Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots.

Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay and a commercial real-time PCR-based inhibition assay (NAB-Sure™). Results: DBS eluates from convalescent individuals were compatible with the spike-ACE2 inhibition assays, but not cell-based pseudovirus assays or PRNT. However, the insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.

Direct Evidence of Powassan Virus Vertical Transmission in Ixodes scapularis in Nature.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.

Active Surveillance of Powassan Virus in Massachusetts Ixodes scapularis Ticks, Comparing Detection Using a New Triplex Real-Time PCR Assay with a Luminex Vector-Borne Panel.

Powassan virus is an emerging tick-borne pathogen capable of causing severe neuroinvasive disease. As the incidence of human Powassan virus grows both in magnitude and geographical range, the development of sensitive detection methods for diagnostics and surveillance is critical. In this study, a Taqman-based triplex real-time PCR assay was developed for the simultaneous and quantitative detection of Powassan virus and Powassan virus lineage II (deer tick virus) in Ixodes scapularis ticks. An exon-exon junction internal control was built-in to allow for accurate detection of RNA quality and the failure of RNA extraction. The newly developed assay was also applied to survey deer tick virus in tick populations at 13 sites on Cape Cod and Martha's Vineyard Island in Massachusetts. The assay's performance was compared with the Luminex xMAP MultiFLEX Vector-borne Panel 2. The results suggested that the real-time PCR method was more sensitive. Powassan virus infection rates among ticks collected from these highly endemic tick areas ranged from 0.0 to 10.4%, highlighting the fine-scale geographic variations in deer tick virus presence in this region. Looking forward, our PCR assay could be adopted in other Powassan virus surveillance systems.

Surveillance and Genetic Analysis of Jamestown Canyon Virus in New York State: 2001-2022.

Jamestown Canyon virus (JCV) (Peribunyavirdae; Orthobunyavirus) is a mosquito-borne pathogen endemic to North America. The genome is composed of three segmented negative-sense RNA fragments designated as small, medium, and large. Jamestown Canyon virus is an emerging threat to public health, and infection in humans can cause severe neurological diseases, including encephalitis and meningitis. We report JCV mosquito surveillance data from 2001 to 2022 in New York state. Jamestown Canyon virus was detected in 12 mosquito species, with the greatest prevalence in Aedes canadensis and Anopheles punctipennis. Detection fluctuated annually, with the highest levels recorded in 2020. Overall, JCV infection rates were significantly greater from 2012 to 2022 compared with 2001 to 2011. Full-genome sequencing and phylogenetic analysis were also performed with representative JCV isolates collected from 2003 to 2022. These data demonstrated the circulation of numerous genetic variants, broad geographic separation, and the first identification of lineage B JCV in New York state in 2022.

Emerging tickborne viruses vectored by Amblyomma americanum (Ixodida: Ixodidae): Heartland and Bourbon viruses.

Heartland (HRTV) and Bourbon (BRBV) viruses are newly identified tick-borne viruses, isolated from serious clinical cases in 2009 and 2014, respectively. Both viruses originated in the lower Midwest United States near the border of Missouri and Kansas, cause similar disease manifestations, and are presumably vectored by the same tick species, Amblyomma americanum Linnaeus (Ixodida: Ixodidae). In this article, we provide a current review of HRTV and BRBV, including the virology, epidemiology, and ecology of the viruses with an emphasis on the tick vector. We touch on current challenges of vector control and surveillance, and we discuss future directions in the study of these emergent pathogens.

Maintenance of a host-specific minority mutation in the West Nile virus NS3.

West Nile virus (WNV), the most prevalent arthropod-borne virus (arbovirus) in the United States, is maintained in a cycle between Culex spp. mosquitoes and birds. Arboviruses exist within hosts and vectors as a diverse set of closely related genotypes. In theory, this genetic diversity can facilitate adaptation to distinct environments during host cycling, yet host-specific fitness of minority genotypes has not been assessed. Utilizing WNV deep-sequencing data, we previously identified a naturally occurring, mosquito-biased substitution, NS3 P319L. Using both cell culture and experimental infection in natural hosts, we demonstrated that this substitution confers attenuation in vertebrate hosts and increased transmissibility by mosquitoes. Biochemical assays demonstrated temperature-sensitive ATPase activity consistent with host-specific phenotypes. Together these data confirm the maintenance of host-specific minority variants in arbovirus mutant swarms, suggest a unique role for NS3 in viral fitness, and demonstrate that intrahost sequence data can inform mechanisms of host-specific adaptation.

Dynamics of eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, similar to previous years, cases were driven by multiple independent but short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019, although the ecology of EEEV is complex and further data is required to explore these in more detail. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Diversification of Bourbon Virus in New York State.

Bourbon virus (BRBV, family Orthomyxoviridae) is a tickborne virus recently detected in the United States (US). BRBV was first identified from a fatal human case in 2014 in Bourbon County, Kansas. Enhanced surveillance in Kansas and Missouri implicated Amblyomma americanum as the primary vector for BRBV. Historically, BRBV was only detected in the lower midwestern US, but since 2020 it has been reported in North Carolina, Virginia, New Jersey, and New York State (NYS). This study aimed to elucidate genetic and phenotypic characteristics of BRBV strains from NYS through whole genome sequencing and the assessment of replication kinetics in mammalian cultures and A. americanum nymphs. Sequence analysis revealed the existence of two divergent BRBV clades circulating in NYS. BRBV NY21-2143 is closely related to the midwestern BRBV strains but has unique substitutions in the glycoprotein. Two other NYS BRBV strains, BRBV NY21-1814 and BRBV NY21-2666, form a distinct clade unique from previously sequenced BRBV strains. Phenotypic diversification was also detected in NYS BRBV strains compared to each other and midwestern BRBV strains, with BRBV NY21-2143 displaying attenuation in rodent-derived cell culture and a fitness advantage in experimentally infected A. americanum. These data suggest genetic and phenotypic diversification of emergent BRBV strains circulating in NYS that could contribute to increased spread of BRBV in the northeastern US.

Structural evolution of an immune evasion determinant shapes pathogen host tropism.

Modern infectious disease outbreaks often involve changes in host tropism, the preferential adaptation of pathogens to specific hosts. The Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the molecular mechanisms of host tropism, because different variants of these tick-transmitted bacteria are distinctly maintained in rodents or bird reservoir hosts. To survive in hosts and escape complement-mediated immune clearance, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants differ in human FH-binding ability. Together with the FH polymorphisms between vertebrate hosts, these findings suggest that minor sequence variation in this bacterial outer surface protein may confer dramatic differences in host-specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, and identifying minor variation localized in the FH-binding interface yielding bird and rodent FH-specific binding activity that impacts infectivity. Swapping the divergent region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. We further linked these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ phylogenetic lineages, elucidating evolutionary mechanisms driving host tropism emergence. Our multidisciplinary work provides a novel molecular basis for how a single, short protein motif could greatly modulate pathogen host tropism.

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 and 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 United States mostly following a south-to-north pattern, with a weighted lineage dispersal velocity of ~3 km/y. 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 United States.

Dynamics of Eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans, and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, like previous years, cases were driven by frequent short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Bourbon Virus Transmission, New York, USA.

In July 2019, Bourbon virus RNA was detected in an Amblyomma americanum tick removed from a resident of Long Island, New York, USA. Tick infection and white-tailed deer (Odocoileus virginianus) serosurvey results demonstrate active transmission in New York, especially Suffolk County, emphasizing a need for surveillance anywhere A. americanum ticks are reported.

Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants.

Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.

Phylogenomic Diversity Elucidates Mechanistic Insights into Lyme Borreliae-Host Association.

Host association-the selective adaptation of pathogens to specific host species-evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi (Bb), the main LD pathogen in North America-a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens' ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host-adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host-association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests that several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host-adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCE Host association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD)-causing agent, B. burgdorferi (Bb), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host-association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC, with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Cellular and immunological mechanisms influence host-adapted phenotypes in a vector-borne microparasite.

Predicting pathogen emergence and spillover risk requires understanding the determinants of a pathogens' host range and the traits involved in host competence. While host competence is often considered a fixed species-specific trait, it may be variable if pathogens diversify across hosts. Balancing selection can lead to maintenance of pathogen polymorphisms (multiple-niche-polymorphism; MNP). The causative agent of Lyme disease, Borrelia burgdorferi (Bb), provides a model to study the evolution of host adaptation, as some Bb strains defined by their outer surface protein C (ospC) genotype, are widespread in white-footed mice and others are associated with non-rodent vertebrates (e.g. birds). To identify the mechanisms underlying potential strain × host adaptation, we infected American robins and white-footed mice, with three Bb strains of different ospC genotypes. Bb burdens varied by strain in a host-dependent fashion, and strain persistence in hosts largely corresponded to Bb survival at early infection stages and with transmission to larvae (i.e. fitness). Early survival phenotypes are associated with cell adhesion, complement evasion and/or inflammatory and antibody-mediated removal of Bb, suggesting directional selective pressure for host adaptation and the potential role of MNP in maintaining OspC diversity. Our findings will guide future investigations to inform eco-evolutionary models of host adaptation for microparasites.

Role of Anopheles Mosquitoes in Cache Valley Virus Lineage Displacement, New York, USA.

Cache Valley virus (CVV) is a mosquitoborne virus that infects livestock and humans. We report results of surveillance for CVV in New York, USA, during 2000-2016; full-genome analysis of selected CVV isolates from sheep, horse, humans, and mosquitoes from New York and Canada; and phenotypic characterization of selected strains. We calculated infection rates by using the maximum-likelihood estimation method by year, region, month, and mosquito species. The highest maximum-likelihood estimations were for Anopheles spp. mosquitoes. Our phylogenetic analysis identified 2 lineages and found evidence of segment reassortment. Furthermore, our data suggest displacement of CVV lineage 1 by lineage 2 in New York and Canada. Finally, we showed increased vector competence of An. quadrimaculatus mosquitoes for lineage 2 strains of CVV compared with lineage 1 strains.

Heartland Virus Transmission, Suffolk County, New York, USA.

During 2018, Heartland virus RNA was detected in an Amblyomma americanum tick removed from a resident of Suffolk County, New York, USA. The person showed seroconversion. Tick surveillance and white-tailed deer (Odocoileus virginianus) serosurveys showed widespread distribution in Suffolk County, emphasizing a need for disease surveillance anywhere A. americanum ticks are established or emerging.

A fatal case report of antibody-dependent enhancement of dengue virus type 1 following remote Zika virus infection.

BACKGROUND: Dengue virus (DENV) is endemic in many parts of the world. Antibody dependent enhancement (ADE) in DENV infections occurs when a person with primary immunity is infected by a second, different DENV strain. Antibodies to Zika virus (ZIKV), which emerged in the Western Hemisphere in 2015, are cross reactive with DENV and theoretically could provoke ADE in a DENV naïve individual. CASE PRESENTATION: DENV infection was suspected in a child who had recently returned from a one-month stay in the Dominican Republic. The child presented with fever, vomiting, abdominal pain, and in hypovolemic shock. Volume and pressor resuscitation were unsuccessful, and the child died less than 24 h after hospitalization. Laboratory results suggested an early acute first DENV infection since serum, plasma, and spinal fluid had DENV1 detected by polymerase chain reaction (PCR), yet the serum lacked IgG antibodies to DENV nonstructural protein 1 (NS1) of all four DENV serotypes. This acute DENV infection occurred in the presence of a remote ZIKV infection as determined by antibodies to ZIKV NS1 envelope by multiplex microsphere immunoassay and an exceptionally high plaque reduction neutralization titer to ZIKV. ZIKV IgG avidity index was high, confirming a past infection. DENV1 RNA was detected in all ten organs and tissues examined by PCR. The severe and fatal complications reported here suggest that a remote ZIKV infection may provoke an exaggerated immune response leading to hypovolemic shock when primarily infected by DENV1. CONCLUSION: We report the first known patient in the United States with a rapidly progressive and fatal case of travel-associated DENV in which prior exposure to ZIKV likely played a role in triggering an ADE phenomenon. This association of prior ZIKV immunity and subsequent new dengue infection is a worrisome phenomenon and an important contribution to the body of knowledge on immunity to flaviviruses.