Innovative approaches to genome editing in avian species

The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal.In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells(PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing(STAGE).The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.

Bats and Viruses:a Brief Review

Bats,probably the most abundant,diverse and geographically dispersed vertebrates on earth,have recently been shown to be the reservoir hosts of a number of emerging viruses responsible for severe human and livestock disease outbreaks. Flying foxes have been demonstrated to be the natural reservoir for Hendra and Nipah viruses. Evidence supporting the possibility of bats as potential reservoirs for SARS coronavirus(SARS-CoV) and Ebola virus has also been reported. The recent discovery of these viruses and other viruses occurring naturally in the bat population provides a unique insight into a diverse pool of potentially emergent and pathogenic viruses. The factors which influence the ability of zoonotic viruses to effectively cross the species barrier from bats to other animal populations are poorly understood. A brief review is provided here on the recently emerged bat viruses and on current and future strategies for research in this area.

Identification of Immunogenic Determinants of the Spike Protein of SARS-like Coronavirus

Bat SARS-Iike coronavirus （SL-CoV） has a genome organization almost identical to that of SARS-CoV, but the N-terminus of the Spike （S） proteins, which interacts with host receptor and is a major target of neutralizing antibodies against CoVs, of the two viruses has only 63-64% sequence identity. Although there have been reports studying the overall immunogenicity of SsL, knowledge on the precise location of immunodominant determinants for SSL is still lacking. In this study, using a series of truncated expressed SsL fragments and SsL specific mouse sera, we identified two immunogenic determinants for SSL. Importantly, one of the two regions seems to be located in a region not shared by known immunogenic determinants of the SSARS. This finding will be of potential use in future monitoring of SL-CoV infection in bats and spillover animals and in development of more effective vaccine to cover broad protection against this new group of coronaviruses.

Reverse genetics: Unlocking the secrets of negative sense RNA viral pathogens

Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cD NA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed withreference to new technologies that may be utilized to improve reverse genetic approaches.

Cultural drivers and health-seeking behaviours that impact on the transmission of pig-associated zoonoses in Lao People’s Democratic Republic

Pig rearing is an important income source in the Lao People’s Democratic Republic(PDR),with many smallholder farmers using traditional free-range pig production systems.Despite the potentially significant health risks posed by pig production regarding pig-associated zoonoses,information on the sociocultural drivers of these zoonoses is significantly lacking.This review summarises the existing sociocultural knowledge on eight pig-associated zoonoses suspected to be endemic in Southeast Asia:brucellosis,Q fever(Coxiella burnetii),trichinellosis,hepatitis E virus,leptospirosis,Japanese encephalitis,Streptococcus suis and Taenia solium taeniasis-cysticercosis.It summarises current knowledge on these diseases grouped according to their clinical manifestations in humans to highlight the propensity for underreporting.A literature search was conducted across multiple databases for publications from 1990 to the present day related to the eight pig-associated zoonoses and the risk and impact connected with them,with Lao PDR as a case study.Many of these pig-associated zoonoses have similar presentations and are often diagnosed as clinical syndromes.Misdiagnosis and underreporting are,therefore,substantial and emphasise the need for more robust diagnostics and appropriate surveillance systems.While some reports exist in other countries in the region,information is significantly lacking in Lao PDR with existing information coming mainly from the capital,Vientiane.The disease burden imposed by these zoonoses is not only characterised by morbidity and mortality,but directly impacts on livelihoods through income reduction and production losses,and indirectly through treatment costs and lost work opportunities.Other factors crucial to understanding and controlling these diseases are the influence of ethnicity and culture on food-consumption practices,pig rearing and slaughter practices,hygiene and sanitation,health-seeking behaviours and,therefore,risk factors for disease transmission.Published information on the knowledge,attitudes and beliefs of people regarding pig zoonoses and their risk factors is also extremely limited in Lao PDR and the broader Southeast Asian region.The need for more transdisciplinary research,using a One Health approach,in order to understand the underlining social determinants of health and their impacts on health-seeking behaviours,disease transmission and,ultimately,disease reporting,cannot be more emphasized.

Transmission ecology of rodent-borne diseases:New frontiers

Rodents are recognized reservoir hosts for many human zoonotic pathogens.The current trends resulting from anthropocene defaunation suggest that in the future they,along with other small mammals,are likely to become the dominant mammals in almost all human-modified environments.Recent intricate studies on bat-borne emerging diseases have highlighted that many gaps exist in our understanding of the zoonotic transmission of rodent-borne pathogens.This has emphasized the need for scientists interested in rodent-borne diseases to integrate rodent ecology into their analysis of rodent-borne pathogen transmission in order to identify in more detail the mechanisms of spillover and chains of transmission.Further studies are required to better understand the true impact of rodent abundance and the importance of pathogen sharing and circulation in multi-host–multi-pathogen communities.We also need to explore in more depth the roles of generalist and abundant species as the potential links between pathogen-sharing,co-infections and disease transmission.

Virology at the Lorne Infection and Immunity Conference 2019

The Lome Infection and Immunity Conference is one of five scientific meetings held during each month of February at the Cumberland resort in the picturesque seaside town of Lome,on the Great Ocean Road in Victoria(Australia).The specific aim of the meeting is to bring together basic,clinical and translational researchers-those who examine microbes and their impact on the innate or adaptive immune response,researchers who study the mechanisms that regulate immune responses,and those who apply this knowledge to preventing and treating infectious and in”ammatory diseases.The average number of attendees is 220,with registrants appreciative of the welcoming and relaxed atmosphere(Fig.1).