Host Predictors of Broadly Cross-Reactive Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants of Concern Differ Between Infection and Vaccination.

BACKGROUND: Following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or vaccination there is significant variability between individuals in protective antibody levels against SARS-CoV-2, and within individuals against different virus variants. However, host demographic or clinical characteristics that predict variability in cross-reactive antibody levels are not well-described. These data could inform clinicians, researchers, and policymakers on the populations most likely to require vaccine booster shots. METHODS: In an institutional review board-approved prospective observational cohort study of staff at St. Jude Children's Research Hospital, we identified participants with plasma samples collected after SARS-CoV-2 infection, after mRNA vaccination, and after vaccination following infection, and quantitated immunoglobulin G (IgG) levels by enzyme-linked immunosorbent assay to the spike receptor binding domain (RBD) from 5 important SARS-CoV-2 variants (Wuhan Hu-1, B.1.1.7, B.1.351, P.1, and B.1.617.2). We used regression models to identify factors that contributed to cross-reactive IgG against 1 or multiple viral variants. RESULTS: Following infection, a minority of the cohort generated cross-reactive antibodies, IgG antibodies that bound all tested variants. Those who did had increased disease severity, poor metabolic health, and were of a particular ancestry. Vaccination increased the levels of cross-reactive IgG levels in all populations, including immunocompromised, elderly, and persons with poor metabolic health. Younger people with a healthy weight mounted the highest responses. CONCLUSIONS: Our findings provide important new information on individual antibody responses to infection/vaccination that could inform clinicians on populations that may require follow-on immunization.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Equine-Like H3 Avian Influenza Viruses in Wild Birds, Chile.

Since their discovery in the United States in 1963, outbreaks of infection with equine influenza virus (H3N8) have been associated with serious respiratory disease in horses worldwide. Genomic analysis suggests that equine H3 viruses are of an avian lineage, likely originating in wild birds. Equine-like internal genes have been identified in avian influenza viruses isolated from wild birds in the Southern Cone of South America. However, an equine-like H3 hemagglutinin has not been identified. We isolated 6 distinct H3 viruses from wild birds in Chile that have hemagglutinin, nucleoprotein, nonstructural protein 1, and polymerase acidic genes with high nucleotide homology to the 1963 H3N8 equine influenza virus lineage. Despite the nucleotide similarity, viruses from Chile were antigenically more closely related to avian viruses and transmitted effectively in chickens, suggesting adaptation to the avian host. These studies provide the initial demonstration that equine-like H3 hemagglutinin continues to circulate in a wild bird reservoir.

Effect of Geographic Isolation on the Nasal Virome of Indigenous Children.

The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with very different levels of geographic isolation. Metagenomic analysis was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in decreasing proportions of children. The number of papillomavirus infections detected was greater among Hispanic children most exposed to outside contacts, while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ among the villages. Closely related variants of rhinovirus A or B were identified in 2 to 4 children from each village, reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including members of the families Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep-encoding single-stranded DNA (CRESS-DNA) virus, were also detected in nasal swabs, possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high levels of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages, indicating ongoing exposure to globally circulating viruses.IMPORTANCE Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population sizes may also result in rapid local viral extinction due to a lack of seronegative subjects to maintain transmission chains for rapidly cleared viruses. We compared the viruses in the nasal passages of young children in three villages with increasing levels of geographic isolation. We found that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each village, likely reflecting recent viral introductions and transmission clusters among epidemiologically linked members of these very small communities. We conclude that, despite their geographic isolation, remote indigenous villages show evidence of ongoing exposure to globally circulating viruses.

Persistent Infections with Diverse Co-Circulating Astroviruses in Pediatric Oncology Patients, Memphis, Tennessee, USA.

Human astroviruses are a major cause of pediatric gastroenteritis, especially in immunocompromised children. We conducted a retrospective study to demonstrate that diverse astrovirus genotypes can co-circulate in pediatric oncology patients. A subset of cases is associated with long-term virus shedding (range 17-183 days).

Swine Influenza Virus (H1N2) Characterization and Transmission in Ferrets, Chile.

Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed to characterize the potential risk to humans, as well as the ecology of influenza in swine in South America.

Type I Interferon Response Limits Astrovirus Replication and Protects against Increased Barrier Permeability In Vitro and In Vivo.

UNLABELLED: Little is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-ß]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar results in vivo using a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesis in vivo. IMPORTANCE: Human astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability both in vitro and in vivo. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis.

Non-Human Primates Harbor Diverse Mammalian and Avian Astroviruses Including Those Associated with Human Infections.

Astroviruses (AstVs) are positive sense, single-stranded RNA viruses transmitted to a wide range of hosts via the fecal-oral route. The number of AstV-infected animal hosts has rapidly expanded in recent years with many more likely to be discovered because of the advances in viral surveillance and next generation sequencing. Yet no study to date has identified human AstV genotypes in animals, although diverse AstV genotypes similar to animal-origin viruses have been found in children with diarrhea and in one instance of encephalitis. Here we provide important new evidence that non-human primates (NHP) can harbor a wide variety of mammalian and avian AstV genotypes, including those only associated with human infection. Serological analyses confirmed that >25% of the NHP tested had antibodies to human AstVs. Further, we identified a recombinant AstV with parental relationships to known human AstVs. Phylogenetic analysis suggests AstVs in NHP are on average evolutionarily much closer to AstVs from other animals than are AstVs from bats, a frequently proposed reservoir. Our studies not only demonstrate that human astroviruses can be detected in NHP but also suggest that NHP are unique in their ability to support diverse AstV genotypes, further challenging the paradigm that astrovirus infection is species-specific.

Human H7N9 and H5N1 influenza viruses differ in induction of cytokines and tissue tropism.

UNLABELLED: Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models. IMPORTANCE: The novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological, and tissue tropism differences that could explain differences in clinical disease and viral transmission.

Crystal structure of the avian astrovirus capsid spike.

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.

Diet-induced obesity affects influenza disease severity and transmission dynamics in ferrets.

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet, the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a diet-induced obese ferret model and tools to demonstrate that, like humans, obesity resulted in notable changes to the lung microenvironment, leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for a longer period, making them more likely to transmit to contacts. These data suggest that the obese ferret model may be crucial to understanding obesity's impact on influenza disease severity and community transmission and a key tool for therapeutic and intervention development for this high-risk population.

Utility of nasal swabs for assessing mucosal immune responses towards SARS-CoV-2.

SARS-CoV-2 has caused millions of infections worldwide since its emergence in 2019. Understanding how infection and vaccination induce mucosal immune responses and how they fluctuate over time is important, especially since they are key in preventing infection and reducing disease severity. We established a novel methodology for assessing SARS-CoV-2 cytokine and antibody responses at the nasal epithelium by using nasopharyngeal swabs collected longitudinally before and after either SARS-CoV-2 infection or vaccination. We then compared responses between mucosal and systemic compartments. We demonstrate that cytokine and antibody profiles differ markedly between compartments. Nasal cytokines show a wound healing phenotype while plasma cytokines are consistent with pro-inflammatory pathways. We found that nasal IgA and IgG have different kinetics after infection, with IgA peaking first. Although vaccination results in low nasal IgA, IgG induction persists for up to 180 days post-vaccination. This research highlights the importance of studying mucosal responses in addition to systemic responses to respiratory infections to understand the correlates of disease severity and immune memory. The methods described herein can be used to further mucosal vaccine development by giving us a better understanding of immunity at the nasal epithelium providing a simpler, alternative clinical practice to studying mucosal responses to infection. Teaser: A nasopharyngeal swab can be used to study the intranasal immune response and yields much more information than a simple viral diagnosis.

Detection and phylogenetic analysis of highly pathogenic A/H5N1 avian influenza clade 2.3.4.4b virus in Chile, 2022.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses continue to pose a significant threat to animal and human health worldwide. In late 2022, the first confirmed case of HPAI A/H5N1 infection in wild birds in Chile near the Chilean-Peruvian border was reported. Active surveillance by our group in the adyacent Lluta river estuary revealed an increase in A/H5N1 prevalence coinciding with the arrival of migratory birds from the Northern Hemisphere. Genomic analysis of A/H5N1-positive samples demonstrated a close genetic relationship to strains detected in Peru during the same period, which originated from A/H5N1 viruses causing outbreaks in North America. Notably, we identified genetic mutations that did not correlate with known enhanced transmission or binding traits to mammalian receptors. In summary, this study provides valuable genomic insights into the A/H5N1 Clade 2.3.4.4b viruses in wild birds in Chile, emphasizing the need for enhanced surveillance and response strategies to mitigate the threat posed by these highly pathogenic avian influenza viruses in South America.

Remote Sensing and Ecological Variables Related to Influenza A Prevalence and Subtype Diversity in Wild Birds in the Lluta Wetland of Northern Chile.

The Lluta River is the northernmost coastal wetland in Chile, representing a unique ecosystem and an important source of water in the extremely arid Atacama Desert. During peak season, the wetland is home to more than 150 species of wild birds and is the first stopover point for many migratory species that arrive in the country along the Pacific migratory route, thereby representing a priority site for avian influenza virus (AIV) surveillance in Chile. The aim of this study was to determine the prevalence of influenza A virus (IAV) in the Lluta River wetland, identify subtype diversity, and evaluate ecological and environmental factors that drive the prevalence at the study site. The wetland was studied and sampled from September 2015 to October 2020. In each visit, fresh fecal samples of wild birds were collected for IAV detection by real-time RT-PCR. Furthermore, a count of wild birds present at the site was performed and environmental variables, such as temperature, rainfall, vegetation coverage (Normalized Difference Vegetation Index-NDVI), and water body size were determined. A generalized linear mixed model (GLMM) was built to assess the association between AIV prevalence and explanatory variables. Influenza positive samples were sequenced, and the host species was determined by barcoding. Of the 4349 samples screened during the study period, overall prevalence in the wetland was 2.07% (95% CI: 1.68 to 2.55) and monthly prevalence of AIV ranged widely from 0% to 8.6%. Several hemagglutinin (HA) and neuraminidase (NA) subtypes were identified, and 10 viruses were isolated and sequenced, including low pathogenic H5, H7, and H9 strains. In addition, several reservoir species were recognized (both migratory and resident birds), including the newly identified host Chilean flamingo (Phoenicopterus chilensis). Regarding environmental variables, prevalence of AIV was positively associated with NDVI (OR = 3.65, p < 0.05) and with the abundance of migratory birds (OR = 3.57, p < 0.05). These results emphasize the importance of the Lluta wetland as a gateway to Chile for viruses that come from the Northern Hemisphere and contribute to the understanding of AIV ecological drivers.

Utility of nasal swabs for assessing mucosal immune responses towards SARS-CoV-2.

SARS-CoV-2 has caused millions of infections worldwide since its emergence in 2019. Understanding how infection and vaccination induce mucosal immune responses and how they fluctuate over time is important, especially since they are key in preventing infection and reducing disease severity. We established a novel methodology for assessing SARS-CoV-2 cytokine and antibody responses at the nasal epithelium by using nasopharyngeal swabs collected longitudinally before and after either SARS-CoV-2 infection or vaccination. We then compared responses between mucosal and systemic compartments. We demonstrate that cytokine and antibody profiles differ between compartments. Nasal cytokines show a wound healing phenotype while plasma cytokines are consistent with pro-inflammatory pathways. We found that nasal IgA and IgG have different kinetics after infection, with IgA peaking first. Although vaccination results in low nasal IgA, IgG induction persists for up to 180 days post-vaccination. This research highlights the importance of studying mucosal responses in addition to systemic responses to respiratory infections. The methods described herein can be used to further mucosal vaccine development by giving us a better understanding of immunity at the nasal epithelium providing a simpler, alternative clinical practice to studying mucosal responses to infection.

Diet-induced obesity impacts influenza disease severity and transmission dynamics in ferrets.

Obesity, and the associated metabolic syndrome, is a risk factor for increased disease severity with a variety of infectious agents, including influenza virus. Yet the mechanisms are only partially understood. As the number of people, particularly children, living with obesity continues to rise, it is critical to understand the role of host status on disease pathogenesis. In these studies, we use a novel diet-induced obese ferret model and new tools to demonstrate that like humans, obesity resulted in significant changes to the lung microenvironment leading to increased clinical disease and viral spread to the lower respiratory tract. The decreased antiviral responses also resulted in obese animals shedding higher infectious virus for longer making them more likely to transmit to contacts. These data suggest the obese ferret model may be crucial to understanding obesity's impact on influenza disease severity and community transmission, and a key tool for therapeutic and intervention development for this high-risk population. Teaser: A new ferret model and tools to explore obesity's impact on respiratory virus infection, susceptibility, and community transmission.

Receptor-binding specificity of the human parainfluenza virus type 1 hemagglutinin-neuraminidase glycoprotein.

The hemagglutinin-neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewis(x) motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin.

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.

Novel Low Pathogenic Avian Influenza H6N1 in Backyard Chicken in Easter Island (Rapa Nui), Chilean Polynesia.

Little is known about the prevalence of avian influenza viruses (AIV) in wildlife and domestic animals in Polynesia. Here, we present the results of active AIV surveillance performed during two sampling seasons in 2019 on Easter Island (Rapa Nui). Tracheal and cloacal swabs as well as sera samples were obtained from domestic backyard poultry, while fresh faeces were collected from wild birds. In addition to detecting antibodies against AIV in 46% of the domestic chickens in backyard production systems tested, we isolated a novel low pathogenic H6N1 virus from a chicken. Phylogenetic analysis of all genetic segments revealed that the virus was closely related to AIV's circulating in South America. Our analysis showed different geographical origins of the genetic segments, with the PA, HA, NA, NP, and MP gene segments coming from central Chile and the PB2, PB1, and NS being closely related to viruses isolated in Argentina. While the route of introduction can only be speculated, our analysis shows the persistence and independent evolution of this strain in the island since its putative introduction between 2015 and 2016. The results of this research are the first evidence of AIV circulation in domestic birds on a Polynesian island and increase our understanding of AIV ecology in region, warranting further surveillance on Rapa Nui and beyond.

Evidence of influenza infection in dogs and cats in central Chile.

As companion animals, dogs and cats live in close contact with humans, generating the possibility of interspecies pathogen transmission events. Equine origin H3N8 and avian origin H5N1 influenza virus have been reported in dogs and cats respectively since 2004 with outbreaks associated with different strains recorded for both species in Asia and North America. To date, there have been no reports of influenza viruses from companion animals in South America. To fill this gap in knowledge, we performed active epidemiological surveillance in shelters that received abandoned animals, backyard production systems and veterinary clinics between May 2017 and January 2019 to estimate the burden of influenza infection in cats and dogs in the central region of Chile. Blood samples, oropharyngeal swabs or both were collected for influenza A virus detection by RT-qPCR, NP-ELISA, and hemagglutination inhibition assay. Logistic regression models were performed to assess the association between NP-ELISA-positivity and variables including sex and animal origin. The percentage of ELISA-positive samples was 43.5 % (95 % CI: 37.0-50.1) and 23.3 % (95 % CI: 10.6-42.7) for dogs and cats, respectively. No association was found between NP-ELISA results and sex or animal origin for either dogs or cats. Two ELISA positive samples showed hemagglutination inhibition titers against pandemic H1N1 influenza. One dog sample tested positive by RT-qPCR, indicating an overall RT-qPCR positivity in dogs of 1.1 % (95 % CI: 0.05-6.7). None of the tested cat samples were positive by this assay.