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.

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.

Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system.

Pathogens possess the ability to adapt and survive in some host species but not in others-an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations.

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.

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.

Temporal Analysis of Serial Donations Reveals Decrease in Neutralizing Capacity and Justifies Revised Qualifying Criteria for Coronavirus Disease 2019 Convalescent Plasma.

BACKGROUND: Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) received an Emergency Use Authorization by the US Food and Drug Administration (FDA). CCP with a signal-to-cutoff ratio of ≥12 using the Ortho VITROS severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) test (OVSARS2IgG) is permitted to be labeled "high titer." Little is known about the relationship between OVSARS2IgG ratio and neutralizing capacity of plasma/sera against genuine SARS-CoV-2. METHODS: Nine hundred eighty-one samples from 196 repeat CCP donors 0-119 days post-initial donation (DPID) were analyzed. Neutralizing capacity was assessed for 50% (PRNT50) and 90% (PRNT90) reduction of infectious virus using the gold standard plaque reduction neutralization test (PRNT). A subset of 91 donations was evaluated by OVSARS2IgG and compared to PRNT titers for diagnostic accuracy. RESULTS: Of donations, 32.7%/79.5% (PRNT90/PRNT50) met a 1:80 titer initially but only 14.0%/48.8% (PRNT90/PRNT50) met this cutoff ≥85 DPID. Correlation of OVSARS2IgG results to neutralizing capacity allowed extrapolation to CCP therapy results. CCP with OVSARS2IgG ratios equivalent to a therapeutically beneficial group had neutralizing titers of ≥1:640 (PRNT50) and/or ≥1:80 (PRNT90). Specificity and positive predictive value of the OVSARS2IgG for qualifying highly neutralizing CCP was optimal using ratios significantly greater than the FDA cutoff. CONCLUSIONS: This information provides a basis for refining the recommended properties of CCP used to treat COVID-19.

Neutralizing Antibody Responses in COVID-19 Convalescent Sera.

Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration's (FDA) guidelines for convalescent plasma initially recommended target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at moderate (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity significantly increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was approximately 93% (PRNT50) and approximately 54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 enzyme-linked immunosorbent assay titers) showed maximal activity, but not all high-titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.

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.

Blood treatment of Lyme borreliae demonstrates the mechanism of CspZ-mediated complement evasion to promote systemic infection in vertebrate hosts.

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne disease in the United States and Europe. The spirochetes are transmitted from mammalian and avian reservoir hosts to humans via ticks. Following tick bites, spirochetes colonize the host skin and then disseminate haematogenously to various organs, a process that requires this pathogen to evade host complement, an innate immune defence system. CspZ, a spirochete surface protein, facilitates resistance to complement-mediated killing in vitro by binding to the complement regulator, factor H (FH). Low expression levels of CspZ in spirochetes cultivated in vitro or during initiation of infection in vivo have been a major hurdle in delineating the role of this protein in pathogenesis. Here, we show that treatment of B. burgdorferi with human blood induces CspZ production and enhances resistance to complement. By contrast, a cspZ-deficient mutant and a strain that expressed an FH-nonbinding CspZ variant were impaired in their ability to cause bacteraemia and colonize tissues of mice or quail; virulence of these mutants was however restored in complement C3-deficient mice. These novel findings suggest that FH binding to CspZ facilitates B. burgdorferi complement evasion in vivo and promotes systemic infection in vertebrate hosts.

Co-infections in Persons with Early Lyme Disease, New York, USA.

In certain regions of New York state, USA, Ixodes scapularis ticks can potentially transmit 4 pathogens in addition to Borrelia burgdorferi: Anaplasma phagocytophilum, Babesia microti, Borrelia miyamotoi, and the deer tick virus subtype of Powassan virus. In a prospective study, we systematically evaluated 52 adult patients with erythema migrans, the most common clinical manifestation of B. burgdorferi infection (Lyme disease), who had not received treatment for Lyme disease. We used serologic testing to evaluate these patients for evidence of co-infection with any of the 4 other tickborne pathogens. Evidence of co-infection was found for B. microti only; 4-6 patients were co-infected with Babesia microti. Nearly 90% of the patients evaluated had no evidence of co-infection. Our finding of B. microti co-infection documents the increasing clinical relevance of this emerging infection.

Use of the immunoglobulin G avidity assay to differentiate between recent Zika and past dengue virus infections.

Zika (ZIKV) and dengue (DENV) virus infections elicit a robust but cross-reactive antibody response against the viral envelope protein, while antibody responses against non-structural proteins (NS) are more virus specific. Building on this premise, we have previously developed a flavivirus multiplex microsphere immunoassay (MIA) for the serologic diagnosis of ZIKV and DENV infections. This assay significantly improved diagnostic accuracy; however, MIA could not differentiate more recent from past infections, which still represents a major diagnostic challenge. Therefore, an immunoglobulin G (IgG) based avidity assay was developed and its diagnostic performance evaluated. Specimens from New York State residents were submitted to the Wadsworth Center New York State Department of Health (NYSDOH) for routine clinical testing by Zika IgM ELISA and plaque reduction neutralization test (PRNT). Using our previously developed flavivirus MIA as a platform, we developed an IgG avidity assay to discriminate recent ZIKV from past DENV infections. Zika IgM positive specimens had an average Zika IgG avidity index of 14.8% (95% CI: 11.0-18.4%), while Zika IgM negative but flavivirus MIA and PRNT positive samples had an average Zika IgG avidity index of 34.9% (95% CI: 31.1-38.7%). Specimens positive for dengue antibodies by flavivirus MIA and PRNT had an average dengue IgG avidity index of 68.7% (95% CI: 62.7-75.0%). The IgG avidity assay accurately distinguished recent ZIKV from past DENV infections in patients who traveled to dengue endemic regions. This assay could be very useful in patients with high risk of Zika complications such as pregnant women and monitoring immune responses in vaccine trials.

Development of Zika Virus Serological Testing Strategies in New York State.

The recent outbreak of Zika virus (ZIKV) in the Americas has challenged diagnostic laboratory testing strategies. At the Wadsworth Center, ZIKV serological testing was performed for over 10,000 specimens, using a combination of an enzyme-linked immunosorbent assay (ELISA) for IgM antibodies (Abs) to ZIKV, a polyvalent microsphere immunoassay (MIA) to detect Abs broadly reactive with flaviviruses, and a plaque reduction neutralization test (PRNT) for further testing. Overall, 42% of patients showed serological evidence of flavivirus infection (primarily past dengue virus [DENV] infection), while 7% possessed IgM Abs to ZIKV and/or DENV. ZIKV IgM Abs typically arose within 3 to 4 days, with only one instance of duration beyond 100 days after reported symptoms. PRNT analysis of 826 IgM-positive specimens showed 7% positive neutralization to ZIKV alone, 9% to DENV alone, and 85% to both ZIKV and DENV. Thus, the extensive Ab cross-reactivity among flaviviruses significantly reduced the value of performing PRNT analysis, especially when a traditional paired serum algorithm with viral neutralization titering was used. Nevertheless, the finding of a negative ZIKV result by PRNT was invaluable for reassuring both physicians and patients. The MIA detected both IgM and IgG, which enabled us to identify patients who presented without IgM anti-ZIKV Abs but still had ZIKV-specific neutralizing Abs. On the basis of these results, a new algorithm, which included an IgM Ab capture (MAC)-ELISA to detect recent infection, a flavivirus MIA to identify patients no longer producing IgM, and a single-dilution PRNT for ZIKV exclusion and occasional discrimination of ZIKV and DENV, was implemented.

Serologic Evidence of Powassan Virus Infection in Patients with Suspected Lyme Disease1.

Powassan virus (POWV) lineage II is an emerging tickborne flavivirus with an unknown seroprevalence in humans. In a Lyme disease-endemic area, we examined the seroreactivity to POWV in 2 patient cohorts and described the clinical features of the POWV-seroreactive patients. POWV disease might be less neuroinvasive than previously thought.

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.

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.

Diagnosis of acute deer tick virus encephalitis.

BACKGROUND: Deer tick virus (DTV) is a tick-borne flavivirus that has only recently been appreciated as a cause of viral encephalitis. We describe the clinical presentation of a patient who had DTV encephalitis diagnosed before death and survived for 8 months despite severe neurologic dysfunction. METHODS: Diagnosis was made from a cerebrospinal fluid specimen, using a flavivirus-specific polymerase chain-reaction assay followed by sequence confirmation, and the phylogeny was analyzed. Serologic testing, including plaque reduction neutralization testing, was also performed. RESULTS: Molecular analysis indicated that the virus was closely related to 2 strains of DTV that had been detected in Ixodes scapularis ticks from Massachusetts in 1996 and in the brain of a patient from New York in 2007. CONCLUSIONS: DTV encephalitis should be considered in the differential diagnosis of encephalitis in geographic areas that are endemic for Lyme disease.

Potential role of deer tick virus in Powassan encephalitis cases in Lyme disease-endemic areas of New York, U.S.A.

Powassan virus, a member of the tick-borne encephalitis group of flaviviruses, encompasses 2 lineages with separate enzootic cycles. The prototype lineage of Powassan virus (POWV) is principally maintained between Ixodes cookei ticks and the groundhog (Marmota momax) or striped skunk (Mephitis mephitis), whereas the deer tick virus (DTV) lineage is believed to be maintained between Ixodes scapularis ticks and the white-footed mouse (Peromyscus leucopus). We report 14 cases of Powassan encephalitis from New York during 2004-2012. Ten (72%) of the patients were residents of the Lower Hudson Valley, a Lyme disease-endemic area in which I. scapularis ticks account for most human tick bites. This finding suggests that many of these cases were caused by DTV rather than POWV. In 2 patients, DTV infection was confirmed by genetic sequencing. As molecular testing becomes increasingly available, more cases of Powassan encephalitis may be determined to be attributable to the DTV lineage.