Tracing the First Days of the SARS-CoV-2 Pandemic in Greece and the Role of the First Imported Group of Travelers.

The first SARS-CoV-2 case in Greece was confirmed on February 26, 2020, and since then, multiple strains have circulated the country, leading to regional and country-wide outbreaks. Our aim is to enlighten the events that took place during the first days of the SARS-CoV-2 pandemic in Greece, focusing on the role of the first imported group of travelers. We used whole-genome SARS-CoV-2 sequences obtained from the infected travelers of the group as well as Greece-derived and globally subsampled sequences and applied dedicated phylogenetics and phylodynamics tools as well as in-house-developed bioinformatics pipelines. Our analyses reveal the genetic variants circulating in Greece during the first days of the pandemic and the role of the group's imported strains in the course of the first pandemic wave in Greece. The strain that dominated in Greece throughout the first wave, bearing the D614G mutation, was primarily imported from a certain group of travelers, while molecular and clinical data suggest that the infection of the travelers occurred in Egypt. Founder effects early in the pandemic are important for the success of certain strains, as those arriving early, several times, and to diverse locations lead to the formation of large transmission clusters that can be estimated using molecular epidemiology approaches and can be a useful surveillance tool for the prioritization of nonpharmaceutical interventions and combating present and future outbreaks. IMPORTANCE The strain that dominated in Greece during the first pandemic wave was primarily imported from a group of returning travelers in February 2020, while molecular and clinical data suggest that the origin of the transmission was Egypt. The observed molecular transmission clusters reflect the transmission dynamics of this particular strain bearing the D614G mutation while highlighting the necessity of their use as a surveillance tool for the prioritization of nonpharmaceutical interventions and combating present and future outbreaks.

Combined monitoring of IgG and IgA anti-Spike and anti-Receptor binding domain long term responses following BNT162b2 mRNA vaccination in Greek healthcare workers.

Studies on the humoral response to homologous BNT162b2 mRNA-vaccination focus mainly on IgG antibody dynamics, while long-term IgA kinetics are understudied. Herein, kinetics of IgG and IgA levels against trimeric-Spike (S) and Receptor-Binding-Domain (RBD) were evaluated by in-house ELISAs in 146 two-dose vaccinated Greek healthcare workers (HCWs) in a 9-month period at six time points (up to 270 days after the first dose). The effect of a homologous booster third dose was also studied and evaluated. The peak of immune response was observed 21 days after the second dose; 100% seroconversion rate for anti-S and anti-RBD IgG, and 99.7% and 96.3% respectively for IgA. IgG antibody levels displayed higher increase compared to IgA. Declining but persistent anti-SARS-CoV-2 antibody levels were detected 9 months after vaccination; IgG and IgA anti-S levels approached those after the first dose, while a more rapid reduction rate for anti-RBD antibodies led to significantly lower levels for both classes, supporting the need for a booster dose. Indeed, a homologous booster third dose resulted in enhanced levels of anti-S of both classes, whereas anti-RBD didn't exceed the peak levels after the second dose. Previous SARS-CoV-2 infection, flu vaccination, BMI<35 and the occurrence of an adverse event upon vaccination, were associated with higher IgG antibody levels over time, which however were negatively affected by age increase and the presence of chronic diseases. Overall, after concurrently using the S and RBD target-antigens in in-house ELISAs, we report in addition to IgG, long-term persistence of IgA antibodies. Regarding antibody levels, homologous mRNA vaccination gives rise to an effective anti-viral protection up to 9 months negatively correlated to age. Considering that COVID-19 is still a matter of public concern, booster vaccine doses remain critical to vulnerable individuals.

Targeted Virome Sequencing Enhances Unbiased Detection and Genome Assembly of Known and Emerging Viruses-The Example of SARS-CoV-2.

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known-up to 2015-vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits' design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits' panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses.

A COVID-19 outbreak among migrants in a hosting facility in Greece, April 2020.

In April 2020, a coronavirus disease 2019 outbreak was identified among migrants/refugees in Greece. Overall, 155 of 450 hosted migrants and two of 46 employees were infected (attack rates: 34.4% and 4.3%, respectively). The mean age of infected migrants was 24.9 years (3 days-68 years). In addition, 177 community contacts were tested negative. Cases were cohorted in separate rooms from people tested negative. Surfaces were cleaned and disinfected daily. The implementation of measures for the containment of the outbreak was challenging due to language barriers and lack of space for cohorting. At that time, there was no official recommendation to the general population regarding the use of masks or other personal protective equipment. Extensive testing of vulnerable populations and building trust in order to report symptoms and comply with the recommendations are essential.

Characterizing Kinetics and Avidity of SARS-CoV-2 Antibody Responses in COVID-19 Greek Patients.

In-depth understanding of the immune response provoked by SARS-CoV-2 infection is necessary, as there is a great risk of reinfection and a difficulty in achieving herd immunity due to a decline in both antibody concentration and avidity. Avidity testing, however, could overcome variability in the immune response associated with sex or clinical symptoms, and thus differentiate between recent and past infections. In this context, here, we analyzed SARS-CoV-2 antibody kinetics and avidity in Greek hospitalized (26%) and non-hospitalized (74%) COVID-19 patients (N = 71) in the course of up to 15 months after their infection to improve the accuracy of the serological diagnosis in dating the onset of the infection. The results showed that IgG-S1 levels decline significantly at four months (p = 0.0239) in both groups of patients and are higher in hospitalized ones (up to 2.1-fold, p < 0.001). Additionally, hospitalized patients' titers drop greatly and are equalized to non-hospitalized ones only at a time-point of twelve to fifteen months. Antibody levels of women in total remain more stable months after infection, compared to men. Furthermore, we examined the differential maturation of IgG avidity after SARS-CoV-2 infection, showing an incomplete maturation of avidity that results in a plateau at four months after infection. We also defined 38.2% avidity (sensitivity: 58.9%, specificity: 90.91%) as an appropriate "cut-off" that could be used to determine the stage of infection before avidity reaches a plateau.

Kinetics of Nucleocapsid, Spike and Neutralizing Antibodies, and Viral Load in Patients with Severe COVID-19 Treated with Convalescent Plasma.

COVID-19 is an ongoing pandemic with high morbidity and mortality. Despite meticulous research, only dexamethasone has shown consistent mortality reduction. Convalescent plasma (CP) infusion might also develop into a safe and effective treatment modality on the basis of recent studies and meta-analyses; however, little is known regarding the kinetics of antibodies in CP recipients. To evaluate the kinetics, we followed 31 CP recipients longitudinally enrolled at a median of 3 days post symptom onset for changes in binding and neutralizing antibody titers and viral loads. Antibodies against the complete trimeric Spike protein and the receptor-binding domain (Spike-RBD), as well as against the complete Nucleocapsid protein and the RNA binding domain (N-RBD) were determined at baseline and weekly following CP infusion. Neutralizing antibody (pseudotype NAb) titers were determined at the same time points. Viral loads were determined semi-quantitatively by SARS-CoV-2 PCR. Patients with low humoral responses at entry showed a robust increase of antibodies to all SARS-CoV-2 proteins and Nab, reaching peak levels within 2 weeks. The rapid increase in binding and neutralizing antibodies was paralleled by a concomitant clearance of the virus within the same timeframe. Patients with high humoral responses at entry demonstrated low or no further increases; however, virus clearance followed the same trajectory as in patients with low antibody response at baseline. Together, the sequential immunological and virological analysis of this well-defined cohort of patients early in infection shows the presence of high levels of binding and neutralizing antibodies and potent clearance of the virus.

Transmission dynamics of SARS-CoV-2 within families with children in Greece: A study of 23 clusters.

There is limited information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clustering within families with children. We aimed to study the transmission dynamics of SARS-CoV-2 within families with children in Greece. We studied 23 family clusters of coronavirus disease 2019 (COVID-19). Infection was diagnosed by reverse-transcriptase polymerase chain reaction in respiratory specimens. The level of viral load was categorized as high, moderate, or low based on the cycle threshold values. There were 109 household members (66 adults and 43 children). The median attack rate per cluster was 60% (range: 33.4%-100%). An adult member with COVID-19 was the first case in 21 (91.3%) clusters. Transmission of infection occurred from an adult to a child in 19 clusters and/or from an adult to another adult in 12 clusters. There was no evidence of child-to-adult or child-to-child transmission. In total 68 household members (62.4%) tested positive. Children were more likely to have an asymptomatic SARS-CoV-2 infection compared to adults (40% vs 10.5%; P = .021). In contrast, adults were more likely to develop a severe clinical course compared with children (8.8% vs 0%; P = .021). In addition, infected children were significantly more likely to have a low viral load while adults were more likely to have a moderate viral load (40.7% and 18.6% vs 13.8% and 51.7%, respectively; P = .016). In conclusion, while children become infected by SARS-CoV-2, they do not appear to transmit infection to others. Furthermore, children more frequently have an asymptomatic or mild course compared to adults. Further studies are needed to elucidate the role of viral load on these findings.

Cutaneous Vasculopathy in a COVID-19 Critically Ill Patient: A Histologic, Immunohistochemical, and Electron Microscopy Study.

We describe a critically ill, SARS-CoV-2 positive patient with respiratory failure and thrombotic/livedoid skin lesions, appearing during the course of the disease. The biopsy of the lesions revealed an occlusive, pauci-inflammatory vasculopathy of the cutaneous small vessels characterized by complement and fibrinogen deposition on vascular walls, pointing to a thrombotic vasculopathy. Transmission electron microscopy of the affected skin failed to reveal any viral inclusions. Clinical evaluation and laboratory findings ruled out systemic coagulopathies and disseminated intravascular coagulation, drug-induced skin reaction, and common viral rashes. Our hypothesis is that the, herein evidenced, microvascular occlusive injury might constitute a significant pathologic mechanism in COVID-19, being a common denominator between cutaneous and pulmonary manifestations.

Accurate SARS-CoV-2 seroprevalence surveys require robust multi-antigen assays.

There is a plethora of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) serological tests based either on nucleocapsid phosphoprotein (N), S1-subunit of spike glycoprotein (S1) or receptor binding domain (RBD). Although these single-antigen based tests demonstrate high clinical performance, there is growing evidence regarding their limitations in epidemiological serosurveys. To address this, we developed a Luminex-based multiplex immunoassay that detects total antibodies (IgG/IgM/IgA) against the N, S1 and RBD antigens and used it to compare antibody responses in 1225 blood donors across Greece. Seroprevalence based on single-antigen readouts was strongly influenced by both the antigen type and cut-off value and ranged widely [0.8% (95% CI 0.4-1.5%)-7.5% (95% CI 6.0-8.9%)]. A multi-antigen approach requiring partial agreement between RBD and N or S1 readouts (RBD&N|S1 rule) was less affected by cut-off selection, resulting in robust seroprevalence estimation [0.6% (95% CI 0.3-1.1%)-1.2% (95% CI 0.7-2.0%)] and accurate identification of seroconverted individuals.

Untuned antiviral immunity in COVID-19 revealed by temporal type I/III interferon patterns and flu comparison.

A central paradigm of immunity is that interferon (IFN)-mediated antiviral responses precede pro-inflammatory ones, optimizing host protection and minimizing collateral damage1,2. Here, we report that for coronavirus disease 2019 (COVID-19) this paradigm does not apply. By investigating temporal IFN and inflammatory cytokine patterns in 32 moderate-to-severe patients with COVID-19 hospitalized for pneumonia and longitudinally followed for the development of respiratory failure and death, we reveal that IFN-λ and type I IFN production were both diminished and delayed, induced only in a fraction of patients as they became critically ill. On the contrary, pro-inflammatory cytokines such as tumor necrosis factor (TNF), interleukin (IL)-6 and IL-8 were produced before IFNs in all patients and persisted for a prolonged time. This condition was reflected in blood transcriptomes wherein prominent IFN signatures were only seen in critically ill patients who also exhibited augmented inflammation. By comparison, in 16 patients with influenza (flu) hospitalized for pneumonia with similar clinicopathological characteristics to those of COVID-19 and 24 nonhospitalized patients with flu with milder symptoms, IFN-λ and type I IFN were robustly induced earlier, at higher levels and independently of disease severity, whereas pro-inflammatory cytokines were only acutely produced. Notably, higher IFN-λ concentrations in patients with COVID-19 correlated with lower viral load in bronchial aspirates and faster viral clearance and a higher IFN-λ to type I IFN ratio correlated with improved outcome for critically ill patients. Moreover, altered cytokine patterns in patients with COVID-19 correlated with longer hospitalization and higher incidence of critical disease and mortality compared to flu. These data point to an untuned antiviral response in COVID-19, contributing to persistent viral presence, hyperinflammation and respiratory failure.

Association Between Upper Respiratory Tract Viral Load, Comorbidities, Disease Severity, and Outcome of Patients With SARS-CoV-2 Infection.

BACKGROUND: There is limited information on the association between upper respiratory tract (URT) viral loads, host factors, and disease severity in SARS-CoV-2-infected patients. METHODS: We studied 1122 patients (mean age, 46 years) diagnosed by polymerase chain reaction (PCR). URT viral load, measured by PCR cycle threshold, was categorized as high, moderate, or low. RESULTS: There were 336 (29.9%) patients with comorbidities; 309 patients (27.5%) had high, 316 (28.2%) moderate, and 497 (44.3%) low viral load. In univariate analyses, compared to patients with moderate or low viral load, patients with high viral load were older, more often had comorbidities, developed Symptomatic disease (COVID-19), were intubated, and died. Patients with high viral load had longer stay in intensive care unit and longer intubation compared to patients with low viral load (P values < .05 for all comparisons). Patients with chronic cardiovascular disease, hypertension, chronic pulmonary disease, immunosuppression, obesity, and chronic neurological disease more often had high viral load (P value < .05 for all comparisons). In multivariate analysis high viral load was associated with COVID-19. Level of viral load was not associated with any other outcome. CONCLUSIONS: URT viral load could be used to identify patients at higher risk for morbidity or severe outcome.

Children and Adolescents With SARS-CoV-2 Infection: Epidemiology, Clinical Course and Viral Loads.

BACKGROUND: There is limited information on severe acute respiratory syndrome virus 2 (SARS-CoV-2) infection in children. METHODS: We retrieved data from the national database on SARS-CoV-2 infections. We studied in-family transmission. The level of viral load was categorized as high, moderate, or low based on the cycle threshold values. RESULTS: We studied 203 SARS-CoV-2-infected children (median age: 11 years; range: 6 days to 18.4 years); 111 (54.7%) had an asymptomatic infection. Among the 92 children (45.3%) with coronavirus disease 2019 (COVID-19), 24 (26.1%) were hospitalized. Infants <1 year were more likely to develop COVID-19 (19.5% of all COVID-19 cases) (P-value = 0.001). There was no significant difference between viral load and age, sex, underlying condition, fever and hospitalization, as well as between type of SARS-CoV-2 infection and age, sex, underlying condition and viral load. Transmission from a household member accounted for 132 of 178 (74.2%) children for whom the source of infection was identified. An adult member with COVID-19 was the first case in 125 (66.8%) family clusters. Child-to-adult transmission was found in one occasion only. CONCLUSIONS: SARS-CoV-2 infection is mainly asymptomatic or mild during childhood. Adults appear to play a key role in spread of the virus in families. Most children have moderate or high viral loads regardless of age, symptoms or severity of infection. Further studies are needed to elucidate the role of children in the ongoing pandemic and particularly in light of schools reopening and the need to prioritize groups for vaccination, when COVID-19 vaccines will be available.

Anti-SARS-CoV-2 Antibody Responses in Convalescent Plasma Donors Are Increased in Hospitalized Patients; Subanalyses of a Phase 2 Clinical Study.

We evaluated the antibody responses in 259 potential convalescent plasma donors for Covid-19 patients. Different assays were used: a commercial ELISA detecting antibodies against the recombinant spike protein (S1); a multiplex assay detecting total and specific antibody isotypes against three SARS-CoV-2 antigens (S1, basic nucleocapsid (N) protein and receptor-binding domain (RBD)); and an in-house ELISA detecting antibodies to complete spike, RBD and N in 60 of these donors. Neutralizing antibodies (NAb) were also evaluated in these 60 donors. Analyzed samples were collected at a median time of 62 (14-104) days from the day of first symptoms or positive PCR (for asymptomatic patients). Anti-SARS-CoV-2 antibodies were detected in 88% and 87.8% of donors using the ELISA and the multiplex assay, respectively. The multivariate analysis showed that age ≥50 years (p < 0.001) and need for hospitalization (p < 0.001) correlated with higher antibody titers, while asymptomatic status (p < 0.001) and testing >60 days after symptom onset (p = 0.001) correlated with lower titers. Interestingly, pseudotype virus-neutralizing antibodies (PsNAbs) significantly correlated with spike and with RBD antibodies by ELISA. Sera with high PsNAb also showed a strong ability to neutralize active SARS-CoV-2 virus, with hospitalized patients showing higher titers. Therefore, convalescent plasma donors can be selected based on the presence of high RBD antibody titers.

SARS-CoV-2 exhibits intra-host genomic plasticity and low-frequency polymorphic quasispecies.

In December 2019, an outbreak of atypical pneumonia (Coronavirus disease 2019 -COVID-19) associated with a novel coronavirus (SARS-CoV-2) was reported in Wuhan city, Hubei province, China. The outbreak was traced to a seafood wholesale market and human to human transmission was confirmed. The rapid spread and the death toll of the new epidemic warrants immediate intervention. The intra-host genomic variability of SARS-CoV-2 plays a pivotal role in the development of effective antiviral agents and vaccines, as well as in the design of accurate diagnostics. We analyzed NGS data derived from clinical samples of three Chinese patients infected with SARS-CoV-2, in order to identify small- and large-scale intra-host variations in the viral genome. We identified tens of low- or higher- frequency single nucleotide variations (SNVs) with variable density across the viral genome, affecting 7 out of 10 protein-coding viral genes. The majority of these SNVs (72/104) corresponded to missense changes. The annotation of the identified SNVs but also of all currently circulating strain variations revealed colocalization of intra-host as well as strain specific SNVs with primers and probes currently used in molecular diagnostics assays. Moreover, we de-novo assembled the viral genome, in order to isolate and validate intra-host structural variations and recombination breakpoints. The bioinformatics analysis disclosed genomic rearrangements over poly-A / poly-U regions located in ORF1ab and spike (S) gene, including a potential recombination hot-spot within S gene. Our results highlight the intra-host genomic diversity and plasticity of SARS-CoV-2, pointing out genomic regions that are prone to alterations. The isolated SNVs and genomic rearrangements reflect the intra-patient capacity of the polymorphic quasispecies, which may arise rapidly during the outbreak, allowing immunological escape of the virus, offering resistance to anti-viral drugs and affecting the sensitivity of the molecular diagnostics assays.

Chemotherapy resumption in ovarian cancer patient diagnosed with COVID-19.

•Chemotherapy resumption after convalescence from COVID-19 is safe and feasible.•No guidelines exist for resumption of chemotherapy in patients with COVID-19.•Cancer patients on chemotherapy may develop SARS-CoV-2 antibodies less frequently.