methods. The association was evaluated by selecting rs2228570 (FokΙ), rs7975232 (ApaΙ), and rs731236 (TaqΙ) polymorphic sites in 102 patients and 100 controls. Genotypes were identified by DNA sequencing and PCR-RFLP. Results. The allelic and genotypic frequencies of FokΙ and ApaI were significantly associated with T1D (p<0.001) development. At the FokΙ site, tryptophan was replaced with arginine due to polymorphism. A novel SNP (GeneBank acc number KT280406) was identified through the sequencing of intron 8, 62 bp downstream from the ApaI polymorphic site, and significantly associated with T1D development. The TaqΙ did not depict any association with T1D at the allelic or genotypic level (p>0.05). CCGC, CCGG, CCTC, and CCTG haplotypes were significantly associated with disease development (p<0.05). However, CTGG haplotype was protective towards T1D (p<0.01). Conclusion. VDR polymorphisms were identified as susceptible regions for T1D development in the Pakistani population.

Background Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools. Methods In this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform. Results Twenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25–30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2–3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample. Conclusions This work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0741-5) contains supplementary material, which is available to authorized users.

The complete genome sequence of a virulent Newcastle disease virus (vNDV) strain isolated from an exotic parakeet ( Melopsittacus undulatus ) is described here. The virulent strain parakeet/Pak/R-Pindi/SFR-16/2016 was isolated from a bird reared as a pet in the province of Punjab in the northern region of Pakistan in 2016. Phylogenetic analysis classified the isolate as a member of NDV class II, subgenotype VIIi, in genotype VII.

Here, we report the complete genome sequence of a virulent Newcastle disease virus (vNDV) strain, duck/Pakistan/Lahore/AW-123/2015, isolated from apparently healthy laying ducks ( Anas platyrhynchos domesticus ) from the province of Punjab, Pakistan. The virus has a genome length of 15,192 nucleotides and is classified as member of subgenotype VIIi, class II.