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

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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,


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


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


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,


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


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


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,


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 early view,


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.
Sánchez-Cordón P J, Jabbar T, Berrezaie M, Chapman D, Reis A, Sastre P, Rueda P, Goatley L, Dixon L K (2018)

Evaluation of protection induced by immunisation of domestic pigs with deletion mutant African swine fever virus Benin MGF by different doses and routes

Vaccine early view,


A live attenuated African swine fever virus (ASFV) vaccine candidate, produced by deletion of several genes belonging to multi-gene families MGF360 and 505 from virulent Benin 97/1 strain (BeninΔMGF), induces protection in pigs against parental virulent strain. In order to better define the safety and efficacy of this attenuated vaccine candidate and to understand protective mechanisms, we extended previous studies by intramuscular immunisation of pigs with the deletion mutant BeninΔMFG at different doses (102, 103, 104 TCID50), together with intranasal immunisation at the 103 dose. Results demonstrated a strong correlation between both doses and routes of immunisation of BeninΔMFG and the percentage of protection achieved, the onset of clinical signs, the viremia levels reached and the onset of death in non-protected pigs. The results show that the intramuscular route using high doses (104 TCID50) is the best option for immunisation. Only transient increase in temperature associated with a peak of virus genome levels was observed in most pigs after immunisation. Then, virus genome levels progressively decreased throughout the experiment until reaching low or undetectable levels in those protected pigs that survived after challenge. The IgM antibody responses following immunisation were detected between day 7–10 post-immunisation and remained at elevated levels for 10–18 days in most pigs before dropping. IgG was detected from day 15 to 21 post-immunisation and maintained at increased levels for the remainder of the experiment in most pigs. Induction of IFNγ and IL-10 was detected by ELISA in sera from some pigs immunised with 103 TCID50 by intramuscular or intranasal route at early times post-immunisation. IL-10 was also detected in serum from some non-protected pigs included in these groups after challenge.

Erickson A, Fisher M, Furukawa-Stoffer T, Ambagala A, Hodko D, Pasick J, King D P, Nfon C, Ortega Polo R, Lung O (2018)

A multiplex reverse transcription PCR and automated electronic microarray assay for detection and differentiation of seven viruses affecting swine

Transboundary and Emerging Diseases early view,


Microarray technology can be useful for pathogen detection as it allows simultaneous interrogation of the presence or absence of a large number of genetic signatures. However, most microarray assays are labour-intensive and time-consuming to perform. This study describes the development and initial evaluation of a multiplex reverse transcription (RT)-PCR and novel accompanying automated electronic microarray assay for simultaneous detection and differentiation of seven important viruses that affect swine (foot-and-mouth disease virus [FMDV], swine vesicular disease virus [SVDV], vesicular exanthema of swine virus [VESV], African swine fever virus [ASFV], classical swine fever virus [CSFV], porcine respiratory and reproductive syndrome virus [PRRSV] and porcine circovirus type 2 [PCV2]). The novel electronic microarray assay utilizes a single, user-friendly instrument that integrates and automates capture probe printing, hybridization, washing and reporting on a disposable electronic microarray cartridge with 400 features. This assay accurately detected and identified a total of 68 isolates of the seven targeted virus species including 23 samples of FMDV, representing all seven serotypes, and 10 CSFV strains, representing all three genotypes. The assay successfully detected viruses in clinical samples from the field, experimentally infected animals (as early as 1 day post-infection (dpi) for FMDV and SVDV, 4 dpi for ASFV, 5 dpi for CSFV), as well as in biological material that were spiked with target viruses. The limit of detection was 10 copies/μl for ASFV, PCV2 and PRRSV, 100 copies/μl for SVDV, CSFV, VESV and 1,000 copies/μl for FMDV. The electronic microarray component had reduced analytical sensitivity for several of the target viruses when compared with the multiplex RT-PCR. The integration of capture probe printing allows custom onsite array printing as needed, while electrophoretically driven hybridization generates results faster than conventional microarrays that rely on passive hybridization. With further refinement, this novel, rapid, highly automated microarray technology has potential applications in multipathogen surveillance of livestock diseases.


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