Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual.

Prolonged infections in immunocompromised individuals may be a source for novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, particularly when both the immune system and antiviral therapy fail to clear the infection and enable within-host evolution. Here we describe a 486-day case of SARS-CoV-2 infection in an immunocompromised individual. Following monotherapy with the monoclonal antibody Bamlanivimab, the individual's virus acquired resistance, likely via the earliest known occurrence of Spike amino acid variant E484T. Recently, E484T has arisen again as a derivative of E484A in the Omicron Variant of Concern, supporting the hypothesis that prolonged infections can give rise to novel variants long before they become prevalent in the human population.

Avian H7N9 influenza viruses are evolutionarily constrained by stochastic processes during replication and transmission in mammals.

H7N9 avian influenza viruses (AIVs) have caused over 1,500 documented human infections since emerging in 2013. Although wild-type H7N9 AIVs can be transmitted by respiratory droplets in ferrets, they have not yet caused widespread outbreaks in humans. Previous studies have revealed molecular determinants of H7N9 AIV host switching, but little is known about potential evolutionary constraints on this process. Here, we compare patterns of sequence evolution for H7N9 AIV and mammalian H1N1 viruses during replication and transmission in ferrets. We show that three main factors-purifying selection, stochasticity, and very narrow transmission bottlenecks-combine to severely constrain the ability of H7N9 AIV to effectively adapt to mammalian hosts in isolated, acute spillover events. We find rare evidence of natural selection favoring new, potentially mammal-adapting mutations within ferrets but no evidence of natural selection acting during transmission. We conclude that human-adapted H7N9 viruses are unlikely to emerge during typical spillover infections. Our findings are instead consistent with a model in which the emergence of a human-transmissible virus would be a rare and unpredictable, though highly consequential, 'jackpot' event. Strategies to control the total number of spillover infections will limit opportunities for the virus to win this evolutionary lottery.

Influenza A virus undergoes compartmentalized replication in vivo dominated by stochastic bottlenecks.

Transmission of influenza A viruses (IAV) between hosts is subject to numerous physical and biological barriers that impose genetic bottlenecks, constraining viral diversity and adaptation. The bottlenecks within hosts and their potential impacts on evolutionary pathways taken during infection are poorly understood. To address this, we created highly diverse IAV libraries bearing molecular barcodes on two gene segments, enabling high-resolution tracking and quantification of unique virus lineages within hosts. Here we show that IAV infection in lungs is characterized by multiple within-host bottlenecks that result in "islands" of infection in lung lobes, each with genetically distinct populations. We perform site-specific inoculation of barcoded IAV in the upper respiratory tract of ferrets and track viral diversity as infection spreads to the trachea and lungs. We detect extensive compartmentalization of discrete populations within lung lobes. Bottleneck events and localized replication stochastically sample individual viruses from the upper respiratory tract or the trachea that become the dominant genotype in a particular lobe. These populations are shaped strongly by founder effects, with limited evidence for positive selection. The segregated sites of replication highlight the jackpot-style events that contribute to within-host influenza virus evolution and may account for low rates of intrahost adaptation.

Anti-membrane Antibodies Persist at Least One Year and Discriminate Between Past Coronavirus Disease 2019 Infection and Vaccination.

BACKGROUND: The consequences of past coronavirus disease 2019 (COVID-19) infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. METHODS: We evaluated anti-membrane antibodies in COVID-19 naive, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. RESULTS: We found that anti-membrane antibodies persist for at least 1 year and are a sensitive and specific marker of past COVID-19 infection. CONCLUSIONS: Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naive states to advance public health and research.

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant.

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

Anti-membrane and anti-spike antibodies are long-lasting and together discriminate between past COVID-19 infection and vaccination.

The consequences of past COVID-19 infection for personal health and long-term population immunity are only starting to be revealed. Unfortunately, detecting past infection is currently a challenge, limiting clinical and research endeavors. Widely available anti-SARS-CoV-2 antibody tests cannot differentiate between past infection and vaccination given vaccine-induced anti-spike antibodies and the rapid loss of infection-induced anti-nucleocapsid antibodies. Anti-membrane antibodies develop after COVID-19, but their long-term persistence is unknown. Here, we demonstrate that anti-membrane IgG is a sensitive and specific marker of past COVID-19 infection and persists at least one year. We also confirm that anti-receptor binding domain (RBD) Ig is a long-lasting, sensitive, and specific marker of past infection and vaccination, while anti-nucleocapsid IgG lacks specificity and quickly declines after COVID-19. Thus, a combination of anti-membrane and anti-RBD antibodies can accurately differentiate between distant COVID-19 infection, vaccination, and naïve states to advance public health, individual healthcare, and research goals.

Evidence of Early Household Transmission of SARS-CoV-2 Involving a School-aged Child.

INTRODUCTION: Little is known about the role of school-aged children and household transmission at the start of the SARS-CoV-2 pandemic. To evaluate for SARS-CoV-2 in school-aged children and assess household transmission, we performed reverse transcription polymerase chain reaction on 670 archived specimens that were collected between September 1, 2019 and June 30, 2020 as part of a community-based study. CASE PRESENTATION: A single SARS-CoV-2 case was detected in an 11-year-old girl on March 18, 2020, resulting in very low prevalence (0.15% [95% CI, 0.03-0.84]) in this population. This case was associated with SARS-CoV-2 detection in all other household members. Symptoms were reported as mild to moderate. Whole genome sequencing supported household transmission of near-identical viruses within the 19B clade. DISCUSSION: This case represents the earliest known household cluster of SARS-CoV2 in Wisconsin. CONCLUSION: This case suggests that household transmission associated with school-aged children may have contributed to wide seeding across populations.

Interventions to Disrupt Coronavirus Disease Transmission at a University, Wisconsin, USA, August-October 2020.

University settings have demonstrated potential for coronavirus disease (COVID-19) outbreaks; they combine congregate living, substantial social activity, and a young population predisposed to mild illness. Using genomic and epidemiologic data, we describe a COVID-19 outbreak at the University of Wisconsin-Madison, Madison, Wisconsin, USA. During August-October 2020, a total of 3,485 students, including 856/6,162 students living in dormitories, tested positive. Case counts began rising during move-in week, August 25-31, 2020, then rose rapidly during September 1-11, 2020. The university initiated multiple prevention efforts, including quarantining 2 dormitories; a subsequent decline in cases was observed. Genomic surveillance of cases from Dane County, in which the university is located, did not find evidence of transmission from a large cluster of cases in the 2 quarantined dorms during the outbreak. Coordinated implementation of prevention measures can reduce COVID-19 spread in university settings and may limit spillover to the surrounding community.

Acute SARS-CoV-2 infections harbor limited within-host diversity and transmit via tight transmission bottlenecks.

The emergence of divergent SARS-CoV-2 lineages has raised concern that novel variants eliciting immune escape or the ability to displace circulating lineages could emerge within individual hosts. Though growing evidence suggests that novel variants arise during prolonged infections, most infections are acute. Understanding how efficiently variants emerge and transmit among acutely-infected hosts is therefore critical for predicting the pace of long-term SARS-CoV-2 evolution. To characterize how within-host diversity is generated and propagated, we combine extensive laboratory and bioinformatic controls with metrics of within- and between-host diversity to 133 SARS-CoV-2 genomes from acutely-infected individuals. We find that within-host diversity is low and transmission bottlenecks are narrow, with very few viruses founding most infections. Within-host variants are rarely transmitted, even among individuals within the same household, and are rarely detected along phylogenetically linked infections in the broader community. These findings suggest that most variation generated within-host is lost during transmission.

Previous exposure to dengue virus is associated with increased Zika virus burden at the maternal-fetal interface in rhesus macaques.

Concerns have arisen that pre-existing immunity to dengue virus (DENV) could enhance Zika virus (ZIKV) disease, due to the homology between ZIKV and DENV and the observation of antibody-dependent enhancement (ADE) among DENV serotypes. To date, no study has examined the impact of pre-existing DENV immunity on ZIKV pathogenesis during pregnancy in a translational non-human primate model. Here we show that macaques with a prior DENV-2 exposure had a higher burden of ZIKV vRNA in maternal-fetal interface tissues as compared to DENV-naive macaques. However, pre-existing DENV immunity had no detectable impact on ZIKV replication kinetics in maternal plasma, and all pregnancies progressed to term without adverse outcomes or gross fetal abnormalities detectable at delivery. Understanding the risks of ADE to pregnant women worldwide is critical as vaccines against DENV and ZIKV are developed and licensed and as DENV and ZIKV continue to circulate.

African-Lineage Zika Virus Replication Dynamics and Maternal-Fetal Interface Infection in Pregnant Rhesus Macaques.

Following the Zika virus (ZIKV) outbreak in the Americas, ZIKV was causally associated with microcephaly and a range of neurological and developmental symptoms, termed congenital Zika syndrome (CZS). The viruses responsible for this outbreak belonged to the Asian lineage of ZIKV. However, in vitro and in vivo studies assessing the pathogenesis of African-lineage ZIKV demonstrated that African-lineage isolates often replicated to high titers and caused more-severe pathology than Asian-lineage isolates. To date, the pathogenesis of African-lineage ZIKV in a translational model, particularly during pregnancy, has not been rigorously characterized. Here, we infected four pregnant rhesus macaques with a low-passage-number strain of African-lineage ZIKV and compared its pathogenesis to those for a cohort of four pregnant rhesus macaques infected with an Asian-lineage isolate and a cohort of mock-inoculated controls. The viral replication kinetics for the two experimental groups were not significantly different, and both groups developed robust neutralizing antibody titers above levels considered to be protective. There was no evidence of significant fetal head growth restriction or gross fetal harm at delivery (1 to 1.5 weeks prior to full term) in either group. However, a significantly higher burden of ZIKV viral RNA (vRNA) was found in the maternal-fetal interface tissues of the macaques exposed to an African-lineage isolate. Our findings suggest that ZIKV of any genetic lineage poses a threat to pregnant individuals and their infants. IMPORTANCE ZIKV was first identified in 1947 in Africa, but most of our knowledge of ZIKV is based on studies of the distinct Asian genetic lineage, which caused the outbreak in the Americas in 2015 to 2016. In its most recent update, the WHO stated that improved understanding of African-lineage ZIKV pathogenesis during pregnancy must be a priority. The recent detection of African-lineage isolates in Brazil underscores the need to understand the impact of these viruses. Here, we provide the first comprehensive assessment of African-lineage ZIKV infection during pregnancy in a translational nonhuman primate model. We show that African-lineage isolates replicate with kinetics similar to those of Asian-lineage isolates and can infect the placenta. However, there was no evidence of more-severe outcomes with African-lineage isolates. Our results highlight both the threat that African-lineage ZIKV poses to pregnant individuals and their infants and the need for epidemiological and translational in vivo studies with African-lineage ZIKV.

Severe Acute Respiratory Syndrome Coronavirus 2 Transmission in Intercollegiate Athletics Not Fully Mitigated With Daily Antigen Testing.

BACKGROUND: High-frequency, rapid-turnaround severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, 2 SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester, despite mandatory directly observed daily antigen testing. METHODS: During the fall 2020 semester, athletes and staff in both programs were tested daily using Quidel's Sofia SARS Antigen Fluorescent Immunoassay, with positive antigen results requiring confirmatory testing with real-time reverse-transcription polymerase chain reaction. We used genomic sequencing to investigate transmission dynamics in these 2 outbreaks. RESULTS: In the first outbreak, 32 confirmed cases occurred within a university athletics program after the index patient attended a meeting while infectious, despite a negative antigen test on the day of the meeting. Among isolates sequenced from that outbreak, 24 (92%) of 26 were closely related, suggesting sustained transmission following an initial introduction event. In the second outbreak, 12 confirmed cases occurred among athletes from 2 university programs that faced each other in an athletic competition, despite receipt of negative antigen test results on the day of the competition. Sequences from both teams were closely related and distinct from viruses circulating in the community for team 1, suggesting transmission during intercollegiate competition in the community for team 2. CONCLUSIONS: These findings suggest that antigen testing alone, even when mandated and directly observed, may not be sufficient as an intervention to prevent SARS-CoV-2 outbreaks in congregate settings, and they highlight the importance of vaccination to prevent SARS-CoV-2 outbreak in congregate settings.

Viral Sequencing to Investigate Sources of SARS-CoV-2 Infection in US Healthcare Personnel.

BACKGROUND: Healthcare personnel (HCP) are at increased risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We posit that current infection control guidelines generally protect HCP from SARS-CoV-2 infection in a healthcare setting. METHODS: In this retrospective case series, we used viral genomics to investigate the likely source of SARS-CoV-2 infection in HCP at a major academic medical institution in the Upper Midwest of the United States between 25 March and 27 December 2020. We obtained limited epidemiological data through informal interviews and review of the electronic health record and combined this information with healthcare-associated viral sequences and viral sequences collected in the broader community to infer the most likely source of infection in HCP. RESULTS: We investigated SARS-CoV-2 infection clusters involving 95 HCP and 137 possible patient contact sequences. The majority of HCP infections could not be linked to a patient or coworker (55 of 95 [57.9%]) and were genetically similar to viruses circulating concurrently in the community. We found that 10.5% of HCP infections (10 of 95) could be traced to a coworker. Strikingly, only 4.2% (4 of 95) could be traced to a patient source. CONCLUSIONS: Infections among HCP add further strain to the healthcare system and put patients, HCP, and communities at risk. We found no evidence for healthcare-associated transmission in the majority of HCP infections evaluated. Although we cannot rule out the possibility of cryptic healthcare-associated transmission, it appears that HCP most commonly become infected with SARS-CoV-2 via community exposure. This emphasizes the ongoing importance of mask wearing, physical distancing, robust testing programs, and rapid distribution of vaccines.

SARS-CoV-2 transmission in intercollegiate athletics not fully mitigated with daily antigen testing.

BACKGROUND: High frequency, rapid turnaround SARS-CoV-2 testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, two SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester despite mandatory directly observed daily antigen testing. METHODS: During the fall 2020 semester, athletes and staff in both programs were tested daily using Quidel's Sofia SARS Antigen Fluorescent Immunoassay (FIA), with positive antigen results requiring confirmatory testing with real-time reverse transcription polymerase chain reaction (RT-PCR). We used genomic sequencing to investigate transmission dynamics in these two outbreaks. RESULTS: In Outbreak 1, 32 confirmed cases occurred within a university athletics program after the index patient attended a meeting while infectious despite a negative antigen test on the day of the meeting. Among isolates sequenced from Outbreak 1, 24 (92%) of 26 were closely related, suggesting sustained transmission following an initial introduction event. In Outbreak 2, 12 confirmed cases occurred among athletes from two university programs that faced each other in an athletic competition despite receiving negative antigen test results on the day of the competition. Sequences from both teams were closely related and unique from strains circulating in the community, suggesting transmission during intercollegiate competition. CONCLUSIONS: These findings suggest that antigen testing alone, even when mandated and directly observed, may not be sufficient as an intervention to prevent SARS-CoV-2 outbreaks in congregate settings, and highlights the importance of supplementing serial antigen testing with appropriate mitigation strategies to prevent SARS-CoV-2 outbreak in congregate settings. SUMMARY: High frequency, rapid turnaround SARS-CoV-2 testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, here we describe two SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester.

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.

Rapid evolution of enhanced Zika virus virulence during direct vertebrate transmission chains.

Zika virus (ZIKV) has the unusual capacity to circumvent natural alternating mosquito-human transmission and be directly transmitted human-to-human via sexual and vertical routes. The impact of direct transmission on ZIKV evolution and adaptation to vertebrate hosts is unknown. Here we show that molecularly barcoded ZIKV rapidly adapted to a mammalian host during direct transmission chains in mice, coincident with the emergence of an amino acid substitution previously shown to enhance virulence. In contrast, little to no adaptation of ZIKV to mice was observed following chains of direct transmission in mosquitoes or alternating host transmission. Detailed genetic analyses revealed that ZIKV evolution in mice was generally more convergent and subjected to more relaxed purifying selection than in mosquitoes or alternate passages. These findings suggest that prevention of direct human transmission chains may be paramount to resist gains in ZIKV virulence.Importance We used experimental evolution to model chains of direct and indirect Zika virus (ZIKV) transmission by serially passaging a synthetic swarm of molecularly barcoded ZIKV within and between mosquitoes and mice. We observed that direct mouse transmission chains facilitated a rapid increase in ZIKV replication and enhanced virulence in mice. These findings demonstrate that ZIKV is capable of rapid adaptation to a vertebrate host and indicate that direct human-to-human transmission could pose a greater threat to public health than currently realized.

Gender Can Influence Student Experiences in MD-PhD Training.

Background: Female physician-scientists have led major advances in medicine broadly and particularly in women's health. Women remain underrepresented in dual MD-PhD degree programs that train many physician-scientists despite gender parity among medical and biomedical research students. Materials and Methods: To explore how the training environment might be experienced differently for male and female students in one MD-PhD program, the authors analyzed gender differences in annual symposium speakers with exact binomial tests, student participation as question-askers at a weekly seminar with logistic regression, and number of publications with quasi-Poisson generalized linear models. They compared male and female students' perceptions of gender-based discrimination using a survey, including qualitative analysis of free text responses. The program consisted of 71 total students in the 2017-2018 and 2018-2019 academic years. Female students comprised 42.0% (81/191) of program matriculants from 1997 to 2019. Results: Male and female students were equally likely to present at the annual program symposium, but faculty (p = 0.001) and keynote (p = 0.012) presenters were more likely to be male. Compared with male counterparts, female students asked fewer seminar questions (p < 0.005) and female speakers received more questions (p = 0.03). Female students perceived less support and differed from men in reasons for asking or not asking seminar questions. Free text responses described repeated small acts of discrimination toward women with cumulative impact. Positive program changes followed presentation of findings to program leaders and students. Conclusions: The authors identified several aspects of one MD-PhD program that could discourage career or training persistence of female students. Increasing awareness of these issues was temporally related to positive programmatic changes.

Transmission of SARS-CoV-2 in domestic cats imposes a narrow bottleneck.

The evolutionary mechanisms by which SARS-CoV-2 viruses adapt to mammalian hosts and, potentially, undergo antigenic evolution depend on the ways genetic variation is generated and selected within and between individual hosts. Using domestic cats as a model, we show that SARS-CoV-2 consensus sequences remain largely unchanged over time within hosts, while dynamic sub-consensus diversity reveals processes of genetic drift and weak purifying selection. We further identify a notable variant at amino acid position 655 in Spike (H655Y), which was previously shown to confer escape from human monoclonal antibodies. This variant arises rapidly and persists at intermediate frequencies in index cats. It also becomes fixed following transmission in two of three pairs. These dynamics suggest this site may be under positive selection in this system and illustrate how a variant can quickly arise and become fixed in parallel across multiple transmission pairs. Transmission of SARS-CoV-2 in cats involved a narrow bottleneck, with new infections founded by fewer than ten viruses. In RNA virus evolution, stochastic processes like narrow transmission bottlenecks and genetic drift typically act to constrain the overall pace of adaptive evolution. Our data suggest that here, positive selection in index cats followed by a narrow transmission bottleneck may have instead accelerated the fixation of S H655Y, a potentially beneficial SARS-CoV-2 variant. Overall, our study suggests species- and context-specific adaptations are likely to continue to emerge. This underscores the importance of continued genomic surveillance for new SARS-CoV-2 variants as well as heightened scrutiny for signatures of SARS-CoV-2 positive selection in humans and mammalian model systems.

Revealing fine-scale spatiotemporal differences in SARS-CoV-2 introduction and spread.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties following the statewide "Safer at Home" order, which went into effect 25 March 2020. Our results suggest patterns of SARS-CoV-2 transmission may vary substantially even in nearby communities. Understanding these local patterns will enable better targeting of public health interventions.