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Goodman, L. B.

Publications and source records attributed to Goodman, L. B..

At least 19 recordsLinked to original sources
bioRxiv2025

Distinct evolutionary patterns of endemic and emerging parvoviruses, and the origin of a new pandemic virus.

Emergence of epidemic viruses in new hosts threatens both human and animal populations, and often involves virus evolution to overcome barriers that normally prevent efficient infection and spread in that host. After transfer the separated viruses will evolve in parallel as they spread within the original and new hosts. Here we examine the details of a virus involved in such a host-jumping event, where we define the natural evolution of feline panleukopenia virus (FPV) over 60 years, clarify the origins of the new pandemic canine parvovirus (CPV) that arose in the 1970s, and compare the separate evolution of those viruses over 47 years in cats or dogs. Several live-attenuated FPV vaccine viruses originated from early 1960s isolates or were a recombinant of an early virus, and many sequences in databases proved to be vaccine-derived. The sequences of wild viruses showed that FPV-like strains evolved at [~]25% the rate seen for CPV in dogs, and the higher rate of CPV evolution was consistent since 1979 when a genetic variant became widespread. The common ancestor of the CPV lineage was related to FPVs from Europe, and contained several unique host-adaptive capsid changes associated with canine transferrin receptor type-1 binding. Although the FPV vaccine strains are around 60 years old, little selection for antigenic variation was observed. The distinct evolutionary patterns of these closely related viruses circulating for decades in different hosts emphasizes the complex evolution associated with viral epidemic emergence and spread in endemic and new hosts. SIGNIFICANCE STATEMENTComparing the evolution of a virus in its reservoir host with that seen in a new host will reveal the special circumstances that allow epidemic emergence. A feline parvovirus (FPV) jumping to dogs in the mid-1970s formed canine parvovirus (CPV), which has circulated world-wide until today. The evolutionary rate of FPV in its original hosts was much lower than that of CPV in dogs, and the mutational patterns seen in the different hosts were also distinct. Early CPV isolates differed from the ancestral FPV clade in several key host range mutations. These results highlight the complex biology associated with epidemic emergence, including host-specific rates of lineage evolution and complex origins of host-adaptive mutations. (113/120)

evolutionary biology↗
bioRxiv2025

Identification of within-host deletions in domain 0 of the spike gene of highly pathogenic feline coronavirus type 2 from the United States

Feline coronavirus (FCoV) is known to gain pathogenicity within-host to cause the lethal disease feline infectious peritonitis (FIP). Most FIP cases are caused by viruses in genotype 1 (FCoV-1) via an internal mutation in the spike gene. However, genotype 2 (FCoV-2) has risen to prominence based on the emergence of FCoV-23, a highly pathogenic novel variant from Cyprus that has a deletion in the N-terminus (domain 0) of spike. Here, we conducted a retrospective molecular study of FCoV-2 detected in three cats in the U.S. during 2013 and 2016. Whole-genome sequencing revealed that the two cats exhibiting long-term signs each had an FCoV-2 with a distinct deletion in domain 0 of spike in all examined tissues. The epidemiologically-linked cat displaying signs for a short duration had an FCoV-2 with an intact spike. Our results suggest that this "internal deletion" in the spike gene is a biomarker of highly pathogenic FCoV-2. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/664542v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1d10d9eorg.highwire.dtl.DTLVardef@1d53c7forg.highwire.dtl.DTLVardef@76ef35org.highwire.dtl.DTLVardef@528d1f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗
bioRxiv2025

TICKHUNTER: A Targeted Hybridization-Capture Sequencing Approach for the Detection and Characterization of Tick-borne Pathogens and Blood Meals

As weather systems quickly change, vector communities and their pathogens evolve faster than assay panels can be redesigned. Additionally, PCRs narrow target range makes it structurally unable to catch divergent or reassorted agents. We developed a hybrid capture next-generation sequencing enrichment platform that provides comprehensive detection and characterization of tick-borne agents alongside ecological vertebrate host identification with low-pass sequencing. Analytical validation demonstrated performance comparable to qPCR with superior variant tolerance and multiplexing capacity. Field deployment in subtropical, metropolitan New York detected Anaplasma phagocytophilum strains (n=3) linked to human granulocytic anaplasmosis and a Babesia microti-like species in urban raccoon (Procyon lotor) populations. Tick vector screening revealed a putatively novel chimeric Flavi-like virus in invasive Haemaphysalis longicornis ticks combining segmented and unsegmented genomic features, with codon adaptation analysis indicating strong human compatibility. Serology revealed high Flavivirus seropositivity in NYC raccoons, suggesting an unrecognized urban reservoir role. Blood meal analysis simultaneously revealed complex ecological pathogen transmission networks spanning multiple vertebrate hosts. This integrated surveillance system enables comprehensive pathogen discovery, real-time evolutionary monitoring, and ecological risk assessment, transforming our capacity to detect emerging tick-borne threats in rapidly changing environments and prevent spillovers.

microbiology↗
bioRxiv2024

The Evolution and Epidemiology of H3N2 Canine Influenza Virus After 20 Years in Dogs

The H3N2 canine influenza virus (CIV) emerged from an avian reservoir in Asia around 2004. As the virus has now been circulating entirely among dogs for 20 years, we here update our understanding of the evolution of virus in its new host. As a host-switched virus, H3N2 CIV will also reveal any host-adaptive changes arising during thousands of infections within its new host, and our analysis showed that the virus has evolved at a constant rate. CIV was first introduced into North America in 2015 from Korea, and we specifically examined the epidemiology of the virus among dogs in North America since then, including local outbreaks, regional die-outs, and repeated reintroduction from Asia. The H3N2 CIV now appears endemic only in China after dying out in South Korea around 2017. Virus lineages circulating in China appear to have seeded the most recent US outbreaks - with 2 or 3 introductions into North America during the past 3 years. Combining clinical reports, diagnostic testing data, and analysis of viral genomes we show that the virus spreads rapidly among dogs in kennels and shelters in different regions - likely dying out locally after all those animals become infected and immune. The overall epidemic therefore requires longer-distance dispersal of virus to initiate outbreaks in new locations. Patterns of spread in the USA may select viruses most adapted to those dense populations, which may lack the properties required for efficient long-distance transfers to other dog populations that would keep the virus in prolonged circulation. IMPORTANCEViruses occasionally jump into new hosts to cause epidemics and may spread widely due to movement of humans or animals, or their viruses, with profound consequences for global health. The emergence and epidemiology of new epidemic viruses in companion animals provides a model for understanding disease dynamics and evolution. The H3N2 canine influenza virus arose from an avian virus, and infected dogs provide many opportunities for human exposure. H3N2 CIV transmission is dominated by fast-moving outbreaks within dense populations in animal shelters or kennels, while sustaining the epidemic likely requires movement of virus to more distant dog populations. Viral spread within North Americahas only been sustained for a few years at a time after which the virus dies out. The epidemiological and evolutionary dynamics of this virus in this structured host population shows how an acute respiratory pathogen can emerge and spread in a new host and population.

microbiology↗
bioRxiv2024

Molecular detection using hybridization capture and next-generation sequencing reveals cross-species transmission of feline coronavirus type-1 between a domestic cat and a captive wild felid

Feline coronavirus (FCoV) infection normally causes mild or subclinical signs and is common in domestic cats. However, in some cats, FCoV infection can also lead to the development of feline infectious peritonitis (FIP)--a typically lethal disease. FCoV has two serotypes or genotypes, FCoV-1 and FCoV-2, both of which can cause FIP. The main difference between the genotypes is the viral spike (S) protein that determines tropism and pathogenicity, crucial mechanisms in the development of FIP. Subclinical infection and FIP have both been reported in wild felids, including in threatened species. Due to the high genetic variability of the S gene and the technical challenges to sequencing it, detection and characterization of FCoV in wild felids have mainly centered on other more conserved genes. Therefore, the genotype causing FIP in most wild felids remains unknown. Here, we report a retrospective molecular epidemiological investigation of FCoV in a zoological institution in the United States. In 2008, a domestic cat (Felis catus) and a Pallas cat (Otocolobus manul) sharing the same room succumbed to FIP. Using hybridization capture and next-generation sequencing, we detected and sequenced the complete 3-end of the viral genome ([~]8.2 Kb), including the S gene, from both felids. Our data show for the first time that FCoV-1 can be transmitted between domestic and wild felids and extends the known host range of FCoV-1. Our findings highlight the importance of identifying the genotype causing FIP, to develop effective control measures.

microbiology↗
bioRxiv2023

Beta-catenin-mediated activation of Wnt target genes utilizes a biomolecular condensate-dependent mechanism

The Wnt/{beta}-catenin signaling pathway plays numerous, essential roles in animal development and tissue/stem cell maintenance. The activation of genes regulated by Wnt/{beta}-catenin signaling requires the nuclear accumulation of {beta}-catenin, a transcriptional co-activator. {beta}-catenin is recruited to many Wnt-regulated enhancers through direct binding to T-cell factor/Lymphoid enhancer factor (TCF/LEF) family transcription factors. {beta}-catenin has previously been reported to form phase-separated biomolecular condensates (BMCs), which was implicated as a component of {beta}-catenins mechanism of action. This function required aromatic amino acid residues in the intrinsically disordered regions (IDRs) at the N- and C-termini of the protein. In this report, we further explore a role for {beta}-catenin BMCs in Wnt target gene regulation. We find that {beta}-catenin BMCs are miscible with LEF1 BMCs in vitro. We characterized a panel of {beta}-catenin mutants with different combinations of aromatic residue mutations in human cell culture and Drosophila melanogaster. Our data support a model in which aromatic residues across both IDRs contribute to BMC formation in vitro and signaling activity in vivo. Although different Wnt targets have different sensitivities to loss of {beta}-catenins aromatic residues, the activation of every target examined was compromised by aromatic substitution. These mutants are not defective in nuclear import, and residues in the N-terminal IDR with no previously known role in signaling are clearly required for the activation of various Wnt readouts. Consistent with this, deletion of the N-terminal IDR results in a loss of signaling activity, which can be rescued by the addition of heterologous IDRs enriched in aromatic residues. Overall, our work supports a model in which the ability of {beta}-catenin to form biomolecular condensates in the nucleus is tightly linked to its function as a transcriptional co-regulator.

molecular biology↗
bioRxiv2023

Survey of white-footed mice in Connecticut, USA reveals low SARS-CoV-2 seroprevalence and infection with divergent betacoronaviruses

Diverse mammalian species display susceptibility to and infection with SARS-CoV-2. Potential SARS-CoV-2 spillback into rodents is understudied despite their host role for numerous zoonoses and human proximity. We assessed exposure and infection among white-footed mice (Peromyscus leucopus) in Connecticut, USA. We observed 1% (6/540) wild-type neutralizing antibody seroprevalence among 2020-2022 residential mice with no cross-neutralization of variants. We detected no SARS-CoV-2 infections via RT-qPCR, but identified non-SARS-CoV-2 betacoronavirus infections via pan-coronavirus PCR among 1% (5/468) of residential mice. Sequencing revealed two divergent betacoronaviruses, preliminarily named Peromyscus coronavirus-1 and -2. Both belong to the Betacoronavirus 1 species and are [~]90% identical to the closest known relative, Porcine hemagglutinating encephalomyelitis virus. Low SARS-CoV-2 seroprevalence suggests white-footed mice may not be sufficiently susceptible or exposed to SARS-CoV-2 to present a long-term human health risk. However, the discovery of divergent, non-SARS-CoV-2 betacoronaviruses expands the diversity of known rodent coronaviruses and further investigation is required to understand their transmission extent.

ecology↗
bioRxiv2023

Intra-host variation in the spike S1/S2 region of a feline coronavirus type-1 in a cat with persistent infection

Feline coronavirus type 1 (FCoV-1) is widely known for causing feline infectious peritonitis (FIP), a systemic infection that is often fatal, with the virus known as the FIPV biotype. However, subclinical disease also occurs, in which cats may not show signs and intermittently shed the virus, including in feces, possibly for long periods of time. This virus is known as the FECV biotype. Progression of FECV to FIPV has been linked to several genomic changes, however a specific region of the viral spike protein at the interface of the spike S1 and S2 domains has been especially implicated. In this study, we followed a cat (#576) for six years from 2017, at which time FCoV-1 was detected in feces and conjunctival swabs, until 2022, when the animal was euthanized based on a diagnosis of alimentary small cell lymphoma. Over this time period, the cat was clinically diagnosed with inflammatory bowel disease and chronic rhinitis, and cardiac problems were also suspected. Using hybridization capture targeting the spike (S) gene of FCoV followed by next-generation sequencing, we screened 27 clinical samples. We detected FCoV-1 in 4 samples taken in 2017 (intestine and nasal tissue, feces, and conjunctiva), and 3 samples taken in 2022 (feces, and intestinal and heart tissue), but not in fecal samples taken in 2019 and 2020. Next, we focused on the S1/S2 region within S, which contains the furin cleavage site (FCS), a key regulator of viral transmission and pathogenesis. We show that the FCoV-1 variants obtained from feces in 2017 and 2022 were identical, while the ones from conjunctiva (2017), heart (2022), and intestine (2017 and 2022) were distinct. Sequence comparison of all the variants obtained showed that most of the non-synonymous changes in the S1/S2 region occur within the FCS. In the heart, we found two variants that differed by a single nucleotide, resulting in distinct FCS motifs that differ in one amino acid. It is predicted that one of these FCS motifs will down-regulate spike cleavability. The variant from the conjunctiva (2017) had a 6-nucleotide in-frame insertion that resulted in a longer and more exposed S1/S2 loop, which is predicted to be more accessible to the furin protease. Our studies indicate that FCoV-1 can independently persist in the gastrointestinal tract and heart of a cat over a long period of time without evidence of typical FIP signs, with intermittent viral shedding from the gastrointestinal and respiratory tracts.

molecular biology↗
bioRxiv2023

Detection and characterization of novel luchacoviruses, genus Alphacoronavirus, in meso-carnivores in the northeastern United States

Small to mid-sized carnivores, or meso-carnivores, comprise a group of diverse mammals, many of which can adapt to anthropogenically disturbed environments. Wild meso-carnivores living in urban areas may get exposed to or spread pathogens to other species, including stray/feral domestic animals. Several coronaviruses (CoVs) have been detected in domesticated and farmed meso-carnivores, but knowledge of CoVs circulating in free-ranging wild meso-carnivores remains limited. In this study, we analyzed 321 samples collected between 2016 and 2022 from 9 species of free-ranging wild meso-carnivores and stray/feral domestic cats in the northeastern United States. Using a pan-CoV PCR, we screened tissues, feces, and saliva, nasal, and rectal swabs. We detected CoV RNA in fecal and saliva samples of animals in four species: fisher (Pekania pennanti), bobcat (Lynx rufus), red fox (Vulpes vulpes), and domestic cat (Felis catus). Next-generation sequencing revealed that all these viruses belonged to the Luchacovirus subgenus (Alphacoronavirus genus), previously reported only in rodents and lagomorphs (i.e., rabbits). Genetic comparison of the 3-end of the genome ([~]12,000bp) revealed that although the viruses detected group with, and have a genetic organization similar to other luchacoviruses, they are genetically distinct from those from rodents and lagomorphs. Genetic characterization of the spike protein revealed that the meso-carnivore luchacoviruses do not have an S1/S2 cleavage motif but do have highly variable structural loops containing cleavage motifs similar to those identified in certain pathogenic CoVs. This study highlights the importance of characterizing the spike protein of CoVs in wild species for further targeted epidemiologic monitoring. ImportanceSeveral coronaviruses (CoVs) have been detected in domesticated, farmed, and wild meso-carnivores, causing a wide range of diseases, and infecting diverse species, highlighting their important but understudied role in the epidemiology of these viruses. Assessing the viral diversity hosted in wildlife species is essential to understand their significance in the cross-species transmission of CoVs. Our focus here was on CoV discovery in meso-carnivores in the Northeast USA as a potential "hotspot" area with high density of humans and urban wildlife. This study identifies novel alphacoronaviruses circulating in multiple free-ranging wild and domestic species in this area and explores their potential epidemiological importance based on regions of the Spike gene that are relevant for virus-host interactions.

microbiology↗
bioRxiv2023

Isolation of Actinomyces cricetomyis sp. nov from orocervicofacial abscesses of African giant pouched rats (Cricetomys ansorgei)

African giant pouched rats are of interest for their unique sense of smell and can be trained for a variety of applications including detection of explosives and infectious diseases. A colony housed at a university animal care facility developed abscesses associated with the jaw and eye in multiple animals. The predominant bacterial species in each case was a catalase-positive Actinomyces-like Gram-positive bacillus. The isolates from different sites and animals matched each other genetically but had sequences and biochemical profiles inconsistent with previously described species of this group. Based on whole genome sequence, biochemical characterization, and fatty acid profile, a novel species of the genus Actinomyces is proposed, namely Actinomyces cricetomyis (type strain 186855T). The type strain is deposited at ATCC (TSD-310) and BCCM/LMG (LMG 32803).

microbiology↗
bioRxiv2023

Understanding the Divergent Evolution and Epidemiology of H3N8 Influenza Viruses in Dogs and Horses

Cross-species virus transmission events can lead to dire public health emergencies in the form of epidemics and pandemics. One example in animals is the emergence of the H3N8 equine influenza virus (EIV), first isolated in 1963 in Miami, Florida, USA, after emerging among horses in South America. In the early 21st century the American lineage of EIV diverged into two Florida clades that persist today, while an EIV transferred to dogs around 1999 and gave rise to the H3N8 canine influenza virus (CIV), first reported in 2004. Here, we compare CIV in dogs and EIV in horses to reveal their host-specific evolution, to determine the sources and connections between significant outbreaks, and to gain insight into the factors controlling their different evolutionary fates. H3N8 CIV only circulated in North America, was geographically restricted after the first few years, and went extinct in 2016. Of the two EIV Florida clades, clade 1 circulates widely and shows frequent transfers between the USA and South America, Europe and elsewhere, while clade 2 was globally distributed early after it emerged, but since about 2018 has only been detected in Central Asia. Any potential zoonotic threat of these viruses to humans can only be determined with an understanding of its natural history and evolution. Our comparative analysis of these three viral lineages reveals distinct patterns and rates of sequence variation yet with similar overall evolution between clades, suggesting epidemiological intervention strategies for possible eradication of H3N8 EIV. (242 words) IMPORTANCEThe emergence of viruses in new hosts is a threat to human and animal health. The H3N8 equine influenza virus (EIV) emerged in 1963 by transfer of an avian influenza virus, and the H3N8 canine influenza virus (CIV) subsequently emerged in 1999 when EIV transferred to dogs. H3N8 CIV persistently circulated in only a few locations in the USA, and has not been detected since 2016. In the same period H3N8 EIV has circulated as two separate clades, one in North America and other regions of the world, while the other currently appears to be found only in Central Asia. By comparing the hosts, epidemiology, and evolution of these influenza viruses we explain how these lineages had different evolutionary fates, and show why elucidating these evolutionary processes is key to understanding zoonotic disease and viral emergence. (137 words)

evolutionary biology↗
bioRxiv2023

Viral capsid, antibody, and receptor interactions: experimental analysis of the antibody escape evolution of canine parvovirus

Canine parvovirus (CPV) is a small non-enveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late-1970s due to a host range switch of a virus similar to the feline panleukopenia virus (FPV) that infected another host. The virus that emerged in dogs had altered capsid receptor- and antibody-binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we use in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bind two distinct epitopes, and one largely overlaps the host receptor binding site. We also engineered antibody variants with altered binding structures. Viruses were passaged with the wild type or mutated antibodies, and their genomes deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the TfR-binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. IMPORTANCEAntibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we use an in vitro model system and deep genome sequencing to reveal the mutations that arise in the virus genome during selection by each of two monoclonal antibodies or their engineered variants. High-resolution structures of each of the Fab: capsid complexes revealed their binding interactions. The engineered forms of the wild-type antibodies or mutant forms allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and likely have parallels for many other viruses.

evolutionary biology↗
bioRxiv2023

Natural selection differences detected in key protein domains between non-pathogenic and pathogenic Feline Coronavirus phenotypes

Feline Coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed Feline Enteric Coronavirus [FECV]), with around 12% developing into deadly Feline Infectious Peritonitis (FIP; Feline Infectious Peritonitis Virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV and FECV specific signals of positive selection. We analyzed full length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site, and the other within the fusion domain of Spike. We also found 15 sites subject to positive selection associated with FIPV within Spike, 11 of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were 14 sites (12 novel) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 furin cleavage site and adjacent C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype, and included 24 positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that is unlikely to be one singular "switch" mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.

evolutionary biology↗
bioRxiv2022

Phylogeographic reconstruction of the emergence and spread of Powassan virus in the northeastern United States

Powassan virus is an emerging tick-borne virus of concern for public health, but very little is known about its transmission patterns and ecology. Here, we expanded the genomic dataset by sequencing 279 Powassan viruses isolated from Ixodes scapularis ticks from the northeastern United States. Our phylogeographic reconstructions revealed that Powassan virus lineage II was likely introduced or emerged from a relict population in the Northeast between 1940-1975. Sequences strongly clustered by sampling location, suggesting a highly focal geographical distribution. Our analyses further indicated that Powassan virus lineage II emerged in the northeastern U.S. mostly following a south to north pattern, with a weighted lineage dispersal velocity of ~3 km/year. Since the emergence in the Northeast, we found an overall increase in the effective population size of Powassan virus lineage II, but with growth stagnating during recent years. The cascading effect of population expansion of white-tailed deer and I. scapularis populations likely facilitated the emergence of Powassan virus in the northeastern U.S. Significance statementOur work provides important fundamental insights in the local transmission dynamics of an emerging tick-borne pathogen of public health concern. Without the availability of vaccines or specific treatments, prevention of Powassan virus infection is dependent on education and control. We identified that Powassan virus is maintained in highly localized transmission foci that have been maintained for several years, without introductions of new virus clades. This provides both opportunities for better education about high risk areas and effective targeted control in Powassan virus foci with a long lasting impact.

genomics↗
bioRxiv2021

Recent zoonotic spillover and tropism shift of a Canine Coronavirus is associated with relaxed selection and putative loss of function in NTD subdomain of spike protein.

A recent study reported the occurrence of Canine Coronavirus (CCoV) in nasopharyngeal swabs from a small number of patients hospitalized with pneumonia during a 2017-18 period in Sarawak, Malaysia. Because the genome sequence for one of these isolates is available, we conducted comparative evolutionary analyses of the spike gene of this strain (CCoV-HuPn-2018), with other available Alphacoronavirus 1 spike sequences. The most N-terminus subdomain (0-domain) of the CCoV-HuPn-2018 spike protein has sequence similarity to Transmissible Gastroenteritis Virus (TGEV) and CCoV2b strains, but not to other members of the type II Alphacoronaviruses (i.e., CCoV2a and Feline CoV2-FCoV2). This 0-domain in CCoV-HuPn-2018 has evidence for relaxed selection pressure, an increased rate of molecular evolution, and a number of unique amino acid substitutions relative to CCoV2b and TGEV sequences. A region of the 0-domain determined to be key to sialic acid binding and pathogenesis in TGEV had clear differences in amino acid sequences in CCoV-HuPn-2018 relative to both CCoV2b (enteric) and TGEV (enteric and respiratory). The 0-domain of CCoV-HuPn-2018 also had several sites inferred to be under positive diversifying selection, including sites within the signal peptide. Downstream of the 0-domain, FCoV2 shared sequence similarity to the CCoV2b and TGEV sequences, with analyses of this larger alignment identifying positively selected sites in the putative Receptor Binding Domain (RBD) and Connector Domain (CD). Recombination analyses strongly implicated a particular FCoV2 strain in the recombinant history of CCoV-HuPn-2018 with molecular divergence times estimated at around 60 years ago. We hypothesize that CCoV-HuPn-2018 had an enteric origin, but that it has lost that particular tropism, because of mutations in the sialic acid binding region of the spike 0-domain. As selection pressure on this region was reduced, the virus evolved a respiratory tropism, analogous to other Alphacoronavirus 1, such as Porcine Respiratory Coronavirus (PRCV), that have lost this region entirely. We also suggest that signals of positive selection in the signal peptide as well as other changes in the 0-domain of CCoV-HuPn-2018 could represent an adaptive role in this new host and that this could be in part due to the different spatial distribution of the N-linked glycan repertoire for this strain.

evolutionary biology↗
bioRxiv2020

DNA extraction and host depletion methods significantly impact and potentially bias bacterial detection in a biological fluid

Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis could reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering primary material, laboratory methods, data analysis, and interpretation. The focus of this study is in gathering the primary material, in this case, DNA. We used bovine milk as a model to 1) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk; 2) evaluate host DNA depletion methods for use with milk, and 3) develop and evaluate a selective lysis-PMA based protocol for host DNA depletion in milk. Our results suggest that magnetic-based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety where the detection of Gram-negative pathogens and indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host. ImportanceTracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of what the relative composition looks like. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around one thousand times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods in the ability of detecting specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.

molecular biology↗
bioRxiv2020

Association of the invasive Haemaphysalis longicornis tick with vertebrate hosts, other native tick vectors, and tick-borne pathogens in New York City

Haemaphysalis longicornis, the Asian longhorned tick, is an invasive ixodid tick that has rapidly spread across the northeastern and southeastern regions of the United States since first reported in 2017. The emergence of H. longicornis presents a potential threat for livestock, wildlife, and human health as the host associations and vector potential of this invasive pest in the United States are poorly understood. Previous field data from the United States has shown that H. longicornis was not associated with natural populations of small mammals or birds, but they show a preference for medium sized mammals in laboratory experiments. Therefore, medium and large sized mammals were sampled on Staten Island, New York to determine H. longicornis host associations and vector potential for a range of human and veterinary pathogens. A total of 97 hosts were sampled and five species of tick (Amblyomma americanum, Dermacentor variabilis, H. longicornis, Ixodes scapularis, Ixodes cookei) were found feeding concurrently on these hosts. Haemaphysalis longicornis was found in the highest proportions compared to other native tick species on raccoons (55.4%), Virginia opossums (28.9%), and white-tailed deer (11.5%). Tissue, blood, and engorged larvae were tested for 17 different pathogens using a nanoscale PCR platform. Infection with five pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia spp., Mycoplasma haemocanis, and Bartonella spp.) was detected in host samples, but no pathogens were found in any larval samples. These results suggest that although large and medium sized mammals feed large numbers of H. longicornis ticks in the environment there is presently a low potential for H. longicornis to acquire pathogens from these wildlife hosts.HighlightsH. longicornis were sampled from seven genera of large and medium-sized mammalsRaccoons, opossums, and white-tailed deer fed a large proportion of H. longicornisH. longicornis did not acquire pathogens through co-feeding with native tick vectorsHost species were infected with a range of pathogens of human and veterinary concernHost-derived H. longicornis engorged larvae were not infected with any pathogensCompeting Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗