Neutralization of SARS-CoV-2 Omicron XBB.1.5 and JN.1 variants after COVID-19 booster-vaccination and infection.

SARS-CoV-2 Omicron lineages continue to emerge and evolve into new sublineages, causing infection waves throughout 2022 and 2023, which has been attributed to immune escape. We examined neutralizing antibody responses to the recently emerged SARS-CoV-2 JN.1 variant in comparison to ancestral D614G and Omicron BA.1, BA.2, BA.5, and XBB.1.5 variants. We tested 79 human sera from cohorts with different combinations of vaccinations and infections, including 23 individuals who had been repeatedly exposed to Omicron. Individuals with a monovalent XBB.1.5 vaccine booster or XBB.1.5 breakthrough infection had robust antibody levels against all variants tested; however, JN.1 evaded antibodies in individuals after single Omicron BA.1, BA.2 or BA.5 breakthrough infections. Moreover, in the non-vaccinated cohort, serum antibodies demonstrated almost no cross-neutralization activities against D614G, XBB.1.5 and JN.1. after infections with earlier Omicron variants. These findings show that SARS-CoV-2-immunity is heterogeneous, depending on different combinations of vaccinations and infections, and emphasize the importance of considering different immune-backgrounds when evaluating novel variants.

Increased cases of influenza C virus in children and adults in Austria, 2022.

Sentinel surveillance of influenza-like illnesses revealed an increase in the cases of influenza C virus in children and adults in Austria, 2022, compared to previous years, following one season (2020/2021), wherein no influenza C virus was detected. Whole-genome sequencing revealed no obvious genetic basis for the increase. We propose that the reemergence is explained by waning immunity from lack of community exposure due to restrictions intended to limit severe acute respiratory syndrome coronavirus 2 spread in prior seasons, pending further investigation.

Respiratory syncytial virus surge in 2022 caused by lineages already present before the COVID-19 pandemic.

In 2022, Austria experienced a severe respiratory syncytial virus (RSV) epidemic with an earlier-than-usual start (Weeks 35/2021-45/2022) and increased numbers of pediatric patients in emergency departments. This surge came 2 years after a season with no cases detected as a result of coronavirus disease 2019 nonpharmaceutical interventions. We analyzed epidemiologic patterns and the phylodynamics of RSV based on approximately 30 800 respiratory specimens collected year-round over 10 years from ambulatory and hospitalized patients from 248 locations in Austria. Genomic surveillance and phylogenetic analysis of 186 RSV-A and 187 RSV-B partial glycoprotein sequences collected from 2018 to 2022 revealed that the 2022/2023 surge was driven by RSV-B in contrast to the surge in the 2021/2022 season that was driven by RSV-A. Whole-genome sequencing and phylodynamic analysis indicated that the RSV-B strain GB5.0.6a was the predominant genotype in the 2022/2023 season and emerged in late 2019. The results provide insight into RSV evolution and epidemiology that will be applicable to future monitoring efforts with the advent of novel vaccines and therapeutics.

Results of a SARS-CoV-2 virus genome detection external quality assessment round focusing on sensitivity of assays and pooling of samples.

OBJECTIVES: Results of earlier external quality assessment (EQA) rounds suggested remarkable differences in the sensitivity of SARS-CoV PCR assays. Although the test systems are intended to detect SARS-CoV-2 in individual samples, screening is often applied to sample pools to increase efficiency and decrease costs. However, it is unknown to what extent these tests actually meet the manufacturer's specifications for sensitivity and how they perform when testing sample pools. METHODS: The sensitivity of assays in routine use was evaluated with a panel of positive samples in a round of a SARS-CoV-2 virus genome detection EQA scheme. The panel consisted of samples at or near the lower limit of detection ("weakly positive"). Laboratories that routinely test sample pools were asked to also analyze the pooled EQA samples according to their usual pool size and dilution method. RESULTS: All participants could detect a highly positive patient-derived sample (>106 copies/mL). Most (96%) of the test systems could detect at least 1,000 copies/mL, meeting the minimum acceptable benchmark, and many (94%) detected the vRNA in a sample with lower concentration (500 copies/mL). The false negative ratio increased to 16 and 26% for samples with 100 and 50 copies/mL, respectively. CONCLUSIONS: The performance of most assays met or exceeded their specification on sensitivity. If assays are to be used to analyze sample pools, the sensitivity of the assay and the number of pooled samples must be balanced.

Inadequate design of mutation detection panels prevents interpretation of variants of concern: results of an external quality assessment for SARS-CoV-2 variant detection.

OBJECTIVES: Mutation-specific PCR assays have quickly found their way into laboratory diagnostics due to their capacity to be a fast, easy to implement and high-throughput method for the detection of known SARS-CoV-2 variants of concern (VoCs). However, little is known about the performance of such assays in routine laboratory analysis. METHODS: The results reported in a recent round of an external quality assessment (EQA) scheme for SARS-CoV-2 mutation-specific PCR were retrospectively analyzed. For the determination of individual variant-specific sequences as well as for the interpretation results for certain virus variants, correct, incorrect, and unreported results were evaluated, and their possible causes were investigated. RESULTS: A total of 34 laboratories participated in this study. For five samples containing the VoC Alpha + E484K, Beta, Gamma, Delta, or B.1.1.318 (as a variant of interest), 848 results for SARS-2-CoV mutation detection were reported, 824 (97.2%, range per sample 88-100%) of which were correct. Melting curve assays gave 99% correct results, real-time RT-qPCR 94%, microarray-based assays 100%, and MALDI-TOF MS 96%. A total of 122/167 (73%) reported results for SARS-CoV-2 variant determination were correct. Of the 45 inconclusive or incorrect results, 33 (73%) were due to inadequate selection of targets that did not allow identification of contemporary VoC, 11 (24%) were due to incorrect results, and one (3%) was due to correct results of mutation-specific PCR. CONCLUSIONS: Careful and up-to-date selection of the targets used in mutation-specific PCR is essential for successful detection of current SARS-CoV-2 variants.

Association of Dromedary Camels and Camel Ticks with Reassortant Crimean-Congo Hemorrhagic Fever Virus, United Arab Emirates.

We previously detected a potentially novel reassortant of Crimean-Congo hemorrhagic fever virus in camels at the largest livestock market in the United Arab Emirates. A broader survey of large mammals at the site indicated zoonotic transmission is associated with dromedaries and camel ticks. Seroprevalence in cattle, sheep, and goats is minimal.

Variability of cycle threshold values in an external quality assessment scheme for detection of the SARS-CoV-2 virus genome by RT-PCR.

OBJECTIVES: The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. METHODS: Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. RESULTS: A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. CONCLUSIONS: In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.

The versatility of external quality assessment for the surveillance of laboratory and in vitro diagnostic performance: SARS-CoV-2 viral genome detection in Austria.

OBJECTIVES: External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. METHODS: Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. RESULTS: A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (Ct ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of Ct values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. CONCLUSIONS: Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.

Crimean-Congo Hemorrhagic Fever Virus Endemicity in United Arab Emirates, 2019.

We conducted a cross-sectional survey of Crimean-Congo hemorrhagic fever virus (CCHFV) in dromedary camels and attached ticks at 3 locations in the United Arab Emirates. Results revealed a high prevalence of CCHFV-reactive antibodies in camels and viral RNA in ticks and camel serum, suggesting the virus is endemic in this country.

Sympatry and habitat associations of sigmodontine rodents in a neotropical forest-savanna interface.

Small mammal communities in the Neotropics are composed largely of sigmodontine rodents. However, many questions regarding these communities remain unanswered, especially those pertaining to fine-scale sympatry and habitat selection. To address this, we examined sigmodontine community structure and vegetation in the western margin of the Upper Paraná Atlantic Forest and the southwestern-most extent of the Cerrado (CE) (an extensive South American savanna ecoregion) of Paraguay. Vegetation classifications were derived from satellite imagery combined with maps based on extensive ground-based surveys. The three most abundant species (Akodon montensis, Hylaeamys megacephalus, and Oligoryzomys nigripes) were found most often in microsympatry with conspecifics, and were negatively associated with other species. Akodon montensis was associated with high forest (HF), and H. megacephalus with bamboo understory (BU), whereas O. nigripes did not exhibit a habitat preference. The first two species' distributions within the landscape were found to be driven primarily by habitat selection, and O. nigripes by a behavioral response (avoidance) to the presence of the other two species. Moreover, habitat influences whether or not a particular species associates with, or avoids, conspecifics or other species.

Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection.

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.

Molecular Confirmation of Frogs (Anura) as Hosts of Corethrellidae (Diptera) in the Southeastern United States.

Flies in the family Corethrellidae Edwards 1932 (Diptera) are known to be attracted to the mating calls of male frogs. For the first time, the hosts of corethrellids were identified to species by analyzing bloodmeals taken from resting female flies. A portion of the cytochrome b gene was amplified and sequenced from blood-engorged flies using vertebrate-specific primers. The flies were collected over 6 yr at two locations in the southeastern United States from resting boxes and natural resting sites (rodent burrows). Potential host abundance focused on frog surveillance, and estimation relied on visual encounters, passive trapping (artificial refugia), and call surveys. This study confirms that corethrellids take blood from tree frogs (Hylidae); however, it was found that true frogs (Lithobates Fitzinger 1843 (Ranidae: Anura) sp.) were the principal host selected by Corethrella brakeleyi (Coquillett 1902) (~73% of identified bloodmeals). These preliminary data suggest that host selection of Corethrella Freeman 1962 sp. is not necessarily correlated with host calling abundance.

Lower Respiratory Tract Infection of the Ferret by 2009 H1N1 Pandemic Influenza A Virus Triggers Biphasic, Systemic, and Local Recruitment of Neutrophils.

UNLABELLED: Infection of the lower respiratory tract by influenza A viruses results in increases in inflammation and immune cell infiltration in the lung. The dynamic relationships among the lung microenvironments, the lung, and systemic host responses during infection remain poorly understood. Here we used extensive systematic histological analysis coupled with live imaging to gain access to these relationships in ferrets infected with the 2009 H1N1 pandemic influenza A virus (H1N1pdm virus). Neutrophil levels rose in the lungs of H1N1pdm virus-infected ferrets 6 h postinfection and became concentrated at areas of the H1N1pdm virus-infected bronchiolar epithelium by 1 day postinfection (dpi). In addition, neutrophil levels were increased throughout the alveolar spaces during the first 3 dpi and returned to baseline by 6 dpi. Histochemical staining revealed that neutrophil infiltration in the lungs occurred in two waves, at 1 and 3 dpi, and gene expression within microenvironments suggested two types of neutrophils. Specifically, CCL3 levels, but not CXCL8/interleukin 8 (IL-8) levels, were higher within discrete lung microenvironments and coincided with increased infiltration of neutrophils into the lung. We used live imaging of ferrets to monitor host responses within the lung over time with [(18)F]fluorodeoxyglucose (FDG). Sites in the H1N1pdm virus-infected ferret lung with high FDG uptake had high levels of proliferative epithelium. In summary, neutrophils invaded the H1N1pdm virus-infected ferret lung globally and focally at sites of infection. Increased neutrophil levels in microenvironments did not correlate with increased FDG uptake; hence, FDG uptake may reflect prior infection and inflammation of lungs that have experienced damage, as evidenced by bronchial regeneration of tissues in the lungs at sites with high FDG levels. IMPORTANCE: Severe influenza disease is characterized by an acute infection of the lower airways that may progress rapidly to organ failure and death. Well-developed animal models that mimic human disease are essential to understanding the complex relationships of the microenvironment, organ, and system in controlling virus replication, inflammation, and disease progression. Employing the ferret model of H1N1pdm virus infection, we used live imaging and comprehensive histological analyses to address specific hypotheses regarding spatial and temporal relationships that occur during the progression of infection and inflammation. We show the general invasion of neutrophils at the organ level (lung) but also a distinct pattern of localized accumulation within the microenvironment at the site of infection. Moreover, we show that these responses were biphasic within the lung. Finally, live imaging revealed an early and sustained host metabolic response at sites of infection that may reflect damage and repair of tissues in the lungs.

The murine model for Hantaan virus-induced lethal disease shows two distinct paths in viral evolutionary trajectory with and without ribavirin treatment.

In vitro, ribavirin acts as a lethal mutagen in Hantaan virus (HTNV)-infected Vero E6 cells, resulting in an increased mutation load and viral population extinction. In this study, we asked whether ribavirin treatment in the lethal, suckling mouse model of HTNV infection would act similarly. The HTNV genomic RNA (vRNA) copy number and infectious virus were measured in lungs of untreated and ribavirin-treated mice. In untreated, HTNV-infected mice, the vRNA copy number increased for 10 days postinfection (dpi) and thereafter remained constant through 26 dpi. Surprisingly, in ribavirin-treated, HTNV-infected mice, vRNA levels were similar to those in untreated mice between 10 and 26 dpi. Infectious virus levels, however, were different: in ribavirin-treated mice, the amount of infectious HTNV was significantly decreased relative to that in untreated mice, suggesting that ribavirin reduced the specific infectivity of the virus (amount of infectious virus produced per vRNA copy). Mutational analysis revealed a ribavirin-associated elevation in mutation frequency in HTNV vRNA similar to that previously reported in vitro. Codon-based analyses of rates of nonsynonymous (dN) and synonymous (dS) substitutions in the S segment revealed a positive selection for codons within the HTNV N protein gene in the ribavirin-treated vRNA population. In contrast, the vRNA population in untreated, HTNV-infected mice showed a lower level of diversity, reflecting purifying selection for the wild-type genome. In summary, these experiments show two different evolutionary paths that Hantavirus may take during infection in a lethal murine model of disease, as well as the importance of the in vivo host environment in the evolution of the virus, which was not apparent in our prior in vitro model system.

Virus sequencing performance during the SARS-CoV-2 pandemic: a retrospective analysis of data from multiple rounds of external quality assessment in Austria.

Introduction: A notable feature of the 2019 coronavirus disease (COVID-19) pandemic was the widespread use of whole genome sequencing (WGS) to monitor severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Countries around the world relied on sequencing and other forms of variant detection to perform contact tracing and monitor changes in the virus genome, in the hopes that epidemic waves caused by variants would be detected and managed earlier. As sequencing was encouraged and rewarded by the government in Austria, but represented a new technicque for many laboratories, we designed an external quality assessment (EQA) scheme to monitor the accuracy of WGS and assist laboratories in validating their methods. Methods: We implemented SARS-CoV-2 WGS EQAs in Austria and report the results from 7 participants over 5 rounds from February 2021 until June 2023. The participants received sample material, sequenced genomes with routine methods, and provided the sequences as well as information about mutations and lineages. Participants were evaluated on the completeness and accuracy of the submitted sequence and the ability to analyze and interpret sequencing data. Results: The results indicate that performance was excellent with few exceptions, and these exceptions showed improvement over time. We extend our findings to infer that most publicly available sequences are accurate within ≤1 nucleotide, somewhat randomly distributed through the genome. Conclusion: WGS continues to be used for SARS-CoV-2 surveillance, and will likely be instrumental in future outbreak scenarios. We identified hurdles in building next-generation sequencing capacity in diagnostic laboratories. EQAs will help individual laboratories maintain high quality next-generation sequencing output, and strengthen variant monitoring and molecular epidemiology efforts.

External quality assessments for SARS-CoV-2 genome detection in Austria : A comparison of the first postpandemic round with results from the pandemic era.

BACKGROUND: External quality assessment (EQA) schemes provide objective feedback to participating laboratories about the performance of their analytical systems and information about overall regional analytical performance. The EQAs are particularly important during pandemics as they also assess the reliability of individual test results and show opportunities to improve test strategies. With the end of the COVID-19 pandemic, the testing frequency significantly decreased in Austria. Here, we analyzed whether this decrease had an effect on participation and/or performance in SARS-CoV­2 virus detection EQAs, as compared to the pandemic era. MATERIAL AND METHODS: Identical samples were sent to all participating laboratories, and the EQA provider evaluated the agreement of the reported results with defined targets. The EQA was operated under two schemes with identical samples and therefore we analyzed it as a single EQA round. The performance of testing was reported as true positive ratios, comparing the post-pandemic data to previous rounds. Furthermore, subgroups of participants were analyzed stratified by laboratory type (medical or nonmedical) and the test system format (fully automated or requiring manual steps). RESULTS: While the frequency of false negative results per sample did not change during the 3 years of the pandemic (5.7%, 95% confidence interval [CI] 3.1-8.4%), an average per sample false negative ratio of 4.3% was observed in the first post-pandemic EQA (0%, 1.8%, and 11% for the 3 positive samples included in the test panel, n = 109 test results per sample). In this first post-pandemic EQA medical laboratories (average 0.4% false negative across 3 samples, n = 90) and automated test systems (average 1.2% false negative, n = 261) had lower false negative ratios than nonmedical laboratories (22.8%, n = 19) and manual test systems (16.7%, n = 22). These lower average ratios were due to a low concentration sample, where nonmedical laboratories reported 36.8% and manual test systems 54.5% true positive results. CONCLUSION: Overall ratios of true positive results were below the mean of all results during the pandemic but were similar to the first round of the pandemic. A lower post-pandemic true positive ratio was associated with specific laboratory types and assay formats, particularly for samples with low concentration. The EQAs will continue to monitor the laboratory performance to ensure the same quality of epidemiological data after the pandemic, even if vigilance has decreased.

Genetic immunization in the lung induces potent local and systemic immune responses.

Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.

Bivalent COVID-19 mRNA booster vaccination (BA.1 or BA.4/BA.5) increases neutralization of matched Omicron variants.

We report SARS-CoV-2 neutralizing antibody titers in sera of triple-vaccinated individuals who received a booster dose of an original monovalent or a bivalent BA.1- or BA.4/BA.5-adapted vaccine or had a breakthrough infection with Omicron variants BA.1, BA.2 or BA.4/BA.5. A bivalent BA.4/BA.5 booster or Omicron-breakthrough infection induced increased Omicron-neutralization titers compared with the monovalent booster. The XBB.1.5 variant effectively evaded neutralizing-antibody responses elicited by current vaccines and/or infection with previous variants.