Publications

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

There were a total of 2071 results for your search.

Abstract

H9N2 avian influenza viruses circulate in poultry throughout much of Asia, the Middle East and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right as well as in the generation of novel zoonotic viruses, for example H7N9. In recent years it has been observed that H9N2 viruses have further adapted to poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titres in various systems and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for the virulent phenotype and extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the soaring morbidity and mortality rates in the field, as well as rapidly expanding geographical range seen in these viruses. Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here we take representative viruses and investigate the genetic basis for this ‘fitness’. A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes in viral replication as well as contribute in the virus accessory functions – however we find that the fitness effect is specifically due to changes in the protein polymerase activity.

Clements A L, Peacock T P, Sealy J E, Hussain S, Sadeyen J-R, Shelton H, Digard P, Iqbal M (2020)

PA-X is an avian virulence factor in H9N2 avian influenza virus

bioRxiv, 114876

Abstract

Influenza A viruses encode several accessory proteins that have host- and strain-specific effects on virulence and replication. The accessory protein PA-X is expressed due to a ribosomal frameshift during translation of the PA gene. Depending on the particular combination of virus strain and host species, PA-X has been described as either acting to either reduce or increase virulence and/or virus replication. In this study, we set out to investigate the role PA-X plays in H9N2 avian influenza viruses, focussing particularly on the natural avian host, chickens. We found H9N2 PA-X induced robust host shutoff in both mammalian and avian cells and increased replication in mammalian, but not avian cells. We further showed that PA-X affected embryonic lethality in ovo and led to more rapid viral shedding and widespread organ dissemination in vivo in chickens. Overall, we conclude PA-X may act as a virulence factor for H9N2 viruses in chickens, allowing faster replication and wider organ tropism.

Abstract

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vectorpathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

Abstract

The aim of this study was to assess the vaccine-matching and antigenic properties of foot-and-mouth disease virus (FMDV) isolates collected from Ethiopia between 2011 and 2014. Samples (n = 51) were collected from cattle and pigs with clinical signs consistent with foot-and-mouth disease (FMD) on farms in Debre-Berhan, Debre-Zeit/Bishoftu, Sidamo, Mekelle, and Addis Ababa. Infectious FMDV was isolated using BHK-21 cell cultures from 38 of the 51 field samples (74.5%). All of these FMDV-positive samples were characterized as serotype O, belonging to two East Africa topotypes (EA-3 and EA-4), and their VP1-encoding sequences demonstrated amino acid sequence variability encompassing 27 positions in comparison to the vaccine strain (O/ETH/38/2005) currently provided by the National Veterinary Institute of Ethiopia. One-dimensional virus neutralization test (1 dm VNT) results showed that O/ETH/38/2005 was antigenically matched to 10 of the 16 serotype O viruses. These findings indicate that the O/ETH/38/2005 vaccine strain can provide protection against outbreaks caused by the O/EA-3 topotype, although poorer vaccine-matching results for the O/EA-4 topotype reinforce the importance of using a good-quality vaccine with high coverage in the susceptible herds with supporting post-vaccination serosurveillance to ensure that sufficient antibody titers are generated in the vaccinated animals.

Abstract

Following short immunization protocols, naturally attenuated African swine fever virus (ASFV) isolate OURT88/3 and deletion mutant BeninΔMGF have previously been shown to induce high percentage of protection in domestic pigs against challenge with virulent virus. Results obtained in the present study showed that a single intramuscular immunization of domestic pigs with OURT88/3 or BeninΔMGF followed by a challenge with virulent Benin 97/1 isolate at day 130 post-immunisation did not trigger the necessary mechanisms to generate immunological memory able to induce long-term protection against disease. All pigs developed acute forms of ASF. IFNgamma producing cells peaked at day 24 post-immunisation, declining thereafter. Surprisingly, levels of T regulatory cells (Tregs) and IL-10 were elevated at the end of the experiment suggesting that regulatory components of the immune system may inhibit effective protection.Importance. Duration of immunity for any vaccine candidate is crucial. In the case of African swine fever virus vaccine candidates, this issue has received little attention. Attenuated viruses have been proven protective following short immunization protocols in which pigs were challenged a few weeks after the first immunization. Here, duration of immunity and immune responses induced over a duration of 130 days were studied during pre-challenge and after challenge of pigs immunised with the naturally attenuated isolate OURT88/3 and an attenuated gene-deleted isolate BeninΔMGF. After a single intramuscular immunization of domestic pigs with the OURT88/3 isolate o BeninΔMGF virus, animals were not protected against challenge with virulent Benin 97/1 ASFV genotype I isolate at day 130 post-immunization. Levels of T regulatory cells and IL-10 were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.

Riffault S, Hagglund S, Guzman E, Naslund K, Jouneau L, Dubuquoy C, Pietralunga V, Laubreton D, Boulesteix O, Gauthier D, Remot A, Boukaridi A, Falk A, Shevchenko G, Lind S B, Vargmar K, Zhang B, Kwong P D, Rodriguez M J, Duran M G, Schwartz-Cornil I, Eleouet J F, Taylor G, Valarcher J F (2020)

A single shot pre-fusion-stabilized bovine RSV F vaccine is safe and effective in newborn calves with maternally derived antibodies

Vaccines 8 (2), 231

Abstract

Achieving safe and protective vaccination against respiratory syncytial virus (RSV) in infants and in calves has proven a challenging task. The design of recombinant antigens with a conformation close to their native form in virus particles is a major breakthrough. We compared two subunit vaccines, the bovine RSV (BRSV) pre-fusion F (preF) alone or with nanorings formed by the RSV nucleoprotein (preF+N). PreF and N proteins are potent antigenic targets for neutralizing antibodies and T cell responses, respectively. To tackle the challenges of neonatal immunization, three groups of six one-month-old calves with maternally derived serum antibodies (MDA) to BRSV received a single intramuscular injection of PreF, preF+N with MontanideTM  ISA61 VG (ISA61) as adjuvant or only ISA61 (control). One month later, all calves were challenged with BRSV and monitored for virus replication in the upper respiratory tract and for clinical signs of disease over one week, and then post-mortem examinations of their lungs were performed. Both preF and preF+N vaccines afforded safe, clinical, and virological protection against BRSV, with little difference between the two subunit vaccines. Analysis of immune parameters pointed to neutralizing antibodies and antibodies to preF as being significant correlates of protection. Thus, a single shot vaccination with preF appears sufficient to reduce the burden of BRSV disease in calves with MDA.

Parry R, Naccache F, Ndiaye E H, Fall G, Castelli I, Luhken R, Medlock J, Cull B, Hesson J C, Montarsi F, Failloux A B, Kohl A, Schnettler E, Diallo M, Asgari S, Dietrich I, Becker S C (2020)

Identification and RNAi profile of a novel iflavirus infecting Senegalese Aedes vexans arabiensis mosquitoes

Viruses 12 (4), 440
Publisher’s version: https://doi.org/10.3390/v12040440

Abstract

The inland floodwater mosquito Aedes vexans (Meigen, 1830) is a competent vector of numerous arthropod-borne viruses such as Rift Valley fever virus (Phenuiviridae) and Zika virus (Flaviviridae). Aedes vexans spp. have widespread Afrotropical distribution and are common European cosmopolitan mosquitoes. We examined the virome of Ae. vexans arabiensis samples from Barkedji village, Senegal, with small RNA sequencing, bioinformatic analysis, and RT-PCR screening. We identified a novel 9494 nt iflavirus (Picornaviridae) designated here as Aedes vexans iflavirus (AvIFV). Annotation of the AvIFV genome reveals a 2782 amino acid polyprotein with iflavirus protein domain architecture and typical iflavirus 5' internal ribosomal entry site and 3' poly-A tail. Aedes vexans iflavirus is most closely related to a partial virus sequence from Venturia canescens (a parasitoid wasp) with 56.77% pairwise amino acid identity. Analysis of AvIFV-derived small RNAs suggests that AvIFV is targeted by the exogenous RNA interference pathway but not the PIWI-interacting RNA response, as ~60% of AvIFV reads corresponded to 21 nt Dicer-2 virus-derived small RNAs and the 24-29 nt AvIFV read population did not exhibit a "ping-pong" signature. The RT-PCR screens of archival and current (circa 2011-2020) Ae. vexans arabiensis laboratory samples and wild-caught mosquitoes from Barkedji suggest that AvIFV is ubiquitous in these mosquitoes. Further, we screened wild-caught European Ae. vexans samples from Germany, the United Kingdom, Italy, and Sweden, all of which tested negative for AvIFV RNA. This report provides insight into the diversity of commensal Aedes viruses and the host RNAi response towards iflaviruses.

Abstract

Equine-origin H3N8 and avian-origin H3N2 canine influenza viruses (CIVs) prevalent in dogs are thought to pose a public health threat arising from intimate contact between dogs and humans. However, our understanding of CIV virulence is still limited. Influenza A virus PA-X is a fusion protein encoded in part by a +1 frameshifted open reading frame (X-ORF) in segment 3. The X-ORF can be translated in full-length (61 amino acids) or truncated (41 amino acids) form. Genetic analysis indicated that the X-ORFs of equine H3N8 and avian H3N2 influenza viruses encoded 61 amino acids but were truncated after introduction into dogs. To determine the effect of PA-X truncation on the biological characteristics of CIVs, we constructed four recombinant viruses on H3N8 and H3N2 CIV backgrounds bearing truncated or full-length PA-Xs. We observed that truncation of PA-X increased growth of both H3N8 and H3N2 CIVs in MDCK cells and suppressed expression from co-transfected plasmids in MDCK cells. Furthermore, truncation of PA-X enhanced viral pathogenicity in dogs as shown by aggravated clinical symptoms and histopathological changes, increased viral replication in the respiratory system, and prolonged virus shedding. Additionally, CIVs with truncated PA-Xs were transmitted more efficiently in dogs. Global gene expression profiling of the lungs of infected dogs revealed that differentially expressed genes were mainly associated with inflammatory responses, which might contribute to the pathogenicity of PA-X-truncated CIVs. Our findings revealed that truncation of PA-X might be important for the adaptation of influenza viruses to dogs. IMPORTANCE Epidemics of equine-origin H3N8 and avian-origin H3N2 influenza viruses in canine populations are examples of successful cross-species transmission of influenza A viruses. Genetic analysis showed that the PA-X genes of equine H3N8 or avian H3N2 influenza viruses were full-length, with X-ORFs encoding 61 amino acids; however, those of equine-origin H3N8 or avian-origin H3N2 CIVs were truncated, suggesting PA-X truncation occurred after transmission to dogs. Here, we extended the PA-X genes of H3N8 and H3N2 CIVs and compared the biological characteristics of CIVs bearing different lengths of PA-X. We demonstrated that for both H3N8 and H3N2 viruses, truncation of PA-X increased virus yields in MDCK cells and enhanced viral replication, pathogenicity and transmission in dogs. These results might reflect enhanced suppression of host gene expression and up-regulation of genes related to inflammatory responses. Collectively, our data partially explain the conservation of truncated PA-X in CIVs.

Abstract

Lumpy Skin Disease (LSD) is an emerging disease of cattle that causes substantial economic loss to affected regions. However, factors favouring transmission under field conditions and farm-level impacts are poorly quantified. This was a retrospective case-control study of cattle farms in Nakuru, Kenya to determine risk factors associated with lumpy skin disease and the farm-level economic impacts of an outbreak. Data were collected using questionnaires administered through personal interview. Collected data included herd sizes, age, and sex structures, breeds, sources of replacement stock, grazing systems, and costs (direct and indirect) incurred when LSD outbreaks occurred. Farm-level risk factors were examined through univariable and multivariable logistic regression and a final model built using backward stepwise regression and likelihood ratio tests. The factors associated with LSD outbreaks on univariable analysis included breed (exotic vs. indigenous, OR = 15.01, P = 0.007), source of replacement stock (outside the herd vs. within the herd, OR = 8.38, P < 0.001) and herd size (large [>10 cattle] vs. small [1–3 cattle], OR = 3.51, P = 0.029). In the multivariable logistic regression model, only breed (exotic vs. indigenous, OR = 14.87, 95% CI 1.94–113.97, P = 0.009) and source of replacement stock (outside the herd vs. within the herd OR = 8.7, 95% CI 2.80–27.0, P < 0.001) were associated with outbreaks. The economic impact was compared between farms keeping purely indigenous (n = 10) or exotic (n = 29) breeds of cattle which indicated mean farm-level losses of 12,431 KSH/123 USD and 76,297 KSH/755 USD, respectively. The mean farm-level losses from reduction in milk yield and mortality were estimated at 4,725 KSH/97 USD and 3,103 KSH/31USD for farms keeping indigenous breeds whilst for farms keeping exotic breeds the equivalent losses were 26,886 KSH/266 USD and 43,557 KSH/431 USD, respectively. The indirect losses from treatments and vaccinations were proportionately much higher on farms with indigenous breeds at 4,603 KSH/46 USD making up ~37% of the total costs compared to ~8% (5,855 KSH/58 USD per farm) of the total costs for farms with exotic breeds. These findings indicate that LSD caused significant economic losses at the farm level in Nakuru County. This justifies implementation of disease control measures including quarantine of cattle post-purchase and the need for effective vaccinations of susceptible cattle herds.

Abstract

Introduction: Fowl adenovirus can cause important diseases in chickens such as inclusion body hepatitis, hepatitis hydropericardium syndrome, and gizzard erosion and ulceration. Inclusion body hepatitis has been regularly reported from many countries. This is the first case report from Turkey, describing an outbreak of inclusion body hepatitis in broiler farms due to fowl adenovirus-8b (FAdV-8b). Material and Methods: Broiler flocks with mortality about 10% were visited in Turkey, and necropsy was performed on dead birds. Samples were subjected to PCR assay to detect FAdV and other viral pathogens. After sequencing, phylogenetic analysis was performed and the nucleotide sequences of hexon genes were compared with the FAdV sequences data available in GenBank. Results: Clinical signs such as anorexia, depression, ruffled feathers, huddling, and greenish diarrhoea were observed. Mortality started at the 8th day of age and ranged from 10% to 14%. Necropsy showed severe hepatitis, jaundice, and pancreatitis. The main necropsy findings included a pale, enlarged, haemorrhagic, and friable liver along with swollen and haemorrhagic kidneys and spleen. PCR and sequence analysis revealed the presence of fowl adenovirus serotype 8b (FAdV-E). Conclusion: This is the first report on characterisation and the pathological lesions associated with FAdV in broilers in Turkey. Our findings suggest that FAdV strains could be an emerging pathogen in Turkish broilers and could actively contribute to hepatitis and immunosuppression.

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