Divergent Strains Research Articles

Development of a quantitative real time RT-PCR assay for sensitive and rapid detection of emerging Atypical Porcine Pestivirus associated with congenital tremor in pigs

Atypical Porcine Pestivirus (APPV) is reported as the etiologic agent for type AII congenital tremors in newborn piglets. Initial PCR-based diagnostic tests to detect APPV were designed based on the limited sequence information and are not capable of detecting the majority of APPV strains. A sensitive and reliable PCR-based diagnostic test is critical for accurate detection of APPV. In this study, a quantitative reverse transcription PCR (RT-qPCR) assay was developed for reliable detection of all currently known APPV strains. The assay design also included swine 18S rRNA gene as an internal control to monitor RNA extraction efficiency. Two APPV gene fragments, one each from NS5b and NS3, were cloned and used to determine the dynamic range of detection, linearity and analytical sensitivity/limit of detection (LOD). Both individual and multiplex assays (duplex and triplex) had correlation coefficients of >0.99 and PCR amplification efficiencies of >90 %. Comparison of detection limit and analytical sensitivity between individual, and multiplex assays indicated no inhibition of PCR sensitivity upon multiplexing. The detection limit for APPV target, based on analytical sensitivity, is 7.75 copies (NS5b) and 5.2 copies (NS3) per reaction. Assay specificity was verified by testing nucleic acids of other closely related pestiviruses and clinical samples that are positive for other common swine pathogens. Assay sensitivity was also assessed on synthesized gene fragments of the most divergent China strains. Testing 339 known APPV-positive and 202 negative clinical samples demonstrated a good diagnostic sensitivity and specificity. Data from six independent runs, including 5 replicates of three clinical samples with three Ct ranges, were utilized to assess inter-assay repeatability and intra-assay reproducibility. This analysis demonstrated intra-assay/inter-assay coefficients of variation of 0.71 % and 0.01 %, respectively, with a PCR efficiency of 92.71 % for the triplex assay. Testing of 1785 clinical samples revealed ∼19 % prevalence of APPV in the US swine herds and oral fluids demonstrates to be a reliable specimen for viral detection. This multiplex RT-qPCR assay offers a rapid and reliable detection of APPV in swine herds and serves as useful tool in APPV surveillance and epidemiological investigations.

Genes, other than Muc5b, play a role in bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an incurable genetic disease that affects 5 million people worldwide. The gain-of-function MUC5B promoter variant rs35705950 is the dominant genetic risk factor for IPF, yet has a low penetrance. This raises the possibility that other genes and transcripts affect the penetrance of MUC5B. Previously, we have shown that the concentration of Muc5b in bronchoalveolar epithelia is directly associated with the extent and persistence of bleomycin-induced lung fibrosis in mice. In this study, we investigated whether bleomycin-induced lung injury is Muc5b dependent in genetically divergent strains of mice. Specifically, mice from the eight Diversity Outbred (DO) founders were phenotyped for Muc5b expression and lung fibrosis 3 wk after intratracheal bleomycin administration. Although we identified strains with low Muc5b expression and minimal lung fibrosis (CAST/EiJ and PWK/PhJ) and strains with high Muc5b expression and extensive lung fibrosis (NZO/H1LtJ and WSB/EiJ), there also were strains that did not demonstrate a clear relationship between Muc5b expression and lung fibrosis (129S1/SvlmJ, NOD/ShiLtJ, and C57BL/6J, A/J). Hierarchical clustering suggests that other factors may work in concert with or potentially independent of Muc5b to promote bleomycin-induced lung injury and fibrosis. This study suggests that these strains and their recombinant inbred crosses may prove helpful in identifying the genes and transcripts that interact with Muc5b and cause lung fibrosis.

Multiple Mammarenaviruses Circulating in Angolan Rodents.

Rodents are a speciose group of mammals with strong zoonotic potential. Some parts of Africa are still underexplored for the occurrence of rodent-borne pathogens, despite this high potential. Angola is at the convergence of three major biogeographical regions of sub-Saharan Africa, each harbouring a specific rodent community. This rodent-rich area is, therefore, strategic for studying the diversity and evolution of rodent-borne viruses. In this study we examined 290 small mammals, almost all rodents, for the presence of mammarenavirus and hantavirus RNA. While no hantavirus was detected, we found three rodent species positive for distinct mammarenaviruses with a particularly high prevalence in Namaqua rock rats (Micaelamys namaquensis). We characterised four complete virus genomes, which showed typical mammarenavirus organisation. Phylogenetic and genetic distance analyses revealed: (i) the presence of a significantly divergent strain of Luna virus in Angolan representatives of the ubiquitous Natal multimammate mouse (Mastomys natalensis), (ii) a novel Okahandja-related virus associated with the Angolan lineage of Micaelamys namaquensis for which we propose the name Bitu virus (BITV) and (iii) the occurrence of a novel Mobala-like mammarenavirus in the grey-bellied pygmy mouse (Mus triton) for which we propose the name Kwanza virus (KWAV). This high virus diversity in a limited host sample size and in a relatively small geographical area supports the idea that Angola is a hotspot for mammarenavirus diversity.

Targeting Antigens for Universal Influenza Vaccine Development.

Traditional influenza vaccines generate strain-specific antibodies which cannot provide protection against divergent influenza virus strains. Further, due to frequent antigenic shifts and drift of influenza viruses, annual reformulation and revaccination are required in order to match circulating strains. Thus, the development of a universal influenza vaccine (UIV) is critical for long-term protection against all seasonal influenza virus strains, as well as to provide protection against a potential pandemic virus. One of the most important strategies in the development of UIVs is the selection of optimal targeting antigens to generate broadly cross-reactive neutralizing antibodies or cross-reactive T cell responses against divergent influenza virus strains. However, each type of target antigen for UIVs has advantages and limitations for the generation of sufficient immune responses against divergent influenza viruses. Herein, we review current strategies and perspectives regarding the use of antigens, including hemagglutinin, neuraminidase, matrix proteins, and internal proteins, for universal influenza vaccine development.

Tissue context determines the penetrance of regulatory DNA variation

Functional assessment of disease-associated sequence variation at non-coding regulatory elements is complicated by their high degree of context sensitivity to both the local chromatin and nuclear environments. Allelic profiling of DNA accessibility across individuals has shown that only a select minority of sequence variation affects transcription factor (TF) occupancy, yet low sequence diversity in human populations means that no experimental assessment is available for the majority of disease-associated variants. Here we describe high-resolution in vivo maps of allelic DNA accessibility in liver, kidney, lung and B cells from 5 increasingly diverged strains of F1 hybrid mice. The high density of heterozygous sites in these hybrids enables precise quantification of effect size and cell-type specificity for hundreds of thousands of variants throughout the mouse genome. We show that chromatin-altering variants delineate characteristic sensitivity profiles for hundreds of TF motifs. We develop a compendium of TF-specific sensitivity profiles accounting for genomic context effects. Finally, we link maps of allelic accessibility to allelic transcript levels in the same samples. This work provides a foundation for quantitative prediction of cell-type specific effects of non-coding variation on TF activity, which will facilitate both fine-mapping and systems-level analyses of common disease-associated variation in human genomes.

Natural variation in fecundity is correlated with species-wide levels of divergence in Caenorhabditis elegans.

Life history traits underlie the fitness of organisms and are under strong natural selection. A new mutation that positively impacts a life history trait will likely increase in frequency and become fixed in a population (e.g., a selective sweep). The identification of the beneficial alleles that underlie selective sweeps provides insights into the mechanisms that occurred during the evolution of a species. In the global population of Caenorhabditis elegans, we previously identified selective sweeps that have drastically reduced chromosomal-scale genetic diversity in the species. Here, we measured the fecundity of 121 wild C. elegans strains, including many recently isolated divergent strains from the Hawaiian islands and found that strains with larger swept genomic regions have significantly higher fecundity than strains without evidence of the recent selective sweeps. We used genome-wide association (GWA) mapping to identify three quantitative trait loci (QTL) underlying the fecundity variation. In addition, we mapped previous fecundity data from wild C. elegans strains and C. elegans recombinant inbred advanced intercross lines that were grown in various conditions and detected eight QTL using GWA and linkage mappings. These QTL show the genetic complexity of fecundity across this species. Moreover, the haplotype structure in each GWA QTL region revealed correlations with recent selective sweeps in the C. elegans population. North American and European strains had significantly higher fecundity than most strains from Hawaii, a hypothesized origin of the C. elegans species, suggesting that beneficial alleles that caused increased fecundity could underlie the selective sweeps during the worldwide expansion of C. elegans.

Intrinsic exercise capacity induces divergent vascular plasticity via arachidonic acid-mediated inflammatory pathways in female rats

Metabolic syndrome prevalence has increased among US adults, particularly among non-hispanic white and black women. Sedentary behavior often leads to chronic inflammation, a triggering factor of metabolic syndrome. Given that intrinsic exercise capacity is genetically inherited, we questioned if low-grade chronic inflammation would be present in a female rat model of low intrinsic exercise capacity-induced metabolic syndrome, while beneficial increase of resolution of inflammation would be present in a female rat model of high intrinsic exercise capacity. In the vascular system, two primary markers for inflammation and resolution of inflammation are cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Our study focused on the novel hypothesis that untrained, inherited exercise capacity induces divergent vascular plasticity via changes in the delicate balance between COX and LOX inflammatory mediators.We used divergent rat strains with low (LCR) and high (HCR) aerobic running capacity. By using animals with contrasting intrinsic exercise capacities, it is possible to determine the exact triggers that lead to inherited vascular plasticity in female rats. We observed that female LCR displayed increased periovarian fat pad and body weight, which is congruent with their obesity-presenting phenotype. Furthermore, LCR presented with vascular hypocontractility and increased COX and LOX-derived pro-inflammatory factors. On the other hand, HCR presented with a “shutdown” of COX-induced vasoconstriction and enhanced resolution of inflammation to maintain vascular tone and homeostasis. In conclusion, LCR display low-grade chronic inflammation via increased COX activity. These results provide mechanistic clues as to why lower intrinsic aerobic capacity correlates with a predisposition to risk of vascular disease. Conversely, being born with higher intrinsic aerobic capacity is a significant factor for improved vascular physiology in female rats.

What Are the Best Parents for Hybrid Progeny? An Investigation into the Human Pathogenic Fungus Cryptococcus

Hybridization between more divergent organisms is likely to generate progeny with more novel genetic interactions and genetic variations. However, the relationship between parental genetic divergence and progeny phenotypic variation remains largely unknown. Here, using strains of the human pathogenic Cryptococcus, we investigated the patterns of such a relationship. Twenty-two strains with up to 15% sequence divergence were mated. Progeny were genotyped at 16 loci. Parental strains and their progeny were phenotyped for growth ability at two temperatures, melanin production at seven conditions, and susceptibility to the antifungal drug fluconazole. We observed three patterns of relationships between parents and progeny for each phenotypic trait, including (i) similar to one of the parents, (ii) intermediate between the parents, and (iii) outside the parental phenotypic range. We found that as genetic distance increases between parental strains, progeny showed increased fluconazole resistance and growth at 37 °C but decreased melanin production under various oxidative and nitrosative stresses. Our findings demonstrate that, depending on the traits, both evolutionarily more similar strains and more divergent strains may be better parents to generate progeny with hybrid vigor. Together, the results indicate the enormous potential of Cryptococcus hybrids in their evolution and adaptation to diverse conditions.

Characterization and Establishment of a Novel EBV Strain Simultaneously Associated With Nasopharyngeal Carcinoma and B-Cell Lymphoma.

Epstein-Barr virus (EBV)—the prototypical human tumor virus—is responsible for 1–2% of the global cancer burden, but divergent strains seem to exist in different geographical regions with distinct predilections for causing lymphoid or epithelial malignancies. Here we report the establishment and characterization of Yu103, an Asia Pacific EBV strain with a highly remarkable provenance of being derived from nasopharyngeal carcinoma biopsy but subsequently propagated in human B-lymphoma cells and xenograft models. Unlike previously characterized EBV strains which are either predominantly B-lymphotropic or epitheliotropic, Yu103 evinces an uncanny capacity to infect and transform both B-lymphocytes and nasopharyngeal epithelial cells. Genomic and phylogenetic analyses indicated that Yu103 EBV lies midway along the spectrum of EBV strains known to drive lymphomagenesis or carcinogenesis, and harbors molecular features which likely account for its unusual properties. To our knowledge, Yu103 EBV is currently the only EBV isolate shown to drive human nasopharyngeal carcinoma and B-lymphoma, and should therefore provide a powerful novel platform for research on EBV-driven hematological and epithelial malignancies.

Synergistic Effect by Combining a gp120-Binding Protein and a gp41-Binding Antibody to Inactivate HIV-1 Virions and Inhibit HIV-1 Infection

Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.

Viral diagnosis in cultivars of Ipomoea batatas (L.) Lam.

Ipomoea batatas (L.) Lam. commonly known as sweet potato, is an important staple food worldwide, mainly due to its high nutritional value and yield. However, vegetative reproduction of sweet potato makes it more susceptible to viral infections, which threatens its productivity, quality, and difficult long-term preservation in germplasm banks. Also, it can act as a virus reservoir infecting the rest of the plant accessions in the bank collections. Hence, this work aimed to screen Begomovirus, Potyvirus, and Carlavirus infections in 16 traditional sweet potato cultivars from the germplasm collection of the ISOPlexis Germplasm Bank, Madeira, Portugal. The infection prevalence by these viruses among cultivars was 81.25%, 25.00%, and 6.25%, respectively; being ISOP1011 the only accession coinfected by Potyvirus and Carlavirus. The accessions ISOP1006, ISOP1010, and ISOP1047 were also coinfected by Begomovirus and Potyvirus, highlighting their vulnerability to viral infections. The ISOP1005 and ISOP1027 accessions were the only ones not infected by any of these viruses. The analysis of the partial sequence obtained from the Carlavirus detected in the accession ISOP1011, revealed the existence of an ORF that encodes for 93 amino acids of the catalytic domain of an RNA-directed RNA polymerase related to the Tymovirus protein family, as could be confirmed by comparison with proteins stored in UniProtKB. Multiple sequence alignment with these proteins showed that Motifs A and B of the catalytic domain were conserved. The search for sequence similarity with sequences deposited in GenBank reported a high sequence identity with Sweet potato yellow mottle virus (SPYMV) and Sweet potato chlorotic fleck virus (SPCFV). However, the 9-11% discrepancy in nucleotide sequence identity and a phylogenetic analysis carried out using the maximum probability method suggests the virus isolated from ISOP1011 is a new divergent strain of the SPCFV species.

Homology modeling in combination of phylogenetic assortment, a new approach to resolve the phylogeny of selected heterocystous cyanobacteria based on phycocyanin encoding cpcBA-IGS locus

The present communication deals with phylogenetic assessment of phycocyanin coding genes, based on their 3-D structures which is still in its infancy. Homology modeling of cpcA and cpcB in conjunction with molecular phylogenetics for 12 strains belonging to the heterocystous cyanobacteria has been performed with an aim to resolve the ambiguities in their phylogenetic positions. 3D structure has been deduced using Discovery studio while CHIMERA, PEROMALS3D and SALIGN tools have been used for studying the structure based diversity of cpcA and cpcB genes respectively. MEME suite has been further used for motif analysis. Calothrix brevissima Ind9 was the most divergent strain. Nostoc and Anabaena were found to be intermixed at structural level also. The phylogeny suggested monophyletic origin of the heterocystous clade. Conserved Signature Indels provides novel means of identification and also supported monophyletic origin of heterocystous cyanobacteria. At the structure level the secondary elements are more conserved. Overall data obtained through the 3D structure based phylogeny affirmed close association and similar origin of the two subsections. This approach provides better resolution and must be used along with molecular phylogenetics for better identification of cyanobacteria.

Strain-Specific Peptide (SSP) Interference Reference Sample: A Genetically Encoded Quality Control for Isobaric Tagging Strategies.

Isobaric tag-based sample multiplexing strategies are extensively used for global protein abundance profiling. However, such analyses are often confounded by ratio compression resulting from the co-isolation, co-fragmentation, and co-quantification of co-eluting peptides, termed "interference." Recent analytical strategies incorporating ion mobility and real-time database searching have helped to alleviate interference, yet further assessment is needed. Here, we present the strain-specific peptide (SSP) interference reference sample, a tandem mass tag (TMT)pro-labeled quality control that leverages the genetic variation in the proteomes of eight phylogenetically divergent mouse strains. Typically, a peptide with a missense mutation has a different mass and retention time than the reference or native peptide. TMT reporter ion signal for the native peptide in strains that encode the mutant peptide suggests interference which can be quantified and assessed using the interference-free index (IFI). We introduce the SSP by investigating interference in three common data acquisition methods and by showcasing improvements in the IFI when using ion mobility-based gas-phase fractionation. In addition, we provide a user-friendly, online viewer to visualize the data and streamline the calculation of the IFI. The SSP will aid in developing and optimizing isobaric tag-based experiments.

Two novel loci underlie natural differences in Caenorhabditis elegans abamectin responses.

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.

Antiviral activity and mechanism of ESC-1GN from skin secretion of Hylarana guentheri against influenza A virus

Development of new and effective antiinfluenza drugs is critical for prophylaxis and treatment of influenza A virus (IAV) infection. A wide range of amphibian skin secretions have been identified to show antiviral activity. Our previously reported ESC-1GN, a peptide from the skin secretion of Hylarana guentheri, displayed good antimicrobial and antiinflammatory effects. Here, we found that ESC-1GN possessed significant antiviral effects against IAVs. Moreover, ESC-1GN could inhibit the entry of divergent H5N1 and H1N1 virus strains with the IC50 values from 1.29 to 4.59 μM. Mechanism studies demonstrated that ESC-1GN disrupted membrane fusion activity of IAVs by interaction with HA2 subunit. The results of site-directed mutant assay and molecular docking revealed that E105, N50 and the residues around them on HA2 subunit could form hydrogen bonds with amino acid on ESC-1GN, which were critical for ESC-1GN binding to HA2 and inhibiting the entry of IAVs. Altogether, these not only suggest that ESC-1GN maybe represent a new type of excellent template designing drugs against IAVs, but also it may shed light on the immune mechanism and survival strategy of H.guentheri against viral pathogens.

Development and evaluation of reverse transcription loop-mediated isothermal amplification for rapid and real-time detection of Kyasanur forest disease virus

SignificanceKyasanur forest disease (KFD), a re-emerging tick-borne viral disease, causes severe hemorrhagic fever in humans and nonhuman primates. KFD virus (KFDV) is a member of the genus Flavivirus. KFD is now increasingly reported outside its endemic zone in India. Rapid and specific detection of the KFDV plays a critical role in containment of the outbreak. The diagnosis of KFD currently relies on real-time RT-PCR, nested RT-PCR, end point RT-PCR, and serodiagnostic assay. These assays are tedious, time-consuming, and cannot be used as a routine screening platform. ObjectiveThe present study was aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for molecular diagnosis of KFD. DesignThe gene amplification reaction was accomplished by incubation at a constant temperature of 63°C for 60min. ResultsThe limit of detection of RT-LAMP assay was 10 copies. KFD RT-LAMP assay was successfully evaluated with diverse host samples including humans, monkeys, and tick. The assay correctly picked up different KFD isolates indicating its applicability for divergent strains. Comparative evaluation of RT-LAMP assay with quantitative TaqMan real-time RT-PCR revealed 100% concordance. No cross-reaction with related flavi and other hemorrhagic fever viruses was observed, indicating its high specificity. Conclusion and relevanceThe RT-LAMP test developed in this study will serve as a rapid, sensitive alternate detection method for KFDV infection and would be useful for high throughput screening of clinical samples in resource limited areas during outbreaks.

Dissemination mechanisms of NDM genes in hospitalized patients.

BackgroundNDM-producing Enterobacteriaceae are a major clinical concern worldwide. We characterized NDM-positive pathogens isolated from patients and assessed the dissemination patterns of the blaNDM genes in a hospital setting.MethodsEleven NDM-positive Enterobacteriaceae (three Enterobacter hormaechei, six Klebsiella pneumoniae and two Escherichia coli) were isolated from nine patients over a 1 year period. Antimicrobial susceptibility was assessed by MICs. A combination of short- and long-read WGS was used for genome analysis. Clinical treatment history of patients was linked with genetic features of individual isolates to investigate the dissemination patterns of the blaNDM genes and NDM-positive strains.Results bla NDM in clonal K. pneumoniae were transmitted between two patients. In other instances, an identical IncC plasmid encoding NDM-1 was transmitted between E. coli and K. pneumoniae isolated from the same patient, and an IncX3 plasmid, carrying blaNDM-1 or blaNDM-5, was harboured in non-clonal E. hormaechei. Varying patterns of IS elements were identified as a critical transmission mechanism in association with blaNDM genes.ConclusionsMultiple transmission patterns were identified in hospitalized patients, including dissemination of clonal bacterial strains carrying resistance genes and horizontal transfer of resistance genes among divergent bacterial strains. Controlling spread of NDM is complex: while attention to standard infection control practices is critically important, this needs to be matched by aggressive efforts to limit unnecessary antimicrobial use, to minimize the selection for and risk of transfer of ‘high mobility’ resistance genes among Enterobacteriaceae.

Genetic characterization and pathogenicity of a divergent broiler-origin orthoreovirus causing arthritis in China.

Since 2013, there have been an increasing number of cases of arthritis in broilers caused by avian orthoreovirus (ARV) in China, and the virus remains highly virulent in chicks with high-level maternal antibodies. However, little information is available about the complete gene analysis and pathogenicity of the epidemic ARVs. In the study, the ARV strain (V-ARV-SD26) was isolated from broilers associated with arthritis in Shandong Province. To genetically characterize the ARV strain, the whole-genome sequencing was conducted by next-generation sequencing (NGS) technique. Sequence analysis demonstrated that V-ARV-SD26 might have acquired its current genomic composition through several homologous and, in case of the λC, μA and σB, divergent reassortment events. To further investigate the pathogenicity of the strain, 160 one-day-old Ross broilers with maternal antibodies were equally divided into four groups (foodpad-, eye mucosa- and intramuscular-inoculated groups and the negative control group), three experimental groups were inoculated separately with the low-dose virus fluid, and the negative control was equally inoculated with sterile PBS. The results showed that the symptoms of broilers in foodpad inoculation group were the most serious, while that of the eye mucosa infection group were the mildest. Meanwhile, the cloacal cotton swabs and organs were collected for qRT-PCR detection to evaluate the infection status. In conclusion, these findings indicate that V-ARV-SD26 is a divergent ARV strain, which provide experimental data for the prevention and control of newly emerged reovirus, and have a certain reference value for the preparation and evaluation of new vaccines.

Serodetection of astroviruses in runted commercial broilers and turkeys in southwest Nigeria.

Infections with divergent strains of astroviruses appear to be endemic in commercial poultry. In order to investigate enteric viruses associated with hatchery condemnations in Nigerian poultry, an indirect immunofluorescence test with CAstV-612- (Group A), CAstV-11672- (Group B) and ANV-1-infected cells was used to screen sera obtained from commercial broilers (n = 164) and turkeys (n = 97) in farms and hatcheries in southwest Nigeria. Of the 261 sera tested, 16 (6.1%) were positive for CAstV antibodies after immunofluorescent staining with CAstV-11672-infected cells. Thirteen (81.3%) of the positive sera were from broilers with three (18.7%) being from turkeys. Conversely, all tested sera were negative for CAstV-612 and ANV-1 antibodies. Since CAstV-11672, a group B CAstV is known to be antigenically and genetically distinct from CAstV-612 that belongs to group A, these findings reveal that the circulating serotype of CAstV in commercial broilers and turkeys in southwest Nigeria belongs to group B of CAstV. Education of veterinary personnel and poultry farmers about this emerging virus and its impact on commercial poultry in Nigeria, as well as continuous monitoring of chicken and turkey flocks for infections caused by it are therefore imperative in order to facilitate the implementation of effective prevention and control measures.

Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense

Microbial plant pathogens secrete effector proteins, which manipulate the host to promote infection. Effectors can be recognized by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognized by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a noncanonical integrated heavy-metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defenses. The host targets of AVR-Pik are also HMA-domain-containing proteins, namely heavy-metal-associated isoprenylated plant proteins (HIPPs) and heavy-metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared with the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19 and compare the interaction to that formed with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF, which do not interact with any characterized Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognized AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.