Ribeiro André Vinicius Costa, Mannarino Camille Ferreira, Dos Santos Leal Thiago, de Oliveira Carla Santos, Bianco Kayo, Clementino Maysa Mandetta, Novo Shênia Patricia Corrêa, Prado Tatiana, de Castro Eduardo da Silva Gomes, Lermontov André, Fumian Tulio Machado, Miagostovich Marize Pereira
Stricto Sensu Graduate Program in Cellular and Molecular Biology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, 21040-360, Brazil.
Department of Sanitation and Environmental Health, Sergio Arouca National School of Public Health, Oswaldo Cruz Foundation, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ, CEP 21040-360, Brazil.
Food Environ Virol. 2025 Jan 7;17(1):13. doi: 10.1007/s12560-024-09620-4.
This study aimed to investigate the dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in water samples obtained during the coronavirus disease 2019 pandemic period, employing cross-assembly phage (crAssphage) as a fecal contamination biomarker and next-generation sequencing protocols to characterize SARS-CoV-2 variants. Raw wastewater and surface water (stream and sea) samples were collected for over a month in Rio de Janeiro, Brazil. Ultracentrifugation and negatively charged membrane filtration were employed for viral concentration of the wastewater and surface water samples, respectively. Viruses were detected and quantified by (RT-)qPCR applying TaqMan® system protocols. SARS-CoV-2 RNA signals were detected in 92.5% (37/40) of the wastewater samples and in 31.25% (10/32) of the stream water samples, but not in seawater samples. CrAssphage was detected in 100% of the wastewater samples, 93.75% (30/32) of the stream samples, and in 2/4 of the seawater samples. CrAssphage detection and high concentrations in stream surface waters (median 8.95 log gc/L) revealed diffuse contamination by domestic wastewater in a region with high sanitary coverage. The correlations detected between SARS-CoV-2 data and the moving averages of clinical cases per capita over the sampling period were moderate to strong when applying a 13-day offset, regardless of normalization by crAssphage data or not. Sequencing of the receptor-binding domain of the spike protein confirmed the detection of SARS-CoV-2, but did not characterize the circulating variant. On the other hand, the whole genome sequencing protocol identified circulation of the Gamma variant, corroborating the sampling period clinical data.
本研究旨在调查严重急性呼吸综合征冠状病毒2（SARS-CoV-2）在2019年冠状病毒病大流行期间采集的水样中的传播情况，采用交叉组装噬菌体（crAssphage）作为粪便污染生物标志物，并使用下一代测序方案来表征SARS-CoV-2变体。在巴西里约热内卢，对原废水和地表水（溪流和海水）样本进行了一个多月的采集。分别采用超速离心和带负电荷的膜过滤对废水和地表水样本进行病毒浓缩。通过应用TaqMan®系统方案的（逆转录）定量聚合酶链反应（RT-qPCR）对病毒进行检测和定量。在92.5%（37/40）的废水样本和31.25%（10/32）的溪流水样本中检测到了SARS-CoV-2 RNA信号，但在海水样本中未检测到。在100%的废水样本、93.75%（30/32）的溪流样本和2/4的海水样本中检测到了crAssphage。crAssphage在溪流地表水（中位数8.95 log gc/L）中的检测和高浓度表明，在卫生覆盖率高的地区，生活污水存在广泛污染。在应用13天的偏移量时，无论是否通过crAssphage数据进行标准化，SARS-CoV-2数据与采样期间人均临床病例移动平均值之间检测到的相关性为中度至强。刺突蛋白受体结合域的测序证实了SARS-CoV-2的检测，但未对循环变体进行表征。另一方面，全基因组测序方案确定了伽马变体的循环，证实了采样期间的临床数据。
