Multimodal Surveillance Model for Enterovirus D68 Respiratory Disease and Acute Flaccid Myelitis among Children in Colorado, USA, 2022.

Surveillance for emerging pathogens is critical for developing early warning systems to guide preparedness efforts for future outbreaks of associated disease. To better define the epidemiology and burden of associated respiratory disease and acute flaccid myelitis (AFM), as well as to provide actionable data for public health interventions, we developed a multimodal surveillance program in Colorado, USA, for enterovirus D68 (EV-D68). Timely local, state, and national public health outreach was possible because prospective syndromic surveillance for AFM and asthma-like respiratory illness, prospective clinical laboratory surveillance for EV-D68 among children hospitalized with respiratory illness, and retrospective wastewater surveillance led to early detection of the 2022 outbreak of EV-D68 among Colorado children. The lessons learned from developing the individual layers of this multimodal surveillance program and how they complemented and informed the other layers of surveillance for EV-D68 and AFM could be applied to other emerging pathogens and their associated diseases.

Multistate Outbreak of Salmonella Thompson Infections Linked to Seafood Exposure - United States, 2021.

In July 2021, the Colorado Department of Public Health and Environment (CDPHE) laboratory identified a cluster of five Salmonella enterica serotype Thompson isolates related to one another within one allele difference, using whole genome multilocus sequence typing (wgMLST). These five isolates, submitted to the public health laboratory as is routine process for confirmatory testing of Salmonella, were highly related to those identified in a 2020 multistate investigation, during which traceback was conducted for sushi-grade tuna and salmon; a common supplier was not identified. The 2021 investigation commenced on August 5, 2021, with five patients living in Colorado, and one each in Missouri, Washington, and Wisconsin. During August-December 2021, CDC, CDPHE, public health and regulatory officials in several states, and the Food and Drug Administration (FDA) conducted epidemiologic, environmental, and laboratory investigations of this multistate outbreak of Salmonella Thompson. Isolates were genetically related to one another and to 2020 isolates within zero to one allele difference. Implicated seafood products were traced to a single seafood distributor, in which the outbreak strain was identified through environmental sampling, and in which inspection identified inadequate sanitization and opportunities for cross-contamination of raw fish. The distributor issued a voluntary recall of 16 seafood items with high potential for contamination and completed remediation actions. This outbreak illustrated the importance of effective cleaning and sanitizing procedures and implementation of controls. When multiple products are recalled during an outbreak investigation, collaboration between public health agencies and implicated facilities can help provide food safety information to restaurants, retailers, and consumers, and to ensure disposal of all recalled products.

Single-molecule Sequencing of an Animal Mitochondrial Genome Reveals Chloroplast-like Architecture and Repeat-mediated Recombination.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus. These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa-SSC-IRb structure revealed the occurrence of a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution.

Identification of and Surveillance for the SARS-CoV-2 Variants B.1.427 and B.1.429 - Colorado, January-March 2021.

The B.1.427 and B.1.429 variants of SARS-CoV-2, the virus that causes COVID-19, were first described in Southern California on January 20, 2021 (1); on March 16 they were designated variants of concern* (2). Data on these variants are limited, but initial reports suggest that, compared with other lineages, they might be more infectious (1,2), cause more severe illness (2), and be less susceptible to neutralizing monoclonal antibody products such as bamlanivimab, an investigational treatment for mild-to-moderate COVID-19 (1-3). On January 24, the Colorado Department of Public Health and Environment (CDPHE) identified the first Colorado case of COVID-19 attributed to these variants. B.1.427 and B.1.429 were considered a single variant described as CAL.20C or B.1.427/B.1.429 in the 20C clade (1,3); in this report "B.1.427/B.1.429" refers to B.1.427 or B.1.429 lineage, including those reported as B.1.427/B.1.429 without further differentiation.

Asexuality Associated with Marked Genomic Expansion of Tandemly Repeated rRNA and Histone Genes.

How does asexual reproduction influence genome evolution? Although is it clear that genomic structural variation is common and important in natural populations, we know very little about how one of the most fundamental of eukaryotic traits-mode of genomic inheritance-influences genome structure. We address this question with the New Zealand freshwater snail Potamopyrgus antipodarum, which features multiple separately derived obligately asexual lineages that coexist and compete with otherwise similar sexual lineages. We used whole-genome sequencing reads from a diverse set of sexual and asexual individuals to analyze genomic abundance of a critically important gene family, rDNA (the genes encoding rRNAs), that is notable for dynamic and variable copy number. Our genomic survey of rDNA in P. antipodarum revealed two striking results. First, the core histone and 5S rRNA genes occur between tandem copies of the 18S-5.8S-28S gene cluster, a unique architecture for these crucial gene families. Second, asexual P. antipodarum harbor dramatically more rDNA-histone copies than sexuals, which we validated through molecular and cytogenetic analysis. The repeated expansion of this genomic region in asexual P. antipodarum lineages following distinct transitions to asexuality represents a dramatic genome structural change associated with asexual reproduction-with potential functional consequences related to the loss of sexual reproduction.

Invasive freshwater snails form novel microbial relationships.

Resident microbes (microbiota) can shape host organismal function and adaptation in the face of environmental change. Invasion of new habitats exposes hosts to novel selection pressures, but little is known about the impact on microbiota and the host-microbiome relationship (e.g., how rapidly new microbial associations are formed, whether microbes influence invasion success). We used high-throughput 16S rRNA sequencing of New Zealand (native) and European (invasive) populations of the freshwater snail Potamopyrgus antipodarum and found that while invaders do carry over some core microbial taxa from New Zealand, their microbial community is largely distinct. This finding highlights that invasions can result in the formation of novel host-microbiome relationships. We further show that the native microbiome is composed of fewer core microbes than the microbiome of invasive snails, suggesting that the microbiota is streamlined to a narrower set of core members. Furthermore, native snails exhibit relatively low alpha diversity but high inter-individual variation, whereas invasive snails have higher alpha diversity but are relatively similar to each other. Together, our findings demonstrate that microbiota comparisons across native and invasive populations can reveal the impact of a long coevolutionary history and specialization of microbes in the native host range, as well as new associations occurring after invasion. We lay essential groundwork for understanding how microbial relationships affect invasion success and how microbes may be utilized in the control of invasive hosts.

A Potent Postentry Restriction to Primate Lentiviruses in a Yinpterochiropteran Bat.

Bats are primary reservoirs for multiple lethal human viruses, such as Ebola, Nipah, Hendra, rabies, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2. The innate immune systems of these immensely abundant, anciently diverged mammals remain insufficiently characterized. While bat genomes contain many endogenous retroviral elements indicative of past exogenous infections, little is known about restrictions to extant retroviruses. Here, we describe a major postentry restriction in cells of the yinpterochiropteran bat Pteropus alecto Primate lentiviruses (HIV-1, SIVmac) were potently blocked at early life cycle steps, with up to 1,000-fold decreases in infectivity. The block was specific, because nonprimate lentiviruses such as equine infectious anemia virus and feline immunodeficiency virus were unimpaired, as were foamy retroviruses. Interspecies heterokaryons demonstrated a dominant block consistent with restriction of incoming viruses. Several features suggested potential TRIM5 (tripartite motif 5) or myxovirus resistance protein 2 (MX2) protein restriction, including postentry action, cyclosporine sensitivity, and reversal by capsid cyclophilin A (CypA) binding loop mutations. Viral nuclear import was significantly reduced, and this deficit was substantially rescued by cyclosporine treatment. However, saturation with HIV-1 virus-like particles did not relieve the restriction at all. P. alecto TRIM5 was inactive against HIV-1 although it blocked the gammaretrovirus N-tropic murine leukemia virus. Despite major divergence in a critical N-terminal motif required for human MX2 activity, P. alecto MX2 had anti-HIV activity. However, this did not quantitatively account for the restriction and was independent of and synergistic with an additional CypA-dependent restriction. These results reveal a novel, specific restriction to primate lentiviruses in the Pteropodidae and advance understanding of bat innate immunity.IMPORTANCE The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of retroviruses from three genera (Lentivirus, Gammaretrovirus, and Spumavirus) to infect cells of the large fruit-eating bat P. alecto and other mammals. We identified a major, specific postentry restriction to primate lentiviruses. HIV-1 and SIVmac are potently blocked at early life cycle steps, but nonprimate lentiviruses and foamy retroviruses are entirely unrestricted. Despite acting postentry and in a CypA-dependent manner with features reminiscent of antiretroviral factors from other mammals, this restriction was not saturable with virus-like particles and was independent of P. alecto TRIM5, TRIM21, TRIM22, TRIM34, and MX2. These results identify a novel restriction and highlight cyclophilin-capsid interactions as ancient species-specific determinants of retroviral infection.

Development of IFN-Stimulated Gene Expression from Embryogenesis through Adulthood, with and without Constitutive MDA5 Pathway Activation.

Pathogen-associated molecular patterns (e.g., dsRNA) activate expression of IFN-stimulated genes (ISGs), which protect hosts from infection. Although transient ISG upregulation is essential for effective innate immunity, constitutive activation typically causes harmful autoimmunity in mice and humans, often including severe developmental abnormalities. We have shown that transgenic mice expressing a picornavirus RNA-dependent RNA polymerase (RdRP) outside the viral context (RdRP mice) exhibit constitutive, MDA5-dependent, and quantitatively dramatic upregulation of many ISGs, which confers broad viral infection resistance. Remarkably, RdRP mice never develop autoinflammation, interferonopathy, or other discernible abnormalities. In this study, we used RNA sequencing and other methods to analyze ISG expression across five time points from fetal development to adulthood in wild-type and RdRP mice. In RdRP mice, the proportion of upregulated ISGs increased during development, with the most dramatic induction occurring 2 wk postnatally. The amplified ISG profile is then maintained lifelong. Molecular pathways and biological functions associated with innate immune and IFN signaling are only activated postnatally, suggesting constrained fetal responsiveness to innate immune stimuli. Biological functions supporting replication of viruses are only inhibited postnatally. We further determined that the RdRP is expressed at low levels and that blocking Ifnar1 reverses the amplified ISG transcriptome in adults. In conclusion, the upregulated ISG profile of RdRP mice is mostly triggered early postnatally, is maintained through adulthood, and requires ongoing type I IFN signaling to maintain it. The model provides opportunities to study the systems biology of innate immunity and to determine how sustained ISG upregulation can be compatible with robust health.

Adaptive phenotypic plasticity in a clonal invader.

Organisms featuring wide trait variability and occurring in a wide range of habitats, such as the ovoviviparous New Zealand freshwater snail Potamopyrgus antipodarum, are ideal models to study adaptation. Since the mid-19th century, P. antipodarum, characterized by extremely variable shell morphology, has successfully invaded aquatic areas on four continents. Because these obligately and wholly asexual invasive populations harbor low genetic diversity compared to mixed sexual/asexual populations in the native range, we hypothesized that (1) this phenotypic variation in the invasive range might be adaptive with respect to colonization of novel habitats, and (2) that at least some of the variation might be caused by phenotypic plasticity. We surveyed 425 snails from 21 localities across northwest Europe to attempt to disentangle genetic and environmental effects on shell morphology. We analyzed brood size as proxy for fitness and shell geometric morphometrics, while controlling for genetic background. Our survey revealed 10 SNP genotypes nested into two mtDNA haplotypes and indicated that mainly lineage drove variation in shell shape but not size. Physicochemical parameters affected both shell shape and size and the interaction of these traits with brood size. In particular, stronger stream flow rates were associated with larger shells. Our measurements of brood size suggested that relatively larger slender snails with relatively large apertures were better adapted to strong flow than counterparts with broader shells and relatively small apertures. In conclusion, the apparent potential to modify shell morphology plays likely a key role in the invasive success of P. antipodarum; the two main components of shell morphology, namely shape and size, being differentially controlled, the former mainly genetically and the latter predominantly by phenotypic plasticity.

Phosphorus availability in the source population influences response to dietary phosphorus quantity in a New Zealand freshwater snail.

We investigated whether previously documented variation among populations in availability of dietary phosphorus (P) is linked to heterogeneity in growth rate of the New Zealand freshwater snail Potamopyrgus antipodarum on a P-limited diet. We chose this system because P. antipodarum inhabits water bodies that vary in P availability and because P. antipodarum growth rate varies considerably in response to low P. We quantified specific growth rate and alkaline phosphatase (AP) expression in a diverse array of juvenile P. antipodarum fed high vs. low-P diets. We found strong associations between P content of epilithon in the source lake and P. antipodarum growth rate on high vs. low-P diets, with snails collected from lakes with relatively low-P epilithon showing the greatest increase in growth rate on the high-P relative to low-P diet. We also found substantial intraspecific variation in growth response to P limitation. Expression of AP also varied among lineages and was negatively associated with C: P of lake epilithon but did not explain the relationship between C: P in the lake of origin and sensitivity to P limitation. Together, our results demonstrate a strong signature of the P environment in the lake of origin on how this snail responds to P limitation as well as preliminary evidence for intraspecific variation of AP expression in animals.

Genomic evidence for population-specific responses to co-evolving parasites in a New Zealand freshwater snail.

Reciprocal co-evolving interactions between hosts and parasites are a primary source of strong selection that can promote rapid and often population- or genotype-specific evolutionary change. These host-parasite interactions are also a major source of disease. Despite their importance, very little is known about the genomic basis of co-evolving host-parasite interactions in natural populations, especially in animals. Here, we use gene expression and sequence evolution approaches to take critical steps towards characterizing the genomic basis of interactions between the freshwater snail Potamopyrgus antipodarum and its co-evolving sterilizing trematode parasite, Microphallus sp., a textbook example of natural coevolution. We found that Microphallus-infected P. antipodarum exhibit systematic downregulation of genes relative to uninfected P. antipodarum. The specific genes involved in parasite response differ markedly across lakes, consistent with a scenario where population-level co-evolution is leading to population-specific host-parasite interactions and evolutionary trajectories. We also used an FST -based approach to identify a set of loci that represent promising candidates for targets of parasite-mediated selection across lakes as well as within each lake population. These results constitute the first genomic evidence for population-specific responses to co-evolving infection in the P. antipodarum-Microphallus interaction and provide new insights into the genomic basis of co-evolutionary interactions in nature.

Genome Expression Balance in a Triploid Trihybrid Vertebrate.

Polyploidy is increasingly recognized as a driver of biological diversity. How and why polyploidization affects gene expression is critical to understanding the link between ploidy elevation and diversification. In polyploid plants, multiple studies have demonstrated that ploidy elevation can confer major but variable consequences for gene expression, ranging from gene-by-gene alterations to entirely silenced genomes. By contrast, animal polyploids remain largely uncharacterized. Accordingly, how animals respond to and manage polyploidy events is not understood. Here, we address this important knowledge gap by analyzing transcriptomes from a triploid hybrid animal, a unisexual Ambystoma salamander, and three sexual Ambystoma species that represent all three parental genomes in the unisexual. We used a novel bioinformatics pipeline that includes competitively mapping triploid sequences to a reference set of orthologous genes in the sexual species to evaluate subgenome expression. Our comparisons of gene expression levels across the three parental genomes revealed that the unisexual triploid displays a pattern of genome balance, where 72% of the genes analyzed were expressed equally among the subgenomes. This result is strikingly different from the genome imbalance typically observed in hybrid polyploid plants. Our analyses represent the first to address gene expression in a triploid hybrid animal and introduce a novel bioinformatic framework for analyzing transcriptomic data.

De Novo Transcriptome Characterization of a Sterilizing Trematode Parasite (Microphallus sp.) from Two Species of New Zealand Snails.

Snail-borne trematodes represent a large, diverse, and evolutionarily, ecologically, and medically important group of parasites, often imposing strong selection on their hosts and causing host morbidity and mortality. Even so, there are very few genomic and transcriptomic resources available for this important animal group. We help to fill this gap by providing transcriptome resources from trematode metacercariae infecting two congeneric snail species, Potamopyrgus antipodarum and P. estuarinus This genus of New Zealand snails has gained prominence in large part through the development of P. antipodarum and its sterilizing trematode parasite Microphallus livelyi into a textbook model for host-parasite coevolutionary interactions in nature. By contrast, the interactions between Microphallus trematodes and P. estuarinus, an estuary-inhabiting species closely related to the freshwater P. antipodarum, are relatively unstudied. Here, we provide the first annotated transcriptome assemblies from Microphallus isolated from P. antipodarum and P. estuarinus We also use these transcriptomes to produce genomic resources that will be broadly useful to those interested in host-parasite coevolution, local adaption, and molecular evolution and phylogenetics of this and other snail-trematode systems. Analyses of the two Microphallus transcriptomes revealed that the two trematode types are more genetically differentiated from one another than are the M. livelyi infecting different populations of P. antipodarum, suggesting that the Microphallus infecting P. estuarinus represent a distinct lineage. We also provide a promising set of candidate genes likely involved in parasitic infection and response to salinity stress.