A robust, scalable, and cost-efficient approach to whole genome sequencing of RSV directly from clinical samples.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses. IMPORTANCE: Respiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.

Different populations of A(H1N1)pdm09 viruses in a patient with hemolytic-uremic syndrome.

Respiratory viral infections may have different impacts ranging from infection without symptoms to severe disease or even death though the reasons are not well characterized. A patient (age group 5-15 years) displaying symptoms of hemolytic uremic syndrome died one day after hospitalization. qPCR, next generation sequencing, virus isolation, antigenic characterization, resistance analysis was performed and virus replication kinetics in well-differentiated airway cells were determined. Autopsy revealed hemorrhagic pneumonia as major pathological manifestation. Lung samples harbored a large population of A(H1N1)pdm09 viruses with the polymorphism H456H/Y in PB1 polymerase. The H456H/Y viruses replicated much faster to high viral titers than upper respiratory tract viruses in vitro. H456H/Y-infected air-liquid interface cultures of differentiated airway epithelial cells did reflect a more pronounced loss of ciliated cells. A different pattern of virus quasispecies was found in the upper airway samples where substitution S263S/F (HA1) was observed. The data support the notion that viral quasispecies had evolved locally in the lung to support high replicative fitness. This change may have initiated further pathogenic processes leading to rapid dissemination of inflammatory mediators followed by development of hemorrhagic lung lesions and fatal outcome.

Preparing for the Next Influenza Season: Monitoring the Emergence and Spread of Antiviral Resistance.

Purpose: The relaxation of pandemic restrictions in 2022 has led to a reemergence of respiratory virus circulation worldwide and anticipation of substantial influenza waves for the 2022/2023 Northern Hemisphere winter. Therefore, the antiviral susceptibility profiles of human influenza viruses circulating in Germany were characterized. Methods: Between October 2019 (week 40/2019) and March 2022 (week 12/2022), nasal swabs from untreated patients with acute respiratory symptoms were collected in the national German influenza surveillance system. A total of 598 influenza viruses were isolated and analyzed for susceptibility to oseltamivir, zanamivir and peramivir, using a neuraminidase (NA) inhibition assay. In addition, next-generation sequencing was applied to assess molecular markers of resistance to NA, cap-dependent endonuclease (PA) and M2 ion channel inhibitors (NAI, PAI, M2I) in 367 primary clinical samples. Furthermore, a genotyping assay based on RT-PCR and pyrosequencing to rapidly assess the molecular resistance marker PA-I38X in PA genes was designed and established. Results: While NAI resistance in the strict sense, defined by a ≥ 10-fold (influenza A) or ≥5-fold (influenza B) increase of NAI IC50, was not detected, a subtype A(H1N1)pdm09 isolate displayed 2.3- to 7.5-fold IC50 increase for all three NAI. This isolate carried the NA-S247N substitution, which is known to enhance NAI resistance induced by NA-H275Y. All sequenced influenza A viruses carried the M2-S31N substitution, which confers resistance to M2I. Of note, one A(H3N2) virus displayed the PA-I38M substitution, which is associated with reduced susceptibility to the PAI baloxavir marboxil. Pyrosequencing analysis confirmed these findings in the original clinical specimen and in cultured virus isolate, suggesting sufficient replicative fitness of this virus mutant. Conclusion: Over the last three influenza seasons, the vast majority of influenza viruses in this national-level sentinel were susceptible to NAIs and PAIs. These findings support the use of antivirals in the upcoming influenza season.

Vaccination against Borna Disease: Overview, Vaccine Virus Characterization and Investigation of Live and Inactivated Vaccines.

(1) Background: Vaccination of horses and sheep against Borna disease (BD) was common in endemic areas of Germany in the 20th century but was abandoned in the early 1990s. The recent occurrence of fatal cases of human encephalitis due to Borna disease virus 1 (BoDV-1) has rekindled the interest in vaccination. (2) Methods: The full genomes of the BD live vaccine viruses "Dessau" and "Giessen" were sequenced and analyzed for the first time. All vaccination experiments followed a proof-of-concept approach. Dose-titration infection experiments were performed in rabbits, based on both cell culture- and brain-derived viruses at various doses. Inactivated vaccines against BD were produced from concentrated cell culture supernatants and investigated in rabbits and horses. The BoDV-1 live vaccine "Dessau" was administered to horses and antibody profiles were determined. (3) Results: The BD live vaccine viruses "Dessau" and "Giessen" belong to clusters 3 and 4 of BoDV-1. Whereas the "Giessen" virus does not differ substantially from field viruses, the "Dessau" virus shows striking differences in the M gene and the N-terminal part of the G gene. Rabbits infected with high doses of cell-cultured virus developed neutralizing antibodies and were protected from disease, whereas rabbits infected with low doses of cell-cultured virus, or with brain-derived virus did not. Inactivated vaccines were administered to rabbits and horses, following pre-defined vaccination schemes consisting of three vaccine doses of either adjuvanted or nonadjuvanted inactivated virus. Their immunogenicity and protective efficacy were compared to the BD live vaccine "Dessau". Seventy per cent of horses vaccinated with the BD live vaccine "Dessau" developed neutralizing antibodies after vaccination. (4) Conclusion: Despite a complex evasion of immunological responses by bornaviruses, some vaccination approaches can protect against clinical disease. For optimal effectiveness, vaccines should be administered at high doses, following vaccination schemes consisting of three vaccine doses as basic immunization. Further investigations are necessary in order to investigate and improve protection against infection and to avoid side effects.

SARS-CoV-2 Omicron variant is attenuated for replication in a polarized human lung epithelial cell model.

SARS-CoV-2 and its emerging variants of concern remain a major threat for global health. Here we introduce an infection model based upon polarized human Alveolar Epithelial Lentivirus immortalized (hAELVi) cells grown at the air-liquid interface to estimate replication and epidemic potential of respiratory viruses in the human lower respiratory tract. hAELVI cultures are highly permissive for different human coronaviruses and seasonal influenza A virus and upregulate various mediators following virus infection. Our analysis revealed a significantly reduced capacity of SARS-CoV-2 Omicron BA.1 and BA.2 variants to propagate in this human model compared to earlier D614G and Delta variants, which extends early risk assessments from epidemiological and animal studies suggesting a reduced pathogenicity of Omicron.

Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021.

BACKGROUND: Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. METHODS: At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2-positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. RESULTS: We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. CONCLUSIONS: SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.

A Genetic Variation of Lipopolysaccharide Binding Protein Affects the Inflammatory Response and Is Associated with Improved Outcome during Sepsis.

LPS binding protein (LBP) is an important innate sensor of microbial cell wall structures. Frequent functionally relevant mutations exist and have been linked to influence susceptibility to and course of bacterial infections. We examined functional properties of a single nucleotide polymorphism resulting in an exchange of phenylalanine to leucine at position 436 of LBP (rs2232618) and compared the frequent variant of the molecule with the rare one in ligand binding experiments. We then stimulated RAW cells with bacterial ligands in the presence of serum obtained from individuals with different LBP genotypes. We, furthermore, determined the potential effects of structural changes in the molecule by in silico modeling. Finally, we analyzed 363 surgical patients for this genetic variant and examined incidence and course of sepsis following surgery. We found that binding of LBP to bacterial ligands was reduced, and stimulation of RAW cells resulted in an increased release of TNF when adding serum from individuals carrying the F436L variant as compared with normal LBP. In silico analysis revealed structural changes of LBP, potentially explaining some of the effects observed for the LBP variant. Finally, patients carrying the F436L variant were found to be similarly susceptible for sepsis. However, we observed a more favorable course of severe infections in this cohort. Our findings reveal new insights into LPS recognition and the subsequent activation of the innate immune system brought about by LBP. The identification of a genetic variant of LBP influencing the course of sepsis may help to stratify individuals at risk and thus reduce clinical complications of patients.

Virological Surveillance and Molecular Characterization of Human Parainfluenzavirus Infection in Children with Acute Respiratory Illness: Germany, 2015-2019.

Human parainfluenza viruses (HPIVs) are important causes of respiratory illness, especially in young children. However, surveillance for HPIV is rarely performed continuously, and national-level epidemiologic and genetic data are scarce. Within the German sentinel system, to monitor acute respiratory infections (ARI), 4463 respiratory specimens collected from outpatients < 5 years of age between October 2015 and September 2019 were retrospectively screened for HPIV 1-4 using real-time PCR. HPIV was identified in 459 (10%) samples. HPIV-3 was the most common HPIV-type, with 234 detections, followed by HPIV-1 (113), HPIV-4 (61), and HPIV-2 (49). HPIV-3 was more frequently associated with age < 2 years, and HPIV-4 was more frequently associated with pneumonia compared to other HPIV types. HPIV circulation displayed distinct seasonal patterns, which appeared to vary by type. Phylogenetic characterization clustered HPIV-1 in Clades 2 and 3. Reclassification was performed for HPIV-2, provisionally assigning two distinct HPIV-2 groups and six clades, with German HPIV-2s clustering in Clade 2.4. HPIV-3 clustered in C1, C3, C5, and, interestingly, in A. HPIV-4 clustered in Clades 2.1 and 2.2. The results of this study may serve to inform future approaches to diagnose and prevent HPIV infections, which contribute substantially to ARI in young children in Germany.

[SARS-CoV-2 transmission routes and implications for self- and non-self-protection]. / SARS-CoV-2-Übertragungswege und Implikationen für den Selbst- und Fremdschutz.

The global spread of the coronavirus SARS-CoV­2 has massively impacted health, economic, and social systems. Although effective vaccines are now available, it is likely that this pathogen will become endemic and stay with us for years. In order to most effectively protect others and oneself from SARS-CoV­2 infection, an understanding of how SARS-CoV­2 is transmitted is of utmost importance.In this review paper, we explain transmission routes with an eye towards protecting others and oneself. We also address characteristics of SARS-CoV­2 transmission in the community. This work will help to clarify the following questions based on the available literature: When and for how long is an infected person contagious? How is the virus excreted? How is the virus taken up? How does the virus spread in society?Human-to-human transmission of SARS-CoV­2 is strongly determined by pathogen molecular characteristics as well as the kinetics of replication, shedding, and infection. SARS-CoV­2 is transmitted primarily via human aerosols, which infected persons can excrete even if symptoms of the disease are not (yet) present. Most infected people cause only a few secondary cases, whereas a few cases (so-called super-spreaders) cause a high number of secondary infections - at the population level one speaks of a so-called "overdispersion." These special characteristics of SARS-CoV­2 (asymptomatic aerosol transmission and overdispersion) make the pandemic difficult to control.

Trends in respiratory virus circulation following COVID-19-targeted nonpharmaceutical interventions in Germany, January - September 2020: Analysis of national surveillance data.

BACKGROUND: During the initial COVID-19 response, Germany's Federal Government implemented several nonpharmaceutical interventions (NPIs) that were instrumental in suppressing early exponential spread of SARS-CoV-2. NPI effect on the transmission of other respiratory viruses has not been examined at the national level thus far. METHODS: Upper respiratory tract specimens from 3580 patients with acute respiratory infection (ARI), collected within the nationwide German ARI Sentinel, underwent RT-PCR diagnostics for multiple respiratory viruses. The observation period (weeks 1-38 of 2020) included the time before, during and after a far-reaching contact ban. Detection rates for different viruses were compared to 2017-2019 sentinel data (15350 samples; week 1-38, 11823 samples). FINDINGS: The March 2020 contact ban, which was followed by a mask mandate, was associated with an unprecedented and sustained decline of multiple respiratory viruses. Among these, rhinovirus was the single agent that resurged to levels equalling those of previous years. Rhinovirus rebound was first observed in children, after schools and daycares had reopened. By contrast, other nonenveloped viruses (i.e. gastroenteritis viruses reported at the national level) suppressed after the shutdown did not rebound. INTERPRETATION: Contact restrictions with a subsequent mask mandate in spring may substantially reduce respiratory virus circulation. This reduction appears sustained for most viruses, indicating that the activity of influenza and other respiratory viruses during the subsequent winter season might be low,whereas rhinovirus resurgence, potentially driven by transmission in educational institutions in a setting of waning population immunity, might signal predominance of rhinovirus-related ARIs. FUNDING: Robert Koch-Institute and German Ministry of Health.

[Recording of SARS-CoV-2 laboratory tests in Germany]. / Erfassung der Labortestungen auf SARS-CoV-2 in Deutschland.

The collection of data on SARS-CoV­2 tests is central to the assessment of the infection rate in the context of the COVID-19 pandemic. At the Robert Koch Institute (RKI), data collected from various laboratory data recording systems are consolidated. First, this article aims to exemplify significant aspects regarding test procedures. Subsequently the different systems for recording laboratory tests are described and test numbers from the RKI test laboratory query and the laboratory-based SARS-CoV­2 surveillance as well as accounting data from the Association of Statutory Health Insurance Physicians for SARS-CoV­2 laboratory tests are shown.Early in the pandemic, the RKI test laboratory query and the laboratory-based SARS-CoV­2 surveillance became available and able to evaluate data on performed tests and test capacities. By recording the positive and negative test results, statements about the total number of tests and the proportion of positive test rates can be made. While the aggregate test numbers are largely representative nationwide, they are not always representative at the state and district level. The billing data of the Association of Statutory Health Insurance Physicians can complement the laboratory data afterwards. In addition, it can provide a retrospective assessment of the total number of SARS-CoV­2 numbers in Germany, because the services provided by statutory health insurers (around 85% of the population in Germany) are included. The various laboratory data recording systems complement one another and the evaluations flow into the recommended measures for the pandemic response.

COVIDStrategyCalculator: A software to assess testing and quarantine strategies for incoming travelers, contact management, and de-isolation.

While large-scale vaccination campaigns against SARS-CoV-2 are rolled out at the time of writing, non-pharmaceutical interventions (NPIs), including the isolation of infected individuals and quarantine of exposed individuals, remain central measures to contain the spread of SARS-CoV-2. Strategies that combine NPIs with innovative SARS-CoV-2 testing strategies may increase containment efficacy and help to shorten quarantine durations. We developed a user-friendly software tool that implements a recently published stochastic within-host viral dynamics model that captures temporal attributes of the viral infection, such as test sensitivity, infectiousness, and the occurrence of symptoms. Based on this model, the software allows to evaluate the efficacy of user-defined, arbitrary NPI and testing strategies in reducing the transmission potential in different contexts. The software thus enables decision makers to explore NPI strategies and perform hypothesis testing, e.g., with regard to the utilization of novel diagnostics or with regard to containing novel virus variants.

An intra-host SARS-CoV-2 dynamics model to assess testing and quarantine strategies for incoming travelers, contact management, and de-isolation.

Non-pharmaceutical interventions (NPIs) remain decisive tools to contain SARS-CoV-2. Strategies that combine NPIs with testing may improve efficacy and shorten quarantine durations. We developed a stochastic within-host model of SARS-CoV-2 that captures temporal changes in test sensitivities, incubation periods, and infectious periods. We used the model to simulate relative transmission risk for (1) isolation of symptomatic individuals, (2) contact person management, and (3) quarantine of incoming travelers. We estimated that testing travelers at entry reduces transmission risks to 21.3% ([20.7, 23.9], by PCR) and 27.9% ([27.1, 31.1], by rapid diagnostic test [RDT]), compared with unrestricted entry. We calculated that 4 (PCR) or 5 (RDT) days of pre-test quarantine are non-inferior to 10 days of quarantine for incoming travelers and that 8 (PCR) or 10 (RDT) days of pre-test quarantine are non-inferior to 14 days of post-exposure quarantine. De-isolation of infected individuals 13 days after symptom onset may reduce the transmission risk to <0.2% (<0.01, 6.0).

Zoonotic infection with swine A/H1avN1 influenza virus in a child, Germany, June 2020.

A zoonotic A/sw/H1avN1 1C.2.2 influenza virus infection was detected in a German child that presented with influenza-like illness, including high fever. There was a history of close contact with pigs 3 days before symptom onset. The child recovered within 3 days. No other transmissions were observed. Serological investigations of the virus isolate revealed cross-reactions with ferret antisera against influenza A(H1N1)pdm09 virus, indicating a closer antigenic relationship with A(H1N1)pdm09 than with the former seasonal H1N1 viruses.

From CD4-Based Initiation to Treating All HIV-Infected Adults Immediately: An Evidence-Based Meta-analysis.

Background: The World Health Organization (WHO) Consolidated antiretroviral therapy (ART) guidelines set the CD4+ T-cell counts threshold to 500 cells/mm3 in 2013, and 2015 guidelines recommend treating all HIV-infected adults regardless of their CD4+ T-cell counts. To inform the decision-making around ART guidelines for people living with HIV, we systematically reviewed the literature to estimate differences in clinical benefits between individuals starting treatment with baseline CD4+ T-cell counts ≥500 cells/mm3 (early initiation) as compared to <500 cells/mm3 (deferred initiation). Methods: We systematically searched the electronic databases and abstracts for randomized controlled trials (RCT) and observational studies. Outcomes were mortality, AIDS progression, AIDS or death, immunologic recovery, and virologic suppression. We pooled data across studies and performed analyses of effect sizes. Results: We identified 13 studies comparing early and deferred treatment. The pooled risk ratio (RR) of mortality of 11 observational studies was 0.90 (95% CI 0.82-0.99), with moderate heterogeneity (I2 = 53%). The pooled RR for progression to AIDS from two observational studies was 0.77 (95% CI 0.47-1.24). Five observational studies found a pooled RR of death or AIDS of 0.94 (95% CI 0.93-0.95). For the outcome of immunologic recovery, defined as CD4+ T-cell counts reaching at least 800 cells/mm3 after ART, one observational study found early initiation of ART had an HR (hazard ratio) of 2.39 (95% CI 1.93-2.96). The pooled RR of viral suppression (a viral load <50 copies/ml) after 9 months from one cohort was 1.04 (95% CI 0.99-1.09). Conclusion: Mortality risk and risk for AIDS appear to be reduced among people living with HIV with early initiation of ART, based on current WHO guidelines, as compared to those with deferred initiation of ART (<500 cells/mm3).