Specific T-cell subsets have a role in anti-viral immunity and pathogenesis but not viral dynamics or onwards vector transmission of an important livestock arbovirus.

Introduction: Bluetongue virus (BTV) is an arthropod-borne Orbivirus that is almost solely transmitted by Culicoides biting midges and causes a globally important haemorrhagic disease, bluetongue (BT), in susceptible ruminants. Infection with BTV is characterised by immunosuppression and substantial lymphopenia at peak viraemia in the host. Methods: In this study, the role of cell-mediated immunity and specific T-cell subsets in BTV pathogenesis, clinical outcome, viral dynamics, immune protection, and onwards transmission to a susceptible Culicoides vector is defined in unprecedented detail for the first time, using an in vivo arboviral infection model system that closely mirrors natural infection and transmission of BTV. Individual circulating CD4+, CD8+, or WC1+ γδ T-cell subsets in sheep were depleted through the administration of specific monoclonal antibodies. Results: The absence of cytotoxic CD8+ T cells was consistently associated with less severe clinical signs of BT, whilst the absence of CD4+ and WC1+ γδ T cells both resulted in an increased clinical severity. The absence of CD4+ T cells also impaired both a timely protective neutralising antibody response and the production of IgG antibodies targeting BTV non-structural protein, NS2, highlighting that the CD4+ T-cell subset is important for a timely protective immune response. T cells did not influence viral replication characteristics, including onset/dynamics of viraemia, shedding, or onwards transmission of BTV to Culicoides. We also highlight differences in T-cell dependency for the generation of immunoglobulin subclasses targeting BTV NS2 and the structural protein, VP7. Discussion: This study identifies a diverse repertoire of T-cell functions during BTV infection in sheep, particularly in inducing specific anti-viral immune responses and disease manifestation, and will support more effective vaccination strategies.

The evaluation of five serological assays in determining seroconversion to peste des petits ruminants virus in typical and atypical hosts.

Peste des petits ruminants (PPR) is an infectious viral disease, primarily of small ruminants such as sheep and goats, but is also known to infect a wide range of wild and domestic Artiodactyls including African buffalo, gazelle, saiga and camels. The livestock-wildlife interface, where free-ranging animals can interact with captive flocks, is the subject of scrutiny as its role in the maintenance and spread of PPR virus (PPRV) is poorly understood. As seroconversion to PPRV indicates previous infection and/or vaccination, the availability of validated serological tools for use in both typical (sheep and goat) and atypical species is essential to support future disease surveillance and control strategies. The virus neutralisation test (VNT) and enzyme-linked immunosorbent assay (ELISA) have been validated using sera from typical host species. Still, the performance of these assays in detecting antibodies from atypical species remains unclear. We examined a large panel of sera (n = 793) from a range of species from multiple countries (sourced 2015-2022) using three tests: VNT, ID VET N-ELISA and AU-PANVAC H-ELISA. A sub-panel (n = 30) was also distributed to two laboratories and tested using the luciferase immunoprecipitation system (LIPS) and a pseudotyped virus neutralisation assay (PVNA). We demonstrate a 75.0-88.0% agreement of positive results for detecting PPRV antibodies in sera from typical species between the VNT and commercial ELISAs, however this decreased to 44.4-62.3% in sera from atypical species, with an inter-species variation. The LIPS and PVNA strongly correlate with the VNT and ELISAs for typical species but vary when testing sera from atypical species.

Laboratory Diagnosis and Quantification of African Swine Fever Virus Using Real-Time Polymerase Chain Reaction.

Real-time polymerase chain reaction (PCR) for the detection of African swine fever virus (ASFV) is the tool of choice for the diagnostic laboratory and is a robust and easily scalable method for the researcher analyzing viral replication both in vitro and in vivo. In this chapter, we describe protocols for both quantitative real-time polymerase chain reactions (qPCR) and non-quantitative real-time polymerase chain reactions (real-time PCR) for the detection of African swine fever virus genome in a range of samples.

A novel strain of lumpy skin disease virus causes clinical disease in cattle in Hong Kong.

Lumpy skin disease virus (LSDV) is an emerging poxviral pathogen of cattle that is currently spreading throughout Asia. The disease situation is of high importance for farmers and policy makers in Asia. In October 2020, feral cattle in Hong Kong developed multi-focal cutaneous nodules consistent with lumpy skin disease (LSD). Gross and histological pathology further supported the diagnosis and samples were sent to the OIE Reference Laboratory at The Pirbright Institute for confirmatory testing. LSDV was detected using quantitative polymerase chain reaction (qPCR) and additional molecular analyses. This is the first report of LSD in Hong Kong. Whole genome sequencing (WGS) of the strain LSDV/Hong Kong/2020 and phylogenetic analysis were carried out in order to identify connections to previous outbreaks of LSD, and better understand the drivers of LSDV emergence. Analysis of the 90 core poxvirus genes revealed LSDV/Hong Kong/2020 was a novel strain most closely related to the live-attenuated Neethling vaccine strains of LSDV and more distantly related to wildtype LSDV isolates from Africa, the Middle East and Europe. Analysis of the more variable regions located towards the termini of the poxvirus genome revealed genes in LSDV/Hong Kong/2020 with different patterns of grouping when compared to previously published wildtype and vaccine strains of LSDV. This work reveals that the LSD outbreak in Hong Kong in 2020 was caused by a different strain of LSDV than the LSD epidemic in the Middle East and Europe in 2015-2018. The use of WGS is highly recommended when investigating LSDV disease outbreaks.

Simultaneous Detection of Bluetongue Virus Serotypes Using xMAP Technology.

Bluetongue is an economically important disease of ruminants caused by the bluetongue virus (BTV). BTV is serologically diverse, which complicates vaccination strategies. Rapid identification of the causative BTV serotypes is critical, however, real-time PCR (RT-qPCR) can be costly and time consuming to perform when the circulating serotypes are unknown. The Luminex xMAP technology is a high-throughput platform that uses fluorescent beads to detect multiple targets simultaneously. We utilized existing BTV serotyping RT-qPCR assays for BTV-1 to BTV-24 and adapted them for use with the xMAP platform. The xMAP assay specifically detected all 24 BTV serotypes when testing reference strains. In all BTV-positive samples, the sensitivity of the BTV xMAP was 87.55% whereas the sensitivity of the serotype-specific RT-qPCR was 79.85%. The BTV xMAP assay allowed for the specific detection of BTV serotypes 1-24 at a lower cost than current RT-qPCR assays. Overall, the assay provides a useful novel diagnostic tool, particularly when analyzing large sample sets. The use of the BTV xMAP assay will allow for the rapid assessment of BTV epidemiology and may inform decision-making related to control and prevention measures.

Identification of novel testing matrices for African swine fever surveillance.

African swine fever (ASF) is a devastating viral disease of pigs and wild boar, and it threatens global food security. We aimed to identify suitable sample matrices for use in ASF surveillance programs. Six pigs inoculated with ASFV were sampled at postmortem. Blood, bone marrow, ear biopsies, and oral, nasal, and rectal swabs were taken from all pigs. All samples were analyzed using 3 real-time PCR (rtPCR) assays and a LAMP assay. ASFV was detected at > 107 genome copies/mL in blood; bone marrow was found to provide the highest viral load. Ct values provided by the rtPCR assays were correlated, and ASFV was detected in all oral, nasal, and rectal swabs and in all ear biopsy samples irrespective of the location from which they were taken. The LAMP assay had lower sensitivity, and detected ASFV in 54 of 66 positive samples, but delivered positive results within 17 min. We identified additional sample matrices that can be considered depending on the sampling situation: bone marrow had a high probability of detection, which could be useful for decomposed carcasses. However, ear biopsies provide an appropriate, high-throughput sample matrix to detect ASFV and may be useful during surveillance programs.

Towards a Sampling Rationale for African Swine Fever Virus Detection in Pork Products.

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.

Outbreak of African horse sickness in Thailand, 2020.

African horse sickness was confirmed in horses in Thailand during March 2020. The virus was determined to belong to serotype 1 and is phylogenetically closely related to isolates from South Africa. This is the first incidence of African horse sickness occurring in South East Asia and of serotype 1 outside of Africa.

BioSAXS Measurements Reveal That Two Antimicrobial Peptides Induce Similar Molecular Changes in Gram-Negative and Gram-Positive Bacteria.

Two highly active short broad-spectrum AMPs (14D and 69D) with unknown mode of action have been investigated in regards to their effect against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria methicillin-resistant Staphylococcus aureus (MRSA). Minimal inhibitory concentration (MIC) measurements using a cell density of 108 cfu/ml resulted in values between 16 and 32 µg/ml. Time-kill experiments using 108 cfu/ml revealed complete killing, except for 69D in combination with MRSA, where bacterial load was reduced a million times. Small-angle X-ray scattering of biological samples (BioSAXS) at 108 cfu/ml was applied to investigate the ultrastructural changes in E. coli and MRSA in response to these two broad-spectrum AMPs. In addition, electron microscopy (EM) was performed to visualize the treated and non-treated bacteria. As expected, the scattering curves generated using BioSAXS show the ultrastructure of the Gram-positive and Gram-negative bacteria to be very different (BioSAXS is not susceptible to the outer shape). After treatment with either peptide, the scattering curves of E. coli and MRSA cells are much more alike. Whereas in EM, it is notoriously difficult to observe changes for spherical Gram-positives; the BioSAXS results are superior and reveal strongly similar effects for both peptides induced in Gram-positive as well as Gram-negative bacteria. Given the high-throughput possibility and robust statistics, BioSAXS can support and speed up mode of action research in AMPs and other antimicrobial compounds, making a contribution toward the development of urgently needed drugs against resistant bacteria.

Evidence of reduced viremia, pathogenicity and vector competence in a re-emerging European strain of bluetongue virus serotype 8 in sheep.

The outbreak of bluetongue virus (BTV) serotype 8 (BTV-8) during 2006-2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV-8 strain re-emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV-8 strain alongside the re-emerging BTV-8 strain isolated from France in 2017. Two groups of eight BTV-naïve British mule sheep were inoculated with 5.75 log10 TCID50 /ml of either BTV-8 strain. BTV RNA was detected by 2 dpi in both groups with peak viraemia occurring between 5-9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0-8.8 log10 genome copies/ml) than the re-emerging BTV-8 strain (2.9-7.9 log10 genome copies/ml). All infected sheep developed BTV-specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV-8-inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV-8. In Culicoides sonorensis feeding on the sheep over the period 7-12 dpi, vector competence was significantly higher for the 2007 strain than the re-emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs. 1/8) and the overall clinical scores (median 5.25 vs. 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re-emerging strain. However, one sheep infected with the re-emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re-emerging BTV-8 to still cause illness in naïve ruminants with concurrent costs to the livestock industry.

Use of Peptide Libraries for Identification and Optimization of Novel Antimicrobial Peptides.

The increasing rates of resistance among bacteria and to a lesser extent fungi have resulted in an urgent need to find new molecules that hold therapeutic promise against multidrug-resistant strains. Antimicrobial peptides have proven very effective against a variety of multidrug-resistant bacteria. Additionally, the low levels of resistance reported towards these molecules are an attractive feature for antimicrobial drug development. Here we summarise information on diverse peptide libraries used to discover or to optimize antimicrobial peptides. Chemical synthesized peptide libraries, for example split and mix method, tea bag method, multi-pin method and cellulose spot method are discussed. In addition biological peptide library screening methods are summarized, like phage display, bacterial display, mRNA-display and ribosomal display. A few examples are given for small peptide libraries, which almost exclusively follow a rational design of peptides of interest rather than a combinatorial approach.

Cationic antimicrobial peptides as potential new therapeutic agents in neonates and children: a review.

PURPOSE OF REVIEW: Antimicrobial resistance towards conventional antibiotics is a serious problem for modern medicine and for our society. Multidrug-resistant bacteria are very difficult to treat and treatment options have begun to run out. Here, we summarize the newest studies of drug development using cationic antimicrobial peptides as lead molecules for novel antimicrobial drugs. RECENT FINDINGS: A new development is the use of antimicrobial peptides not only as direct antimicrobial lead structures but also using their ability to influence the immune system. Such approaches can be used to develop drugs that influence the immune system in a unique way, supporting specific branches of immune cells in order to clear infection. Applying such an 'immune boost' would also minimize the danger of new resistance emerging in bacteria. In addition, searching for and testing substances that trigger the production of host antimicrobial peptides is still ongoing and opens up a totally new avenue for the use of antimicrobial peptides against infections. Currently, more than 10 clinical trials, phase 2 or 3, using antimicrobial peptides are in progress or have been recently completed. SUMMARY: Multidrug resistance is an urgent problem for modern medicine and novel antimicrobials are needed. Despite some drawbacks, antimicrobial peptides seem now to appear more numerous in clinical trials, indicating the success in developing peptides into novel therapeutics. This can be critical especially for neonates and children, as treatment options for infections with Gram-negatives in neonatal ICUs are becoming rare.