Virological Characterization of the First 2 COVID-19 Patients Diagnosed in Italy: Phylogenetic Analysis, Virus Shedding Profile From Different Body Sites, and Antibody Response Kinetics

Abstract

Background: The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unclear. We report the detection of viral RNA from different anatomical districts and the antibody profile in the first 2 COVID-19 cases diagnosed in Italy.

Methods: We tested for SARS-CoV-2 RNA clinical samples, either respiratory and nonrespiratory (ie, saliva, serum, urine, vomit, rectal, ocular, cutaneous, and cervico-vaginal swabs), longitudinally collected from both patients throughout the hospitalization. Serological analysis was carried out on serial serum samples to evaluate IgM, IgA, IgG, and neutralizing antibody levels.

Results: SARS-CoV-2 RNA was detected since the early phase of illness, lasting over 2 weeks in both upper and lower respiratory tract samples. Virus isolate was obtained from acute respiratory samples, while no infectious virus was rescued from late respiratory samples with low viral RNA load, collected when serum antibodies had been developed. Several other specimens came back positive, including saliva, vomit, rectal, cutaneous, cervico-vaginal, and ocular swabs. IgM, IgA, and IgG were detected within the first week of diagnosis, with IgG appearing earlier and at higher titers. Neutralizing antibodies developed during the second week, reaching high titers 32 days after diagnosis.

Conclusions: Our longitudinal analysis showed that SARS-CoV-2 RNA can be detected in different body samples, which may be associated with broad tropism and different spectra of clinical manifestations and modes of transmission. Profiling antibody response and neutralizing activity can assist in laboratory diagnosis and surveillance actions.

Keywords: COVID-19; Italy; SARS-CoV-2; antibody response; phylogenesis; viral culture; virus shedding.

Figure 1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolation in cell culture. Mock-infected Vero E6 cells (left) and cells inoculated with sputum from Pt1 (right) observed after 24 hours postseed. Magnification insets (100×) of selected regions are shown. Virus-induced cytopathic effect is evident in inoculated Vero E6 cells. Real-time reverse transcription polymerase chain reaction test on spent cell growth medium confirmed SARS-CoV-2 replication (inoculum cycle threshold [Ct] value = 16.73 vs 24 hours postinoculum Ct value = 8.15). Images captured by Cytation 5, Biotek.

Figure 2.

Estimated Bayesian maximum-clade-credibility tree of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequences. Red dots correspond to nodes with >85% posterior probability. The INMI-1 Pt1 sequence is highlighted in red. The nodes leading to the INMI-1 sequence segregation are shown in red. Chains were conducted for at least 100×10⁶ generations with sampling every 10 000 steps and burn-in 10×10⁶ generations. The convergence of the Markov chain Monte Carlo was assessed by calculating for each parameter the ESS (accepted if ESS > 250). A maximum clade credibility tree was obtained from the trees’ posterior distributions with the Tree-Annotator software, version 1.10.4.

Figure 3.

Kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in different clinical samples and of antibody response in the first 2 coronavirus disease 2019 patients diagnosed in Italy. Viral RNA levels detected in respiratory tract secretions (A) and in non–respiratory tract samples (B) and antibody titers (C). Pt1 is shown on the left; pt2 is shown on the right. Antibody titers for IgM, IgG, IgA, and neutralizing antibodies (NT Ab) are expressed as the reciprocal of serum dilution and are shown on a log2 scale; viral RNA levels are expressed as cycle threshold values (Ct) of E gene amplification. Dashed lines represent the limits of detection of immunofluorescence assay (1:20 in (C) and (F)) and of real-time reverse transcription polymerase chain reaction (Ct: 45 in (A), (B), (D), and (E)).