Oladipo E.K.*1,2, Adeniji J.A.3, Oloke J.K2

1Department of Microbiology, Laboratory of Molecular Biology and Bioinformatics, Adeleke University, Ede, Osun State, Nigeria; 2Department of Pure and Applied Biology (Microbiology / Virology Unit), Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria; 3Department of Virology, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria

Introduction: Pigs are susceptible to infections with both avian and mammalian Influenza A viruses. Pigs play an important role in the ecology of influenza virus. The changing epidemiology of influenza has a significant implications for the circulation of viruses in pigs. Little is known about the circulating strains in this area. To understand the current the current situation regarding influenza viruses circulating among pigs in Ogbomoso, a surveillance study was conducted.

Methods: Viral isolation from nasal swabs collected from one hundred pigs was performed using egg inoculation, MDCK and haemagglutination assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was carried out by amplifying the matrix gene for identification. The sequence was determined by Sanger dideoxy sequencing. The homology analysis was implemented by Influenza Research database (IRD) and phylogenetic analysis was performed by Maximum – Likelihood methods using MEGA 7.10 software package.

Results: Multiple sequence alignment showed that the matrix (M) genes of A/H5N1/Ogbomoso/2014 influenza virus showed highest nucleotide identity with A/Pigeon/Sichuan/NCXN29/2014(H5N1) and A/Duck/Sichuan/NCXN11/2014 (H5N1) {98% and 97% respectively}. The phylogenetic analysis of the matrix gene of A/H5N1/Ogbomoso/2014 indicated that this virus is closely related to H5N1 strains circulating in southwest China.

Conclusion: This is the first report of genetic characterization of influenza A virus H5N1 of swine origin isolate from Ogbomoso. The presented results can further promote Influenza A virus surveillance and epidemiology insight in this community. The potential role of pigs in interspecies transmission remains important.

Keywords: Pigs, H5N1, Ogbomoso, Influenza A, Genetic Characterization.

Elizabeth Marincola

African Academy of Sciences, Nairobi, Kenya

Scientists everywhere in the world experience similar frustrations in disseminating the results of their research: delays of months or more; access constraints; bias resulting from opaque peer review; incomplete availability of data, and the tyranny of the Journal Impact Factor, which induces scientists to chase acceptance in a succession of journals, hoping to publish in the one with the highest possible JIF – exacerbating the delay problem. African scientists are subject to all these barriers, with amplified effects resulting from the status of many as relatively unknown, often from unknown institutions. This presents researchers in Africa and other low- and middle-income countries to an uneven playing field and thus even higher barriers to entry to compete in what is increasingly a global science landscape. Yet because of relative lack of legacy and the acceleration of quality research output on the Continent, African STM publishing is positioned to “leap frog” to a better global solution for science communication. These conditions have shown to lead to extraordinary leadership in Africa in other industries, such as mobile money and energy. We believe that open publishing platforms represent a welcome evolution to sharing knowledge and discovery without waste and unnecessary delay. African scientists are positioned to not just "catch up" with more developed countries, but to lead globe in enlightened science communication.

Irene Bassano1, Swee Hoe Ong2, Maximo Sanz-Hernandez3, Michal Vinkler4, Olivier Hanotte5, Ebele Onuigbo6, Paul Kellam1,7, Thomas Whitehead8, Mark Fife8

1Imperial College London, Department of Medicine, Division of Infectious Diseases, Wright Fleming Wing, St Mary's Campus, Norfolk Place, London, UK; 2Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK; 3Imperial College London, Department of Life Sciences, South Kensington, London UK; 4Charles University, Faculty of Science, Department of Zoology, Prague, Czech Republic; 5 International Livestock Research Institute (ILRI), Ethiopia, Addis Ababa, Ethiopia; 6Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, Enugu State, Nigeria; 7Kymab Ltd, Babraham Research Campus, Cambridge; 8The Pirbright Institute, Woking, UK

The chicken IFITM (chIFITM) locus is clustered on chromosome 5 and contains five genes, of which three are known to be interferon stimulated genes (ISGs) with potent antiviral activity, namely chIFITM1, 2, 3. These proteins restrict viral infections by blocking fusion of the viral and host membranes, thereby interfering with viral entry and replication. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood. Here we report the complete sequence of the IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus. We have generated chIFITM sequences from commercial breeds, indigenous chickens from Nigeria and Ethiopia, European breeds and inbred chicken lines from The Pirbright Institute, totalling of 211 chickens. Our data reveal that the chIFITM locus does not show structural variation across the populations analysed. However, SNPs in functionally important regions of the proteins were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria, revealing some SNPs were simultaneously under positive selection. Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.

This research was supported by the BBSRC (Animal Health Research Club) grant Number BB/L003996/1 and BBSRC grant BBS/OS/GC/000015/2.

Keywords: variant calling, SNPs, INDELs, GATK, positive selection

*Nwosu Onyeka .K. and Ngozi Miracle

National Biosafety Management Agency (NBMA), Abuja, Nigeria

With African rising population and considering its commensurate demand for more food; deficiencies of vital dietary nutrients and vulnerability to the negative impacts of climate change, the continent’s full application of genetic diversity and modern biotechnology to mitigate against these impediments remains consequential. This review explains the current standpoint of Africa towards leveraging the genetic diversity, and application of modern biotechnology in agriculture and the effectiveness of biosafety regulation towards crop improvement and food security in the continent. Genetic diversity is the pillar of biodiversity and diversity within species, between species and of ecosystem aimed to serve as a cushion for the adaptation and resilience of plants in the face of environmental challenge. Therefore genomic projects for conserving plant genetic resources (PGR) in most of the stable food crops in Africa will be vital for crop improvement and food production and will provide tools for population monitoring and assessment that can be used for conservation planning and genetic engineering (Agro-Modern Biotechnology). The transfer of a desirable gene from one species to another has proved to be a way of agro revival and economic growth which benefits both the farmers and consumers in terms of yield and improved nutrition respectively. Genetically engineered (GE) maize grown in 2015 by just three countries generated US$ 8.1 billion and over 7000 peer reviewed studies has shown that GE crops has lower concentrations of toxins that are likely carcinogenic in human and animals and there was no significant differences in grain quality such as fiber, lipids and proteins when compared with non-GE plants. Despite all these benefits of agro-modern biotechnology, there are still ill-fated public perceptions of the technology centralized towards environmental, health and socio-economic concerns. However, biosafety regulation that involves the legal and technical efforts and thorough scientific evidence to eliminate the potential risks that may result from modern biotechnology, genetically modified organisms (GMOs) and products thereof which addresses the public concerns is the assurance of the safety. Africa should develop a thoughtful agenda for an effective germplasm for agro-modern biotechnology together with an efficient biosafety system that will meet human increasing demands and improve existing conditions prevalent in our environment.

Keywords: Genetic diversity, Modern biotechnology, Biosafety, Genetic engineering, Genetically modified organisms, Germplasm.

Vivien J Chebii1,3*, Emmanuel Mpolya1, Samuel O. Oyola3, Jean-Baka Domelevo Entfellner 3, J. Musembi Mutuku3, Morris Agaba1,4.

1Nelson Mandela Africa Institution of Science and Technology, Arusha, Tanzania, 3International Livestock Research Institute, Nairobi, Kenya , 4Sarissa Limited, Kampala, Uganda

Capra nubiana is a wild goat species inhabiting the Sahara and Arabia deserts characterized by extreme temperatures, scarcity of feed and water. The genetic basis of its' adaptation to these environments remains unknown. In this study, we explored the possible role of copy number variations in the evolution of Capra species with a focus in the adaptation of Capra nubiana to its' environments. Copy number variations (CNV) are gain or loss of genomic segments greater than 1,000 bp between genomes. CNV have been implicated in phenotypic variations between species and might have a role in species differentiation and adaptations.

Based on Capra nubiana paired-end Illumina Hiseq reads sequenced to a coverage of 30x, we detected CNV relative to the domestic goat reference genome using a combination of read depth and split read approach. We identified 1,622 CNV loci overlapping with 343 protein-coding genes. Functional annotation of the protein-coding genes overlapping with the CNV showed that majority of them were immune response, signal transduction, and metabolism genes. Interferons, major histocompatibility complex proteins, and UL16 binding protein 21 which confer resistance against microbial infections were high copy number variable and significantly enriched. Lower density lipoprotein receptor-related protein 11 (Lrp11) which plays a role in stress adaptation resulting from extreme temperatures, water deprivation, and starvation were also high copy number variable. We suggest that the copy number variations of the immune response genes and Lrp11 possibly have a role in Capra nubiana adaptation to their environments.

This study is the first to shed insights on the possible role of the CNV in Capra nubiana adaptation to its' environments. The candidate copy number variable genes identified could be used upon functional confirmation as markers in animal breeding studies aimed at improving livestock health and productivity.

Keywords: Capra nubiana, gene copy number variations, adaptations, harsh environments, whole genome sequencing, Read depth method

Mohamed Z. Alimohamed, Lennart F. Johansson, Ludolf G. Boven, Krista van Dijk, Paul van der Zwaag, Richard J. Sinke, Rolf H. Sijmons, Birgit Sikkema-Raddatz, Jan D H Jongbloed

Departments of Genetics, University Medical Center Groningen, Groningen, The Netherlands

Background:

Next generation sequencing is increasingly used for clinical evaluation of patients with cardiomyopathies because it allows for simultaneous evaluation of multiple genes known to be associated with the disease. However, the diagnostic yield is variable in routine clinical practice. Furthermore, analysis of copy number variations is still not routinely performed.

Objectives:

(1) To determine the diagnostic yield of our custom targeted NGS gene panel used in routine clinical diagnostics in a Dutch cohort of over 2000 cardiomyopathy patients. (2) Examine the impact on the yield of analyzing this cohort for single or multiple exon duplications and deletions.

Methods:

Up to 61 genes known to be implicated in cardiomyopathies were selected for enrichment and analysis on DNA isolated from peripheral blood from patients as a routine procedure. Patients directed for genetic testing to our clinical genetics laboratory having a referral diagnosis for various types of cardiomyopathies were included in this study [N=2002]. A written informed consent was obtained for all patients referred to our clinical genetics laboratory. Classification of variants was based on guidelines for variant interpretation recommended by the American College of Medical Genetics and Genomics. Diagnostic yield was calculated using cases where a likely pathogenic or pathogenic variant was detected. Putative exonic deletions/duplications were analyzed using CoNVaDING and XHMM tools using settings previously described.

Results and Conclusion:

An overall diagnostic yield of 23% was achieved. CNVs were detected in 16 patients. Variants of unknown clinical significance were identified in 39% patients. These results illustrate the need to further reassess disease variant classification.

Key words:

Cardiomyopathy, NGS-panel, Diagnostic-yield

Ryan K. Cheu1,2,3, Avid Mohammadi4, Tiffany Hensley-Mcbain1,2, Jennifer Manuzak1,2,3, Alexander S. Zevin1,2, Charlene Miller3, Mark Yudin5, Rupert Kaul4, Nichole R. Klatt1,2,3

1Department of Pharmaceutics, University of Washington, Seattle, WA, USA

2Washington National Primate Research Center, University of Washington, Seattle, WA, USA

3Department of Pediatrics, University of Miami Miller School of Medicine, Miami, FL, USA

4Department of Immunology, University of Toronto, Toronto, Canada

5Core Obstetrics & Gynecology, University of Toronto, Toronto, Canada

Background: Bacterial vaginosis (BV) is associated with an increased HIV transmission rate. Inflammatory cells such as neutrophils are critical for innate immune responses but can also contribute to barrier damage and inflammation. Currently, the role of neutrophils in HIV transmission and BV status is unknown. Here, we hypothesize that BV-associated bacteria increase HIV transmission rates by inducing activation and accumulation of neutrophils within the female reproductive tract (FRT), resulting in epithelial barrier damage.

Methods: In order to elucidate the mechanisms for the negative outcomes of BV, we collected cervicovaginal cytobrushes from 11 women with BV and 10 women without BV. We used flow cytometry to assess phenotype and functionality of neutrophils and performed in vitro whole blood co-cultures with bacteria associated with BV (G. vaginalis), healthy commensals such as L. iners and L. crispatus, media alone (negative control) and lipopolysaccharide and peptidoglycan (positive controls). To determine the impact neutrophils have on epithelial integrity, we isolated neutrophils from healthy human whole blood to co-culture with FRT bacteria across a vaginal epithelial cellular monolayer and used a transepithelial electrical resistance (TEER) set-up to assess barrier damage.

Results: We demonstrated increased neutrophil activation (p=0.0022), prolonged lifespan (p=0.0022), and more total neutrophils (p=0.0022) in the cytobrushes from women with non-Lactobacillus dominant (nLD) communities. Similarly, our co-culture experiments showed more neutrophil activation (p<0.0001), prolonged lifespan (p=0.008), and more total neutrophils (p<0.0001) in cultures with G. vaginalis compared with our negative controls and Lactobacillus cultures. Our 16S rRNA analysis identified two distinct community groups one dominated by Lactobacillus and the other a more diverse community consisting of G. vaginalis. Our TEER assays demonstrated significant barrier damage in cultures with neutrophils and G. vaginalis when compared with neutrophils alone (p<0.0001) or with neutrophils and Lactobacillus spp. (p<0.0001).

Conclusions: Here, we demonstrate that BVAB induce neutrophil activation, delay apoptosis leading to accumulation within the FRT, and jeopardize epithelial integrity. This study provides potential mechanistic insights into how BV may lead to FRT inflammation and increased HIV transmission.

Heleen Schuster MDa,b, Annelot Breedveld MScc, Andries Budding MD PhDa, Paul Savelkoul PhDa,d

aDepartment of Medical Microbiology and Infection Control, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; bDepartment of Obstetrics and Gynecology, Amsterdam UMC, Univ of Amsterdam, Amsterdam, Netherlands; cDepartment of Molecular Cell Biology and Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; dDepartment of Medical Microbiology, Maastricht University Medical Centre, Maastricht, Netherlands

As knowledge on the role and composition of the vaginal microbiome is accumulating, more interest is directed to microbe:immune crosstalk. Local immunoglobulins (Igs) play an important role as mediator of mucosal immunity and homeostasis. In the vaginal mucosa, IgG is the dominating antibody, while at other mucosal surfaces IgA is most prevalent. In the gut, alterations in IgA coating of commensal bacteria are associated with inflammatory bowel disease and Clostridium difficile infection. Less is known about the role of local Igs in the female genital tract. We aim to provide insight in the IgA and IgG coating of vaginal bacteria using a combination of flow cytometry based techniques and microbiota profiling.

Vaginal swabs were collected from healthy volunteers. Fluorescence-Activated Cell Sorting (FACS) was used to sort micro-organisms according to IgA and IgG coating. The bacterial composition was analyzed with bacterial profiling technique IS-pro, using fragment variability of the 16S-2S rDNA intergenic spacer region.

Lactobacillus crispatus and L. iners dominated the vaginal microbiota of healthy volunteers and show large numbers of IgA and IgA/IgG coated Lactobacilli. Bacteria with only IgG on their surface were not detected. The proportions of Ig coated bacteria varied among individuals with similar microbial compositions.

Vaginal bacteria show varying amounts of IgA and IgA/IgG coating between different healthy volunteers. The high amount of Ig coated vaginal commensal bacteria demonstrate the importance of local Igs in the mucosal environment. Disturbances of the coating might lead to increased local inflammation and can possibly identify women at risk for diseases of the reproductive tract and adverse pregnancy outcomes. These promising results indicate a putative role of Igs in the maintenance of a healthy microbiome.

Mariya I. Petrova

KU Leuven, Belgium

The vaginal microbial community is typically characterized by abundant lactobacilli. Lactobacillus iners, a fairly recently detected species, is frequently present in the vaginal niche. However, the role of this species in vaginal health is unclear, since it can be identified in normal conditions as well as during vaginal dysbiosis, such as bacterial vaginosis, a condition characterized by an abnormal increase in bacterial diversity and lack of typical lactobacilli. Compared to other Lactobacillus species, L. iners has more complex nutritional requirements and a Gram-variable morphology. L. iners has an unusually small genome, indicative of a symbiotic or parasitic lifestyle, in contrast to other lactobacilli that show niche flexibility and genomes of up to 3-4 Mbp. The presence of specific L. iners genes, such as those encoding iron-sulfur proteins and unique σ-factors, reflects a high degree of niche specification. The genome of L. iners strains also encodes inerolysin, a pore-forming toxin related to vaginolysin of Gardnerella vaginalis. Possibly, this organism may have clonal variants that in some cases promote a healthy vagina, and in other cases are associated with dysbiosis and disease. Future research should examine the exact role of this species and its relationship with the host.

Praveen Thumbikat1, Stephen F. Murphy1, Jonathan F. Anker1, Daniel J. Mazur1, Anthony J. Schaeffer1, Christel Hall1

1Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

Gram-positive bacteria are considered an ubiquitous but incidental finding in urine and prostatic secretion cultures from male patients with chronic pelvic pain syndrome (CPPS) However, we hypothesized that gram-positive traditional non-uropathogenic bacteria can play a direct role in CPPS pathogenesis. Staphylococcus epidermidis, Staphylococcus haemolyticus, and Enterococcus faecalis comprised 62% of prostate-localizing bacteria from CPPS patients. A subset of these induced high NFκB expression, contained pathogenic virulence factors, and were associated with increased clinical symptoms. Here we show that an immunogenic subset of the most common gram-positive prostate-localizing bacterial strains can directly cause pelvic tactile allodynia and voiding dysfunction. Intra-urethral infection of mice with immunogenic bacterial isolates from each strain, but not an S. epidermidis isolate from a healthy control, recapitulated symptoms seen in patients. Mechanistically, these pathogenic gram-positive bacteria induced unique prostate inflammation, involving T cells, NK cells, and cytokine production. We demonstrate for the first time that the prevalent, yet untreated traditional non-uropathogenic gram-positive bacteria can play a causative role in CPPS pathogenesis.