Publications

Publications

The Pirbright Institute publication directory contains details of selected publications written by our researchers.

There were a total of 1662 results for your search.
Chang P, Yao Y, Tang N, Sadeyen J-R, Sealy J, Clements A, Bhat S, Munir M, Bryant J, Iqbal M (2018)

The application of NHEJ-CRISPR/Cas9 and Cre-Lox system in the generation of bivalent duck enteritis virus vaccine against avian influenza virus

Viruses 10 (2), 81

Abstract

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Jankovich J K, Chapman D, Hansen D T, Robida M D, Loskutov A, Craciunescu F, Borovkov A, Kibler K, Goatley L, King K, Netherton C L, Taylor G, Jacobs B, Sykes K, Dixon L K (2018)

Immunisation of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African swine fever virus immunogenic and protective proteins

Journal of Virology early view,

Abstract

African swine fever virus (ASFV) causes an acute haemorrhagic fever in domestic pigs with high socio-economic impact. No vaccine is available limiting options for control. Although live attenuated ASFV can induce up to one hundred percent protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma ELISpot assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost and pigs were challenged with a lethal dose of Georgia 2007/1 ASFV isolate. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunised with vectors expressing ASFV antigens compared with control pigs.ImportanceThe lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The response in immunized pigs to these individual antigens was compared to identify the most immunogenic. Lethal challenge of pigs immunised with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to pigs immunised with control vectors. Novel immunogenic ASFV proteins have been identified to test further as vaccine candidates.

Qian K, Cheng X, Zhang D, Shao H, Yao Y, Nair V, Qin A (2018)

Antiviral effect of lithium chloride on replication of avian leukosis virus subgroup J in cell culture

Archives of Virology early view,

Abstract

Lithium chloride (LiCl) has been reported to possess antiviral activity against several viruses. In the current study, we assessed the antiviral activity effect of LiCl on ALV-J infection in CEF cells by using real-time PCR, Western blot analysis, IFA and p27 ELISA analysis. Our results showed that both viral RNA copy number and protein level decreased significantly in a dose and time dependent manner. Time-course analysis revealed that the antiviral effect was more pronounced when CEFs were treated at the post infection stage rather than at early absorption or pre-absorption stages. Further experiments demonstrated that LiCl did not affect virus attachment or entry, but rather affected early virus replication. We also found that inhibition of viral replication after LiCl treatment was associated with reduced mRNA levels of pro-inflammatory cytokines. These results demonstrate that LiCl effectively blocked ALV-J replication in CEF cells and may be used as an antiviral agent against ALV-J.

Marotta C R, Dos Santos P N, Cordeiro M D, Matos P C M, Barros J H D S, Madeira M D F, Bell-Sakyi L, Fonseca A H (2018)

Trypanosoma rhipicephalis sp. nov. (Protozoa: Kinetoplastida) isolated from Rhipicephalus microplus (Acari: Ixodidae) ticks in Rio de Janeiro, Brazil

Parasitology Open 4, e2

Abstract

Parasites of the genus Trypanosoma are unicellular flagellated microorganisms of the Trypanosomatidae. This study describes an isolate of the genus Trypanosoma naturally infecting Rhipicephalus microplus ticks, characterized through molecular, morphological and biological analysis. Trypanosome cultures, designated strain P1RJ, were obtained by isolation from R. microplus haemolymph in cultures of the tick cell line IDE8. After isolation, strain P1RJ grew well axenically in L15B medium at temperatures of 30, 32 and 34 °C. The new trypanosome remained stable in axenic culture over 14 passages in L15B at 30 °C and was successfully cryopreserved and resuscitated. Morphometric analysis was performed on randomly selected developmental forms. 18S rRNA and 24Sα rDNA sequence analyses confirmed that strain P1RJ is a new species of the genus Trypanosoma. The nucleotide sequences described were submitted to Genbank. Pathogenicity, involvement in vertebrate hosts, epidemiology, developmental cycle and transmission mechanisms of strain P1RJ are still unknown. Therefore, more studies will be necessary to determine life cycle aspects of this trypanosome, for which we propose the name Trypanosoma rhipicephalis sp. nov.

Cozens D, Grahame E, Sutherland E, Taylor G, Berry C C, Davies R L (2018)

Development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract

Scientific Reports 8 (1), 853

Abstract

Cattle are subject to economically-important respiratory tract infections by various bacterial and viral pathogens and there is an urgent need for the development of more realistic in vitro models of the bovine respiratory tract to improve our knowledge of disease pathogenesis. In the present study, we have optimized the culture conditions in serum-free medium that allow bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface to differentiate into a three-dimensional epithelium that is highly representative of the bovine airway. Epidermal growth factor was required to trigger both proliferation and differentiation of BBECs whilst retinoic acid was also essential for mucociliary differentiation. Triiodothyronine was demonstrated not to be important for the differentiation of BBECs. Oxygen concentration had a minimal effect although optimal ciliation was achieved when BBECs were cultured at 14% oxygen tension. Insert pore-density had a significant effect on the growth and differentiation of BBECs; a high-pore-density was required to trigger optimum differentiation. The established BBEC model will have wide-ranging applications for the study of bacterial and viral infections of the bovine respiratory tract; it will contribute to the development of improved vaccines and therapeutics and will reduce the use of cattle in in vivo experimentation.

Klassmann A, Ferretti L (2018)

The third moments of the site frequency spectrum

Theoretical Population Biology early view,

Abstract

The analysis of patterns of segregating (i.e. polymorphic) sites in aligned sequences is routine in population genetics. Quantities of interest include the total number of segregating sites and the number of sites with mutations of different frequencies, the so-called site frequency spectrum. For neutrally evolving sequences, some classical results are available, including the expected value and variance of the spectrum in the Kingman coalescent model without recombination as calculated by Fu (1995). In this work, we use similar techniques to compute the third moments of the frequencies of three linked sites. Based on these results, we derive analytical results for the bias of Tajima’s D and other neutrality tests. As a corollary, we obtain the second moments of the frequencies of two linked mutations conditional on the presence of a third mutation with a certain frequency. These moments can be used for the normalisation of new neutrality tests relying on these spectra.

Glidden C K, Beechler B, Buss P E, Charleston B, de Klerk-Lorist L-M, Maree F F, Muller T, Pérez-Martin E, Scott K A, van Schalkwyk O L, Jolles A (2018)

Detection of pathogen exposure in African buffalo using non-specific markers of inflammation

Frontiers in Immunology 8, 1944

Abstract

Detecting exposure to new or emerging pathogens is a critical challenge to protecting human, domestic animal and wildlife health. Yet current techniques to detect infections typically target known pathogens of humans or economically important animals. In the face of the current surge in infectious disease emergence, non-specific disease surveillance tools are urgently needed. Tracking common host immune responses indicative of recent infection may have potential as a non-specific diagnostic approach for disease surveillance. The challenge to immunologists is to identify the most promising markers, which ideally should be highly conserved across pathogens and host species, become upregulated rapidly and consistently in response to pathogen invasion, and remain elevated beyond clearance of infection. This study combined an infection experiment and a longitudinal observational study to evaluate the utility of non-specific markers of inflammation (NSMI; two acute phase proteins (haptoglobin and serum amyloid A), two pro-inflammatory cytokines (IFNγ and TNF-α)) as indicators of pathogen exposure in a wild mammalian species, African buffalo (Syncerus caffer). Specifically, in the experimental study we asked (1) How quickly do buffalo mount NSMI responses upon challenge with an endemic pathogen, foot-and-mouth disease virus; (2) for how long do NSMI remain elevated after viral clearance and; (3) how pronounced is the difference between peak NSMI concentration and baseline NSMI concentration? In the longitudinal study, we asked (4) Are elevated NSMI associated with recent exposure to a suite of bacterial and viral respiratory pathogens in a wild population? Among the four NSMI that we tested, haptoglobin showed the strongest potential as a surveillance marker in African buffalo: Concentrations quickly and consistently reached high levels in response to experimental infection, remaining elevated for almost a month. Moreover, elevated haptoglobin was indicative of recent exposure to two respiratory pathogens assessed in the longitudinal study. We hope this work motivates studies investigating suites of non-specific markers of inflammation as indicators for pathogen exposure in a broader range of both pathogen and host species, potentially transforming how we track disease burden in natural populations.

Li H, Bradley K C, Long J S, Frise R, Ashcroft J W, Hartgroves L C, Shelton H, Makris S, Johansson C, Cao B, Barclay W S (2018)

Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice

PLoS Pathog 14 (1), e1006821

Abstract

The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology.

Sánchez-Cordón P J, Montoya M, Reis A L, Dixon L K (2018)

African swine fever: A re-emerging viral disease threatening the global pig industry

Veterinary Journal early view,

Abstract

African swine fever (ASF) recently has spread beyond sub-Saharan Africa to the Trans-Caucasus region, parts of the Russian Federation and Eastern Europe. In this new epidemiological scenario, the disease has similarities, but also important differences, compared to the situation in Africa, including the substantial involvement of wild boar. A better understanding of this new situation will enable better control and prevent further spread of disease. In this article, these different scenarios are compared, and recent information on the pathogenesis of ASF virus strains, the immune response to infection and prospects for developing vaccines is presented. Knowledge gaps and the prospects for future control are discussed.

Tang N, Zhang Y, Pedrera M, Chang P, Baigent S, Moffat K, Shen Z, Nair V, Yao Y (2018)

A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system

Vaccine 36 (5), 716-722

Abstract

Herpesvirus of turkeys (HVT) has been successfully used as live vaccine against Marek's disease (MD) worldwide for more than 40 years either alone or in combination with other serotypes. HVT is also widely used as a vector platform for generation of recombinant vaccines against a number of avian diseases such as infectious bursal disease (IBD), Newcastle disease (ND) and avian influenza (AI) using conventional recombination methods or recombineering tools on cloned viral genomes. In the present study, we describe the application of CRISPR/Cas9-based genome editing as a rapid and efficient method of generating HVT recombinants expressing VP2 protein of IBDV. This approach offers an efficient method to introduce other viral antigens into the HVT genome for rapid development of recombinant vaccines.

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