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Porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious threat to the pig industry worldwide. Increasing evidence suggests that microevolution within a quasispecies population can give rise to high sequence heterogeneity in PRRSV; potentially impacting the pathogenicity of the virus. Here, we report on micro-evolutionary events taking place within the viral quasispecies population in lung and lymph node 3 days post infection (dpi) following experimental in vivo infection with the prototypical Lelystad PRRSV (LV). Sequence analysis revealed 16 high frequency single nucleotide variants (SNV) or differences from the reference LV genome which are assumed to be representative of the consensus inoculum genome. Additionally, 49 other low frequency SNVs were also found in the inoculum population. At 3 dpi, a total of 9 and 10 SNVs of varying frequencies could already be detected in the LV population infecting the lung and lymph nodes, respectively. Interestingly, of these, three and four novel SNVs emerged independently in the two respective tissues when compared to the inoculum. The remaining variants, though already present at lower frequencies in the inoculum, were positively selected and their frequency increased within the quasispecies population. Hence, we were able to determine directly from tissues infected with PRRSV the repertoire of genetic variants within the viral quasispecies population. Our data also suggest that microevolution of these variants is rapid and some may be tissue-specific.

Background The advent and continual improvement of high-throughput sequencing technologies has made immunoglobulin repertoire sequencing accessible and informative regardless of study species. However, to fully map dynamic changes in polyclonal responses precise framework and complementarity determining region annotation of rearranging genes is pivotal. Most sequence annotation tools are designed primarily for use with human and mouse antibody sequences which use databases with fixed species lists, applying very specific assumptions which select against unique structural characteristics. For this reason, data agnostic tools able to learn from presented data can be very useful with new species or with novel datasets. Results We have developed IgMAT, which utilises a reduced amino acid alphabet, that incorporates multiple HMM alignments into a single consensus to automatically annotate immunoglobulin sequences from most organisms. Additionally, the software allows the incorporation of user defined databases to better represent the species and/or antibody class of interest. To demonstrate the accuracy and utility of IgMAT, we present analysis of sequences extracted from structural data and immunoglobulin sequence datasets from several different species. Conclusions IgMAT is fully open-sourced and freely available on GitHub ( https://github.com/TPI-Immunogenetics/igmat ) for download under GPLv3 license. It can be used as a CLI application or as a python module to be integrated in custom scripts.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the main cause of viral encephalitis in Asia. However, with changing climate JEV has the potential to emerge in novel temperate regions. Here, we have assessed the vector competence of the temperate mosquito Culex pipiens f. pipiens to vector JEV genotype III at temperatures representative of those experienced, or predicted in the future during the summer months, in the United Kingdom. Our results show that Cx. pipiens is susceptible to JEV infection at both temperatures. In addition, at 25 °C, JEV disseminated from the midgut and was recovered in saliva samples, indicating the potential for transmission. At a lower temperature, 20 °C, following an incubation period of fourteen days, there were reduced levels of JEV dissemination and virus was not detected in saliva samples. The virus present in the bodies of these mosquitoes was restricted to the posterior midgut as determined by microscopy and viable virus was successfully recovered. Apart from the influence on virus dissemination, mosquito mortality was significantly increased at the higher temperature. Overall, our results suggest that temperature is a critical factor for JEV vector competence and infected-mosquito survival. This may in turn influence the vectorial capacity of Cx. pipiens to vector JEV genotype III in temperate areas.

Worldwide, arthropod-borne disease transmission represents one of the greatest threats to public and animal health. For the British Isles, an island group on the north-western coast of continental Europe consisting of the United Kingdom (UK) and the Republic of Ireland, physical separation offers a barrier to the introduction of many of the pathogens that affect animals on the rest of the continent. Added to this are strict biosecurity rules at ports of entry and the depauperate vector biodiversity found on the islands. Nevertheless, there are some indigenous arthropod-borne pathogens that cause sporadic outbreaks, such as the tick-borne louping ill virus, found almost exclusively in the British Isles, and a range of piroplasmid infections that are poorly characterized. These provide an ongoing source of infection whose emergence can be unpredictable. In addition, the risk remains for future introductions of both exotic vectors and the pathogens they harbor, and can transmit. Current factors that are driving the increases of both disease transmission and the risk of emergence include marked changes to the climate in the British Isles that have increased summer and winter temperatures, and extended the period over which arthropods are active. There have also been dramatic increases in the distribution of mosquito-borne diseases, such as West Nile and Usutu viruses in mainland Europe that are making the introduction of these pathogens through bird migration increasingly feasible. In addition, the establishment of midge-borne bluetongue virus in the near continent has increased the risk of wind-borne introduction of infected midges and the inadvertent importation of infected cattle. Arguably the greatest risk is associated with the continual increase in the movement of people, pets and trade into the UK. This, in particular, is driving the introduction of invasive arthropod species that either bring disease-causing pathogens, or are known competent vectors, that increase the risk of disease transmission if introduced. The following review documents the current pathogen threats to animals transmitted by mosquitoes, ticks and midges. This includes both indigenous and exotic pathogens to the UK. In the case of exotic pathogens, the pathway and risk of introduction are also discussed.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy–light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy–light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.

The advent and continual improvement of high-throughput sequencing technologies has made immunoglobulin repertoire sequencing accessible and informative regardless of study species. However, to fully map changes in polyclonal dynamics, precise annotation of these constantly rearranging genes is pivotal. For this reason, data agnostic tools able to learn from presented data are required. Most sequence annotation tools are designed primarily for use with human and mouse antibody sequences which use databases with fixed species lists, applying very specific assumptions which select against unique structural characteristics. We present IgMAT, which utilises a reduced amino acid alphabet, incorporates multiple HMM alignments into a single consensus and enables the incorporation of user defined databases to better represent their species of interest. Competing Interest Statement The authors have declared no competing interest.

Abstract Sustainable modern poultry production depends on effective protection against infectious diseases and a diverse range of antibodies is key for an effective immune response. In the domestic chicken, somatic gene conversion is the dominant process in which the antibody immunoglobulin genes are diversified. Affinity maturation by somatic hypermutation (SHM) also occurs, but the relative contribution of gene conversion versus somatic hypermutation to immunoglobulin (Ig) gene diversity is poorly understood. In this study, we use high throughput long-read sequencing to study immunoglobulin diversity in multiple immune-associated tissues in Rhode Island Red chickens. To better understand the impact of genetic diversification in the chicken, a novel gene conversion identification software was developed (BrepConvert). In this study, BrepConvert enabled the identification of over 1 million gene conversion events. Mapping the occurrence of putative somatic gene conversion (SGC) events throughout the variable gene region revealed repetitive and highly restricted patterns of genetic insertions in both the antibody heavy and light chains. These patterns coincided with the locations of genetic variability in available pseudogenes and align with antigen binding sites, predominately the complementary determining regions (CDRs). We found biased usage of pseudogenes during gene conversion, as well as immunoglobulin heavy chain diversity gene (IGHD) preferences during V(D)J gene rearrangement, suggesting that antibody diversification in chickens is more focused than the genetic potential for diversity would suggest. Graphical Abstract Graphical Abstract