Roy Neal Platt

The nuclear, internal transcribed spacer (ITS) and mitochondrial cox1 markers are widely used to differentiate Schistosoma haematobium from its livestock counterparts, S. bovis and S. curassoni . Schistosoma isolated from humans with ITS and cox 1 alleles from livestock parasites are typically inferred to be zoonotic infections and those with heterozygous ITS alleles (suggesting mixed species ancestry) are classified as recent hybrids. These classifications assume that the ITS and cox1 markers accurately reflect genome-wide ancestry. Here, we evaluated the reliability of this classification scheme by genotyping ITS and cox1 from 132 parasites isolated from human urine, and from 37 adult schistosomes collected from cattle at 14 Nigerian locations. We also genome sequenced each sample to empirically determine livestock schistosome ancestry. ITS/ cox1 genotyping suggested extensive recent hybridization and zoonotic infection. Among parasites from humans, 10.1% carried both S. curassoni and S. haematobium ITS, consistent with F1 or early generation hybrids, 21% had livestock schistosome markers at both cox1 and ITS suggesting zoonotic infection, while 13.7% carried S. bovis cox1 alongside mixed S. curassoni and S. haematobium ITS, suggesting more complex ancestry. Genome sequencing revealed a very different picture. All parasites from humans formed a tight cluster regardless of ITS or cox1 genotype, while all worms from cattle were well differentiated. We found no schistosomes containing 50% livestock parasite ancestry consistent with F1s. Instead, we observed regionally varying levels of S. bovis introgression, with modest levels in southern Nigeria (mean = 4.9%) and low levels in northern Nigeria (mean = 0.06%). These results demonstrate that: (i) two-locus genotyping is uninformative for detecting zoonotic infection or recent hybridization between S. haematobium and livestock schistosomes and (ii) previous data generated using this approach requires reinterpretation. These findings reveal the limitations of widely-used approaches for documenting zoonotic infection and hybridization between S. haematobium and livestock schistosome species.

Since its emergence in 2020, multiple genotypes of the H5N1 clade 2.3.4.4b have been identified, with B3.13 and D1.1 emerging in the USA as two major and concerning genotypes. However, their relative pathogenicity and transmissibility in mammals have not been fully elucidated. We compared the pathogenicity and transmissibility of the first two human H5N1 clade 2.3.4.4b cases caused by B3.13 in Texas (A/Texas/37/2024; HPhTX B3.13) and D1.1 in Louisiana (A/Louisiana/12/2024; HPhLA D1.1) in a ferret model of infection and transmission. HPhTX B3.13 infection resulted in more severe clinical disease and enhanced viral shedding, with evidence of increased transmission relative to HPhLA D1.1. Histopathological analysis revealed more extensive lung pathology in animals infected with HPhTX B3.13, consistent with increased viral loads and inflammatory responses. Importantly, both genotypes showed no significant differences in reactivity to ferret sera raised against candidate vaccine virus (CVV) strains, receptor binding properties, or neuraminidase (NA) activity and thermostability. Whole-genome sequencing revealed no adaptive mutations in HPhTX B3.13 following infection or transmission. In contrast, HPhLA D1.1 showed rapid acquisition of the mammalian-adaptive mutation E627K in infected ferrets and both E627K and Q194K in the only fatal contact animal. Both mutations were associated with enhanced polymerase activity and computational analyses suggested that they enhance interactions with the mammalian host factors ANP32A and B. Our findings indicate that B3.13 is already well adapted for mammalian infection and transmission whereas D1.1 retains evolutionary potential through the rapid acquisition of adaptive mutations, highlighting important genotype-specific differences relevant to zoonotic risk assessment and pandemic preparedness.

The nuclear, internal transcribed spacer (ITS) and mitochondrial cox1 markers are widely used to differentiate Schistosoma haematobium from its livestock counterparts, S. bovis and S. curassoni . Schistosoma isolated from humans that have ITS and cox1 markers from livestock schistosomes are typically inferred as zoonotic infections, those with mixed species, heterozygous ITS are classified as F1s or recent hybrids, while those with discordant ITS and cox1 markers are considered to reflect older hybridization events. We evaluated the reliability of this classification scheme by genotyping ITS and cox1 from 132 parasites isolated from human urine, and from 37 adult schistosomes collected from cattle at 14 Nigerian locations. We also genome sequenced each sample to empirically determine livestock schistosome ancestry. ITS/ cox1 genotyping suggested extensive recent hybridization and zoonotic infection. Among parasites from humans, 10.1% carried both S. curassoni and S. haematobium ITS, consistent with F1 or early generation hybrids, 21% had livestock schistosome markers at both cox1 and ITS suggesting zoonotic infection, while 13.7% carried S. bovis cox1 alongside mixed S. curassoni and S. haematobium ITS, suggesting more complex ancestry. Genome sequencing revealed a very different picture. All parasites from humans formed a tight cluster regardless of ITS or cox1 genotype, while all worms from cattle were well differentiated. We found no schistosomes containing 50% livestock parasite ancestry consistent with F1s. Instead, we observed regionally varying levels of S. bovis introgression, with modest levels in southern Nigeria (mean = 4.9%) and low levels in northern Nigeria (mean = 0.06%). These results demonstrate that: (i) two-locus genotyping is uninformative for detecting zoonotic infection or recent hybridization between S. haematobium and livestock schistosomes and (ii) previous data generated using this approach requires reinterpretation. These findings reveal the limitations of widely-used approaches for documenting zoonotic infection and hybridization between S. haematobium and livestock schistosome species.

Genetic markers for detecting hybridization and measuring population genetic parameters must be informative and cost-effective. Most population genetic studies within the Schistosoma haematobium species group rely on either a two-marker system consisting of the mitochondrial cytochsniromeoxidase 1 (cox1) and the nuclear internal transcribed spacer (ITS) markers or, at the other extreme, millions of single nucleotide variants (SNVs) from whole genome/exome sequencing. cox1 and ITS studies contain minimal population genetic information, but whole genome sequencing is cost-prohibitive. We examined approximately 38 million previously published, whole genome SNVs genotyped in 162 S. haematobium and Schistosoma bovis sampled across Africa. We compared population genetic parameters from 4000 panels of 10–100 000 randomly sampled SNVs to results from the whole genome dataset to test the resolution of reduced representation sequencing in schistosomes. We found that panels of 500 SNVs captured >99% of the population genetic information contained in the whole genome dataset by using Procrustes transformed principal component analyses and ancestry estimates (r² = 0.85). Additionally, the costs of genotyping parasites with an amplicon panel are two to three times less than whole genome sequencing. Our results show that moderately sized amplicon panels targeting random SNVs provide an efficient approach to large-scale, field-based schistosome surveillance. This article is part of the Science+ meeting issue ‘Parasite evolution and impact in action: exploring the importance and control of hybrid schistosomes’.

Schistosomes are obligately sexual blood flukes that can be maintained in the laboratory using freshwater snails as intermediate and rodents as definitive hosts. The genetic composition of laboratory schistosome populations is poorly understood: whether genetic variation has been purged due to serial inbreeding or retained is unclear. We sequenced 19 – 24 parasites from each of five laboratory Schistosoma mansoni populations and compared their genomes with published exome data from four S. mansoni field populations. We found abundant genomic variation (0.897 – 1.22 million variants) within laboratory populations: these carried on average 62% (π = 1.52e-04 – 7.15e-04) less nucleotide diversity than the four field parasite populations (π = 9.06e-03 – 2.24e-03). However, the pattern of variation was very different in laboratory and field populations. Tajima’s D was positive in all laboratory populations (except SmBRE), indicative of recent population bottlenecks, but negative in all field populations. Current effective population size estimates of laboratory populations were lower (2 – 258) compared to field populations (3,174 – infinity). The distance between markers at which linkage disequilibrium (LD) decayed to 0.5 was longer in laboratory populations (59 bp – 271 kb) compared to field populations (9 bp – 17.1 kb). SmBRE was the least variable laboratory population; this parasite also shows low fitness across the lifecycle, consistent with inbreeding depression. The abundant genetic variation present in most laboratory schistosome populations has several important implications: (i) measurement of parasite phenotypes, such as drug resistance, using laboratory parasite populations will determine average values and underestimate trait variation; (ii) genome-wide association studies (GWAS) can be conducted in laboratory schistosome populations by measuring phenotypes and genotypes of individual worms; (iii) genetic drift may lead to divergence in schistosome populations maintained in different laboratories. We conclude that the abundant genetic variation retained within many laboratory schistosome populations can provide valuable, untapped opportunities for schistosome research.

Genetic markers for detecting hybridization and measuring population genetic parameters must be informative and cost-effective. Most population genetic studies within the Schistosoma haematobium species group rely on either a two-marker system consisting of the mitochondrial, c ytochrome ox idase 1 ( cox 1) and the nuclear internal transcribed s pacer (ITS) markers or, at the other extreme, millions of s ingle n ucleotide v ariants (SNVs) from whole genome/exome sequencing. cox 1 and ITS studies contain minimal population genetic information but whole genome sequencing is cost-prohibitive. We examined ∼38 million previously published, whole genome SNVs genotyped in 162 S. haematobium and S. bovis sampled across Africa. We compared population genetic parameters from 4,000 panels of 10-100,000 randomly sampled SNVs to results from the whole genome dataset to test the resolution of reduced representation sequencing in schistosomes. We found that panels of 500 SNVs captured >99% of the population genetic information contained in the whole genome dataset by using Procrustes transformed principal component analyses and ancestry estimates ( r² = 0.85). Additionally, the costs of genotyping parasites with an amplicon panel is two to three times less than whole genome sequencing. Our results show that moderately-sized amplicon panels targeting random SNVs provide an efficient approach to large scale, field-based schistosome surveillance.

Influenza A viruses (IAV) infect a wide range of mammal and bird species and are responsible for seasonal outbreaks and occasional pandemics. Studying IAV requires methods to detect the presence of the virus in infected cells or animal models. Recombinant IAV expressing fluorescent proteins have allowed monitoring viral infection in cultured cells and ex vivo in the organs of infected animals. However, fluorescent-expressing IAV are often attenuated and are not suited for the imaging of infected animals using in vivo imaging systems (IVIS). To overcome this limitation, we generated a recombinant A/California/04/2009 H1N1 (pH1N1) expressing nanoluciferase (Nluc) from the non-structural (NS) viral segment (pH1N1-Nluc) that replicates efficiently in vitro , with growth kinetics and plaque morphology comparable to wild-type pH1N1 (pH1N1-WT). We used this pH1N1-Nluc to demonstrate its ability to identify neutralizing antibodies and antivirals, with neutralization and inhibition results comparable to pH1N1-WT. In mice, pH1N1-Nluc was able to induce similar body weight loss and mortality, and viral titers comparable to pH1N1-WT, results that were recapitulated in a ferret model of IAV infection. Using IVIS, pH1N1-Nluc enabled non-invasive, real-time tracking of viral infection in vivo and ex vivo following infection of mice with viral titers comparable to pH1N1-WT. The flexibility of this approach was further demonstrated by the generation of a Nluc-expressing recombinant A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Altogether, our results demonstrate that Nluc-expressing recombinant IAV represent a valuable tool for in vitro and in vivo studies, including the identification of antivirals and/or neutralizing antibodies, and to assess protective efficacy of vaccines. IMPORTANCE: Despite the availability of recombinant influenza A viruses (IAV) expressing fluorescent reporter genes to track viral infections in vitro and ex vivo , these viruses are often attenuated and do not represent the best option for imaging entire animals using in vivo imaging systems (IVIS). To solve this limitation, we generated recombinant influenza pandemic A/California/04/2009 H1N1 expressing nanoluciferase (pH1N1-Nluc) from the viral non-structural (NS) segment and demonstrate how expression of Nluc does not affect viral replication in vitro or viral pathogenesis in vivo. Importantly, we demonstrate the feasibility of detecting pH1N1-Nluc infection in vivo using IVIS. We also validate the flexibility of this approach by generating an influenza A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Our results support the feasibility of using these recombinant IAVs expressing Nluc from the NS segment for in vitro and in vivo studies, including the identification of neutralizing antibodies and/or antivirals, and to assess protective efficacy of vaccines.

Pooled sequencing provides a rapid cost-effective approach to assess genetic variation segregating within populations of organisms. However, such studies are typically limited to single nucleotide variants and small indels (≤50 bp), and have not been used for structural variants (SVs > 50 bp) which impact large portions of most genomes and may significantly impact phenotype. Here, we examined SVs circulating in five laboratory populations of the human parasite Schistosoma mansoni by generating long-read sequences from pools of worms (92−152 per population). We were able to identify and genotype 17,446 SVs, representing 6.5% of the genome despite challenges in identifying low-frequency variants. SVs included deletions (n = 8,525), duplications (n = 131), insertions (n = 8,410), inversions (n = 311), and translocations (n = 69) and were enriched in repeat regions. More than half (59%) of the SVs were shared between ≥4 populations, but 12% were found in only one of the five populations. Within this subset, we identified 168 population-specific SVs that were at-or-near fixation (>95% alternate allele frequency) in one population but missing (<5%) in the other four populations. Five of these variants impact the coding sequence of six genes. We also identified eight SVs with extreme allele frequency differences between populations within quantitative trait loci for biomedically important pathogen phenotypes (drug resistance, larval stage production) identified in prior genetic mapping studies. These results demonstrate that long-read sequence data from pooled individuals is a viable method to quickly catalogue SVs circulating within populations. Furthermore, some of these variants may be responsible for, or linked to, regions experiencing, population-specific directional selection.

The human parasitic fluke, Schistosoma haematobium hybridizes with the livestock parasite S. bovis in the laboratory, but the frequency of hybridization in nature is unclear. Here, we analyze 34.6 million single nucleotide variants in 162 samples from 18 African countries, revealing a sharp genetic discontinuity between northern and southern S. haematobium. We find no evidence for recent hybridization. Instead the data reveal admixture events that occurred 257–879 generations ago in northern S. haematobium populations. Fifteen introgressed S. bovis genes are approaching fixation in northern S. haematobium with four genes potentially driving adaptation. Further, we identify 19 regions that are resistant to introgression; these are enriched on the sex chromosomes. These results (i) suggest strong barriers to gene flow between these species, (ii) indicate that hybridization may be less common than currently envisaged, but (iii) reveal profound genomic consequences of rare interspecific hybridization between schistosomes of medical and veterinary importance.

A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in dairy cows was first reported on March 25, 2024, in the United States (US), marking the first discovery of HPAIV H5N1 in cattle. Soon after, a dairy worker on an affected dairy farm became the first human case linked directly to this outbreak. Studies with influenza A virus (IAV) require secondary methods to detect the virus in infected cells or animal models of infection. We modified the non-structural (NS) genome segment of the human A/Texas/37/2024 (HPhTX) H5N1 virus to create a recombinant virus expressing nanoluciferase (HPhTX NSs-Nluc), enabling the tracking of virus in cultured cells and mice via in vitro , ex vivo , and in vivo imaging systems (IVIS). In vitro , HPhTX NSs-Nluc showed growth and plaque characteristics similar to its wild-type (WT) counterpart. In vivo , HPhTX NSs-Nluc allowed tracking viral infection in the entire animals and in the organs of infected animals using in vivo and ex vivo IVIS, respectively. Importantly, the morbidity, mortality, and replication titers of HPhTX NSs-Nluc were comparable to those of the WT HPhTX. In vitro , HPhTX NSs-Nluc was inhibited by Baloxavir acid (BXA) to levels observed with WT HPhTX. We also demonstrate the feasibility of using HPhTX NSs-Nluc to evaluate the antiviral activity of BXA in vivo . Our findings support that HPhTX NSs-Nluc represents an excellent tool for tracking viral infections, including the identification of prophylactics or therapeutics for the treatment of the HPAIV H5N1 responsible of the outbreak in dairy cows.