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

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Turan N, Ozsemir C, Yilmaz A, Cizmecigil U Y, Aydin O, Bamac O E, Gurel A, Kutukcu A, Ozsemir K, Tali H E, Tali B H, Yilmaz S G, Yaramanoglu M, Tekelio?lu B K, Ozsoy S, Richt J A, Iqbal M, Yilmaz H (2020)

Identification of Newcastle disease virus subgenotype VII.2 in wild birds in Turkey

BMC Veterinary Research 16 (1), 277


Background: Newcastle disease viruses (NDVs) can spread across continents via migratory birds. Hence, we investigated the frequency of NDV in both non-migratory and birds migrating on the Black Sea-Mediterranean flyway, in Istanbul, Turkey. Birds were trapped using nets placed around the Kucukcekmece lake Avcilar, Istanbul, in spring seasons of 2016 and 2018. In total, 297 birds belonging to 42 different species were trapped, categorized according to species and sex, and flocked oropharyngeal swabs were collected. In addition, flocked swabs were also collected from 115 mallards caught by hunters around Edirne and from 207 birds which had been treated in the Veterinary Faculty of Istanbul university-Cerrahpasa. Tissue samples were taken from dead wild birds brought by public to Veterinary Faculty. A total of 619 flocked oropharyngeal swabs were pooled into 206 samples. RNA was extracted from swabs and tissue samples. Real-time RT-PCR prob. assay was used to detect NDV-RNA in samples.

Results: There was no amplification in real time RT-PCR in samples taken from wild birds caught by traps. However, amplification of NDV-F gene was observed in oropharyngeal swabs taken from 2 waterfowls (Common Moorhen and Mallard), and in tissue samples taken from 2 little owls and 1 common kestrel. Sequencing and phylogenetic analyses of these 5 samples for NDV-F gene showed great similarity with NDV subgenotype VII.2 viruses. Analysis also showed that there is a high similarity with the F gene sequences previously reported from Turkey in 2012 and as well as the sequences from neighbouring countries Bulgaria and Georgia and geographically close country such as Pakistan. Although the strains found in this study are closely related, there is a relatively small degree of molecular divergence within 543 bp of F gene of the Turkish NDV isolate and strains detected in Israel, Pakistan, Iran, United Arab Emirates and Belgium.

Conclusions: Our findings revealed the presence of subgenotype VII.2 of NDVs in wild birds in north west of Turkey and demonstrated some degree of molecular evolution when compared to the earlier NDV-VII.2 isolate in Turkey.


The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.


Immune tolerance induced by avian leukosis virus subgroup J (ALV-J) is a prerequisite for tumorigenesis. Although we had reported that B cell anergy induced by ALV-J was the main reason for immune tolerance, the molecular mechanism still remains unclear. Here, we found SU protein of ALV-J interacted with tyrosine kinase Lyn (a key protein in BCR signaling pathway) by confocal laser scanning microscopy and co-immunoprecipitation test, which suggested that Lyn might play an important role in B cell anergy induced by ALV-J. Correspondingly, the mRNA and protein level of Lyn was significantly up-regulated in B cells after ALV-J infection. Subsequently, the phosphorylated protein levels of Lyn at Tyr507 site were significantly up-regulated in ALV-J-infected B cells after BCR signal activation, but the phosphorylated protein level of Syk (a direct substrate of Lyn) at Tyr525/526 site, Ca2+ flux, and NF-κB p65 protein level were significantly down-regulated. Interestingly, the phosphorylated protein level of Syk at Tyr525/526 site, Ca2+ flux, and NF-κB p65 protein level were both significantly retrieved after the shLyn treatment in B cells infected by ALV-J. In summary, these results indicated that ALV-J activated the negative regulatory effect of phosphorylated Lyn protein at 507 site in BCR signal transduction pathway and then mediated B cell anergy, which will provide a new insight for revealing the pathogenesis of immune tolerance induced by ALV-J.

Flannery J, Moore R, Marsella L, Harris K, Ashby M, Rajko-Nenow P, Roberts H, Gubbins S, Batten C (2020)

Towards a sampling rationale for African swine fever virus detection in pork products

Foods 9 (9), 1148


African swine fever (ASF) is a highly lethal disease of pigs caused by the ASF virus (ASFV), which presents a serious threat to global food security. The movement of contaminated pork products has previously been postulated as contributing to the introduction of ASF into new areas. To evaluate the performance of ASFV detection systems in multi-component pork products, we spiked sausage meat with four different ASFV-containing materials (ASFV cell culture, pork loin, meat juice and bone marrow). DNA was extracted using two manual systems (MagMAX CORE, Qiagen) and one automated (MagMAX CORE) one, and three qPCR assays (VetMAX, King, UPL) were used. The performance of the DNA extraction systems was as follows; automated MagMAX > manual MagMAX > manual Qiagen. The commercial VetMAX qPCR assay yielded significantly lower CT values (p < 0.001), showing greater sensitivity than the World Organization for Animal Health (OIE)-prescribed assays (King, UPL). Detection probability was the highest for matrices contaminated with bone marrow compared with pork loin or meat juice. An estimated minimum sample size of one 1-g sample is sufficient to detect ASFV in a homogenous pork product if bone marrow from infected pigs comprises 1 part in 10,000. We demonstrated that existing ASFV detection systems are appropriate for use in a food-testing capacity, which can provide an additional control measure for ASF.


Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.

Canini L, Holzer B, Morgan S, Hemmink J D, Clark B, sLoLa Dynamics Consortium, Woolhouse M E J, Tchilian E, Charleston B (2020)

Timelines of infection and transmission dynamics of H1N1pdm09 in swine

PLoS Pathogens 16 (7), e1008628


Influenza is a major cause of mortality and morbidity worldwide. The relationship between the time course of influenza infection and virus shedding and onward transmission of the virus remains poorly characterized. Pigs are a natural host for influenza infection with shedding patterns similar to humans. Therefore we experimentally infected pigs with the H1N1pdm09 influenza A virus using direct contact challenge and then mixed the infected pigs with a different naïve pig each day to understand when transmission occurred. Using mathematical modeling, we found that transmission events occurred on 60% of occasions when the infected pigs were shedding virus and that the risk of transmission increased with the quantity of virus shed. Also it was clear the incontact pigs started to shed virus later after exposure when the infected pigs were shedding low quantities of virus. Our study therefore provides quantitative information on the time lines of influenza virus infection and the dynamics of transmission. This is important to understand the spread of influenza viruses through animal populations and, potentially, in humans.


Neuraminidase inhibitors (NAIs) are antiviral agents recommended worldwide to treat or prevent influenza virus infections in humans. Past influenza virus pandemics seeded by zoonotic infection by avian influenza viruses (AIV) as well as the increasing number of human infections with AIV have shown the importance of having information about resistance to NAIs by avian NAs that could cross the species barrier. In this study we introduced four NAI resistance-associated mutations (N2 numbering) previously found in human infections into the NA of three current AIV subtypes of the H5Nx genotype that threaten the poultry industry and human health: highly pathogenic H5N8, H5N6 and H5N2. Using the established MUNANA assay we showed that a R292K substitution in H5N6 and H5N2 viruses significantly reduced susceptibility to three licenced NAIs: oseltamivir, zanamivir and peramivir. In contrast the mutations E119V, H274Y and N294S had more variable effects with NAI susceptibility being drug- and strain-specific. We measured the replicative fitness of NAI resistant H5N6 viruses and found that they replicated to comparable or significantly higher titres in primary chicken cells and in embryonated hens' eggs as compared to wild type - despite the NA activity of the viral neuraminidase proteins being reduced. The R292K and N294S drug resistant H5N6 viruses had single amino acid substitutions in their haemagglutinin (HA): Y98F and A189T, respectively (H3 numbering) which reduced receptor binding properties possibly balancing the reduced NA activity seen. Our results demonstrate that the H5Nx viruses can support drug resistance mutations that confer reduced susceptibility to licenced NAIs and that these H5N6 viruses did not show diminished replicative fitness in avian cell cultures. Our results support the requirement for on-going surveillance of these strains in bird populations to include motifs associated with human drug resistance.

Keep S, Oade M S, Lidzbarski-Silvestre F, Bentley K, Stevenson-Leggett P, Freimanis G L, Tennakoon C, Sanderson N, Hammond J A, Jones R C, Britton P, Bickerton E (2020)

Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus

Journal of General Virology early view


Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.


The most sensitive cell culture system for the isolation of foot-and-mouth disease virus (FMDV) is primary bovine thyroid (BTY) cells. However, BTY cells are seldom used because of the challenges associated with sourcing thyroids from FMDV-negative calves (particularly in FMD endemic countries), and the costs and time required to regularly prepare batches of cells. Two continuous cell lines, a fetal goat tongue cell line (ZZ-R 127) and a fetal porcine kidney cell line (LFBK-αVβ6), have been shown to be highly sensitive to FMDV. Here, we assessed the sensitivity of ZZ-R 127 and LFBK-αVβ6 cells relative to primary BTY cells by titrating a range of FMDV original samples and isolates. Both the ZZ-R 127 and LFBK-αVβ6 cells were susceptible to FMDV for >100 passages, and there were no significant differences in sensitivity relative to primary BTY cells. Notably, the LFBK-αVβ6 cell line was highly sensitive to the O/CATHAY porcine-adapted FMDV strain. These results support the use of ZZ-R 127 and LFBK-αVβ6 as sensitive alternatives to BTY cells for the isolation of FMDV, and highlight the use of LFBK-αVβ6 cells as an additional tool for the isolation of porcinophilic viruses.

Fletcher N F, Meredith L W, Tidswell E L, Bryden S R, Gonçalves-Carneiro D, Chaudhry Y, Shannon-Lowe C, Folan M A, Lefteri D A, Pingen M, Bailey D, McKimmie C S, Baird A W (2020)

A novel antiviral formulation inhibits a range of enveloped viruses

Journal of General Virology early view


Some free fatty acids derived from milk and vegetable oils are known to have potent antiviral and antibacterial properties. However, therapeutic applications of short- to medium-chain fatty acids are limited by physical characteristics such as immiscibility in aqueous solutions. We evaluated a novel proprietary formulation based on an emulsion of short-chain caprylic acid, ViroSAL, for its ability to inhibit a range of viral infections in vitro and in vivo. In vitro, ViroSAL inhibited the enveloped viruses Epstein-Barr, measles, herpes simplex, Zika and orf parapoxvirus, together with Ebola, Lassa, vesicular stomatitis and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) pseudoviruses, in a concentration- and time-dependent manner. Evaluation of the components of ViroSAL revealed that caprylic acid was the main antiviral component; however, the ViroSAL formulation significantly inhibited viral entry compared with caprylic acid alone. In vivo, ViroSAL significantly inhibited Zika and Semliki Forest virus replication in mice following the inoculation of these viruses into mosquito bite sites. In agreement with studies investigating other free fatty acids, ViroSAL had no effect on norovirus, a non-enveloped virus, indicating that its mechanism of action may be surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for the prevention and/or treatment of a broad range of enveloped viruses, particularly those of the skin and mucosal surfaces.


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