MHC class II genotype-by-pathogen genotype interaction for infection prevalence in a natural rodent-Borrelia system.

MHC genes are extraordinarily polymorphic in most taxa. Host-pathogen coevolution driven by negative frequency-dependent selection (NFDS) is one of the main hypotheses for the maintenance of such immunogenetic variation. Here, we test a critical but rarely tested assumption of this hypothesis-that MHC alleles affect resistance/susceptibility to a pathogen in a strain-specific way, that is, there is a host genotype-by-pathogen genotype interaction. In a field study of bank voles naturally infected with the tick-transmitted bacterium Borrelia afzelii, we tested for MHC class II (DQB) genotype-by-B. afzelii strain interactions for infection prevalence between 10 DQB alleles and seven strains. One allele (DQB*37) showed an interaction, such that voles carrying DQB*37 had higher prevalence of two strains and lower prevalence of one strain than individuals without the allele. These findings were corroborated by analyses of strain composition of infections, which revealed an effect of DQB*37 in the form of lower ß diversity among infections in voles carrying the allele. Taken together, these results provide rare support at the molecular genetic level for a key assumption of models of antagonistic coevolution through NFDS.

Screening of Eurasian Tundra Reindeer for Viral Sequences by Next-Generation Sequencing.

Reindeer husbandry is essential for the livelihood and culture of indigenous people in the Arctic. Parts of the herding areas are also used as pastures for farm animals, facilitating potential transmission of viruses between species. Following the Covid-19 pandemic, viruses circulating in the wild are receiving increased attention, since they might pose a potential threat to human health. Climate change will influence the prevalence of infectious diseases of both humans and animals. The aim of this study was to detect known and previously unknown viruses in Eurasian tundra reindeer. In total, 623 nasal and 477 rectal swab samples were collected from reindeer herds in Fennoscandia, Iceland, and Eastern Russia during 2016-2019. Next-generation sequencing analysis and BLAST-homology searches indicated the presence of viruses of domesticated and wild animals, such as bovine viral diarrhea virus, bovine papillomavirus, alcephaline herpesvirus 1 and 2, deer mastadenovirus B, bovine rotavirus, and roe deer picobirnavirus. Several viral species previously found in reindeer and some novel species were detected, although the clinical relevance of these viruses in reindeer is largely unknown. These results indicate that it should be possible to find emerging viruses of relevance for both human and animal health using reindeer as a sentinel species.

Seroprevalence of pestivirus in Eurasian tundra reindeer in Finland, Sweden, Norway, Iceland and Russian Federation.

Reindeer herding is of great importance for the indigenous people of the Fennoscandia peninsula and northern Russia. There are also free-ranging feral populations of reindeer in Finland, Iceland, Norway and Russian Federation. The genus Pestivirus contains several viral species that infect ungulates and often show capacity to transmit between different host species. Sera from 520 Eurasian tundra reindeer (Rangifer tarandus tarandus) from Finland, Sweden, Norway, Iceland and Russian Federation were analysed and the prevalence of pestivirus-specific antibodies was determined. Seropositivity proportion was 48.5% for Sweden and 41.2% for Norway, but only 1.6% for Iceland and 2.5% for Finland. All Russian reindeer investigated were seronegative. Pan-pestivirus RT-PCR of seronegative animals (n = 156) from seropositive herds confirmed their negative status. These results indicate unexpectedly non-uniform circulation of an as yet uncharacterised pestivirus in Eurasian reindeer populations. The high seroprevalence in some regions warrants further studies of pestivirus infection dynamics, effects on reindeer health and population dynamics.

Chlamydia pecorum Associated With an Outbreak of Infectious Keratoconjunctivitis in Semi-domesticated Reindeer in Sweden.

Infectious keratoconjunctivitis (IKC), the most common ocular disease in ruminants worldwide, has affected semi-domesticated Eurasian reindeer (Rangifer tarandus tarandus) for over 100 years, both as individual cases and in outbreaks affecting tens to hundreds of animals. Recurrent IKC outbreaks have been affecting a semi-domesticated reindeer herd in Östra Kikkejaure (Norrbotten county, Sweden) from 2014. The latest episode of these recurrent outbreaks, in winter 2016/2017, was investigated in this study. Clinical findings were in line with previous reports of IKC in semi-domesticated reindeer and the clinical signs displayed by the affected animals (n = 30) included increased lacrimation, follicular conjunctivitis, purulent secretions around the affected eyes and corneal edema. Laboratory analyses of the samples revealed the presence of Chlamydiaceae in most samples obtained from the clinically affected animals (98.3%, n = 60), but also a high seroprevalence of cervid herpesvirus 2 (CvHV2) antibodies (56.6%, n = 53). Moraxella bovoculi was isolated from nine IKC-affected animals during the outbreak (45.0%, n = 20). All affected animals were treated with long-acting antibiotics and recovered from the disease, testing negative for the presence of Chlamydiaceae DNA by PCR 16 days and 3 months after the initial treatment. For the first time, Chlamydia pecorum was identified in semi-domesticated reindeer, and the involvement of Chlamydiaceae in a clinical outbreak of IKC is reported. The CvHV2 seroprevalence (56.6%) and the data obtained from a previous outbreak in 2014 also suggest the involvement of the reindeer alphaherpesvirus in the recurrent outbreaks.

Epimerisation of chiral hydroxylactones by short-chain dehydrogenases/reductases accounts for sex pheromone evolution in Nasonia.

Males of all species of the parasitic wasp genus Nasonia use (4R,5S)-5-hydroxy-4-decanolide (RS) as component of their sex pheromone while only N. vitripennis (Nv), employs additionally (4R,5R)-5-hydroxy-4-decanolide (RR). Three genes coding for the NAD+-dependent short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 are linked to the ability of Nv to produce RR. Here we show by assaying recombinant enzymes that SDRs from both Nv and N. giraulti (Ng), the latter a species with only RS in the pheromone, epimerise RS into RR and vice versa with (4R)-5-oxo-4-decanolide as an intermediate. Nv-derived SDR orthologues generally had higher epimerisation rates, which were also influenced by NAD+ availability. Semiquantitative protein analyses of the pheromone glands by tandem mass spectrometry revealed that NV10127 as well as NV10128 and/or NV10129 were more abundant in Nv compared to Ng. We conclude that the interplay of differential expression patterns and SDR epimerisation rates on the ancestral pheromone component RS accounts for the evolution of a novel pheromone phenotype in Nv.

Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2.

The wine and beer yeast Dekkera bruxellensis thrives in environments that are harsh and limiting, especially in concentrations with low oxygen and high ethanol. Its different strains' chromosomes greatly vary in number (karyotype). This study isolates two novel centromeric loci (CEN1 and CEN2), which support both the yeast's autonomous replication and the stable maintenance of plasmids. In the sequenced genome of the D. bruxellensis strain CBS 2499, CEN1 and CEN2 are each present in one copy. They differ from the known "point" CEN elements, and their biological activity is retained within ~900-1300 bp DNA segments. CEN1 and CEN2 have features of both "point" and "regional" centromeres: They contain conserved DNA elements, ARSs, short repeats, one tRNA gene, and transposon-like elements within less than 1 kb. Our discovery of a miniature inverted-repeat transposable element (MITE) next to CEN2 is the first report of such transposons in yeast. The transformants carrying circular plasmids with cloned CEN1 and CEN2 undergo a phenotypic switch: They form fluffy colonies and produce three times more biofilm. The introduction of extra copies of CEN1 and CEN2 promotes both genome rearrangements and ploidy shifts, with these effects mediated by homologous recombination (between circular plasmid and genome centromere copy) or by chromosome breakage when integrated. Also, the proximity of the MITE-like transposon to CEN2 could translocate CEN2 within the genome or cause chromosomal breaks, so promoting genome dynamics. With extra copies of CEN1 and CEN2, the yeast's enhanced capacities to rearrange its genome and to change its gene expression could increase its abilities for exploiting new and demanding niches.

Molecular Diagnosis of Hemorrhagic Fever with Renal Syndrome Caused by Puumala Virus.

Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS.

A semi-automated magnetic capture probe based DNA extraction and real-time PCR method applied in the Swedish surveillance of Echinococcus multilocularis in red fox (Vulpes vulpes) faecal samples.

BACKGROUND: Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012-2013 on more than 2000 faecal samples. METHODS: We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic. RESULTS: Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR. The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7-100). CONCLUSIONS: The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.

Laboratory assessment of sensitive molecular tools for detection of low levels of Echinococcus multilocularis-eggs in fox (Vulpes vulpes) faeces.

BACKGROUND: In endemic areas with very low infection prevalence, the frequency and intensity of Echinococcus multilocularis can be extremely low. This necessitates efficient, specific and sensitive molecular tools. We wanted to compare the existing molecular tools, used in the Norwegian national surveillance programme, and compare these with new techniques for detection of this zoonotic pathogen in fox faeces. Here we present the results of screening samples containing a known level of E. multilocularis eggs with two highly sensitive DNA isolation and extraction methods combined with one conventional PCR and three real-time PCR methods for detection. METHODS: We performed a comparison of two extraction protocols; one based on sieving of faecal material and one using targeted DNA sampling. Four methods of molecular detection were tested on E. multilocularis-egg spiked fox faeces. RESULTS: There were significant differences between the multiplex PCR/egg sieving DNA extraction methods compared to the new DNA fishing method and the three real-time PCR assays. Results also indicate that replicates of the PCR-reactions improve detection sensitivity when egg numbers are low. CONCLUSIONS: The results indicate that the use of real-time PCR combined with targeted DNA extraction, improves the sensitivity of E. multilocularis detection in faecal samples containing low numbers of E. multilocularis eggs. Results also indicate the importance of replicates of the PCR-reactions when pathogen levels are low.

A novel fatty acyl desaturase from the pheromone glands of Ctenopseustis obliquana and C. herana with specific Z5-desaturase activity on myristic acid.

Sexual communication in the Lepidoptera typically involves a female-produced sex pheromone that attracts males of the same species. The most common type of moth sex pheromone comprises individual or blends of fatty acyl derivatives that are synthesized by a specific enzymatic pathway in the female's pheromone gland, often including a desaturation step. This reaction is catalyzed by fatty acyl desaturases that introduce double bonds at specific locations in the fatty acid precursor backbone. The two tortricid moths, Ctenopseustis obliquana and C. herana (brown-headed leafrollers), which are endemic in New Zealand, both use (Z)-5-tetradecenyl acetate as part of their sex pheromone. In C. herana, (Z)-5-tetradecenyl acetate is the sole component of the pheromone. Labeling experiments have revealed that this compound is produced via an unusual Δ5-desaturation of myristic acid. Previously six desaturases were identified from the pheromone glands of Ctenopseustis and its sibling genus Planotortrix, with one differentially regulated to produce the distinct blends used by individual species. However, none were able to conduct the Δ5-desaturation observed in C. herana, and presumably C. obliquana. We have now identified an additional desaturase gene, desat7, expressed in the pheromone glands of both Ctenopseustis species, which is not closely related to any previously described moth pheromone desaturase. The encoded enzyme displays Δ5-desaturase activity on myristic acid when heterologously expressed in yeast, but is not able to desaturate any other fatty acid (C8-C16). We conclude that desat7 represents a new group of desaturases that has evolved a role in the biosynthesis of sex pheromones in moths.

A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory.

BACKGROUND: Moths (Lepidoptera) are highly dependent on chemical communication to find a mate. Compared to conventional unselective insecticides, synthetic pheromones have successfully served to lure male moths as a specific and environmentally friendly way to control important pest species. However, the chemical synthesis and purification of the sex pheromone components in large amounts is a difficult and costly task. The repertoire of enzymes involved in moth pheromone biosynthesis in insecta can be seen as a library of specific catalysts that can be used to facilitate the synthesis of a particular chemical component. In this study, we present a novel approach to effectively aid in the preparation of semi-synthetic pheromone components using an engineered vector co-expressing two key biosynthetic enzymes in a simple yeast cell factory. RESULTS: We first identified and functionally characterized a ∆11 Fatty-Acyl Desaturase and a Fatty-Acyl Reductase from the Turnip moth, Agrotis segetum. The ∆11-desaturase produced predominantly Z11-16:acyl, a common pheromone component precursor, from the abundant yeast palmitic acid and the FAR transformed a series of saturated and unsaturated fatty acids into their corresponding alcohols which may serve as pheromone components in many moth species. Secondly, when we co-expressed the genes in the Brewer's yeast Saccharomyces cerevisiae, a set of long-chain fatty acids and alcohols that are not naturally occurring in yeast were produced from inherent yeast fatty acids, and the presence of (Z)-11-hexadecenol (Z11-16:OH), demonstrated that both heterologous enzymes were active in concert. A 100 ml batch yeast culture produced on average 19.5 µg Z11-16:OH. Finally, we demonstrated that oxidized extracts from the yeast cells containing (Z)-11-hexadecenal and other aldehyde pheromone compounds elicited specific electrophysiological activity from male antennae of the Tobacco budworm, Heliothis virescens, supporting the idea that genes from different species can be used as a molecular toolbox to produce pheromone components or pheromone component precursors of potential use for control of a variety of moths. CONCLUSIONS: This study is a first proof-of-principle that it is possible to "brew" biologically active moth pheromone components through in vitro co-expression of pheromone biosynthetic enzymes, without having to provide supplementary precursors. Substrates present in the yeast alone appear to be sufficient.

Subcellular localization of the fatty acyl reductase involved in pheromone biosynthesis in the tobacco budworm, Heliothis virescens (Noctuidae: Lepidoptera).

Sex pheromone components are produced in specialized glands of female moths via well-characterized biosynthetic pathways, where a Fatty Acyl Reductase (FAR) is often essential for producing the specific ratio of the different pheromone components. The subcellular localization and membrane topology of FARs is important for understanding how pheromones are synthesized and exported to the exterior for release. We investigated the subcellular localization of HvFAR from the noctuid moth Heliothis virescens by producing recombinant fusion proteins with green fluorescent protein (GFP) in yeast. A C-terminally tagged construct was localized to the endoplasmic reticulum (ER) and retained full reductive activity on a broad range of saturated and unsaturated fatty acyl precursors. In contrast, an N-terminally-tagged construct was poorly expressed in the cytoplasm and was not enzymatically active, indicating that HvFAR requires a free N-terminal for both proper targeting and catalytic activity. A series of truncations of the N-and C-termini of HvFAR was conducted based on in silico-predicted hydrophobic domains and transmembrane regions. The N-terminally truncated protein was found in the cytoplasm and did not retain activity, emphasizing the importance of the N-terminal for FAR function. In addition, the orientation in the membrane of the C-terminus-tagged HvFAR-GFP construct was analyzed using a fluorescence protease protection (FPP) assay, implying that the C-terminal of HvFAR is orientated towards the cytoplasm. These results, together with previous data on the localization of desaturases, confirm the importance of the ER as a subcellular site of pheromone production.

Semi-selective fatty acyl reductases from four heliothine moths influence the specific pheromone composition.

BACKGROUND: Sex pheromones are essential in moth mate communication. Information on pheromone biosynthetic genes and enzymes is needed to comprehend the mechanisms that contribute to specificity of pheromone signals. Most heliothine moths use sex pheromones with (Z)-11-hexadecenal as the major component in combination with minor fatty aldehydes and alcohols. In this study we focus on four closely related species, Heliothis virescens, Heliothis subflexa, Helicoverpa armigera and Helicoverpa assulta, which use (Z)-11-hexadecenal, (Z)-9-tetradecanal, and (Z)-9-hexadecenal in different ratios in their pheromone blend. The components are produced from saturated fatty acid precursors by desaturation, ß-oxidation, reduction and oxidation. RESULTS: We analyzed the composition of fatty acyl pheromone precursors and correlated it to the pheromone composition. Next, we investigated whether the downstream fatty-acyl reduction step modulates the ratio of alcohol intermediates before the final oxidation step. By isolating and functionally characterizing the Fatty Acyl Reductase (pgFAR) from each species we found that the pgFARs were active on a broad set of C8 to C16 fatty acyl substrates including the key pheromone precursors, Z9-14, Z9-16 and Z11-16:acyls. When presenting the three precursors in equal ratios to yeast cultures expressing any of the four pgFARs, all reduced (Z)-9-tetradecenoate preferentially over (Z)-11-hexadecenoate, and the latter over (Z)-9-hexadecenoate. Finally, when manipulating the precursor ratios in vitro, we found that the pgFARs display small differences in the biochemical activity on various substrates. CONCLUSIONS: We conclude that a pgFAR with broad specificity is involved in heliothine moth pheromone biosynthesis, functioning as a semi-selective funnel that produces species-specific alcohol product ratios depending on the fatty-acyl precursor ratio in the pheromone gland. This study further supports the key role of these in pheromone biosynthesis and emphasizes the interplay between the pheromone fatty acyl precursors and the Lepidoptera specific pgFARs in shaping the pheromone composition.

Evolution of multicomponent pheromone signals in small ermine moths involves a single fatty-acyl reductase gene.

Fatty-acyl CoA reductases (FAR) convert fatty acids into fatty alcohols in pro- and eukaryotic organisms. In the Lepidoptera, members of the FAR gene family serve in the biosynthesis of sex pheromones involved in mate communication. We used a group of closely related species, the small ermine moths (Lepidoptera: Yponomeutidae) as a model to investigate the role of FARs in the biosynthesis of complex pheromone blends. Homology-based molecular cloning in three Yponomeuta species led to the identification of multiple putative FAR transcripts homologous to FAR genes from the Bombyx mori genome. The expression of one transcript was restricted to the female pheromone-gland tissue, suggesting a role in pheromone biosynthesis, and the encoded protein belonged to a recently identified Lepidoptera-specific pgFAR gene subfamily. The Yponomeuta evonymellus pgFAR mRNA was up-regulated in sexually mature females and exhibited a 24-h cyclic fluctuation pattern peaking in the pheromone production period. Heterologous expression confirmed that the Yponomeuta pgFAR orthologs in all three species investigated [Y. evonymellus (L.), Yponomeuta padellus (L.), and Yponomeuta rorellus (Hübner)] encode a functional FAR with a broad substrate range that efficiently promoted accumulation of primary alcohols in recombinant yeast supplied with a series of biologically relevant C14- or C16-acyl precursors. Taken together, our data evidence that a single alcohol-producing pgFAR played a critical function in the production of the multicomponent pheromones of yponomeutids and support the hypothesis of moth pheromone-biosynthetic FARs belonging to a FAR gene subfamily unique to Lepidoptera.