Astrovirus in the Brazilian Amazon: First detection of non-classical astroviruses (MLB-3) in the Americas

Human astrovirus (HAstV) is a viral agent responsible for acute gastroenteritis (AGE), primarily affecting children and the elderly worldwide. Belonging to the Astroviridae family, HAstV is classified into eight classical serotypes (HAstV 1-8) and two other divergent non-classical clades: Melbourne (MLB 1-3) and Virginia (VA 1-6), which have been associated with gastroenteritis, central nervous system complications, and acute respiratory disease. This study aimed to investigate the frequency of classical and non-classical HAstV in fecal specimens collected from children up to 14 years of age in northern Brazil, within the Amazon region, between 2013 and 2022. A total of 560 samples, all previously tested negative for other gastroenteric viruses such as rotavirus and norovirus, were analyzed using reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) and conventional RT-PCR. For classical HAstV, 10.7% (60/560) of the samples were positive by RT-qPCR and 2.0% (11/560) to 3.0% (17/560) by conventional RT-PCR using different primers set. Non-classical HAstV was detected in 0.2% (1/560) of the samples. Diarrhea was present in 91.7% of positive cases, vomiting in 71.7%, and fever in 53.3%. The most affected age group was children aged >5-10 years (25.0%), with no significant association between infection rate and sex. A higher number of infections occurred during the Amazon winter (11.6%), with Roraima identified as the federative unit with the highest number of cases. Fifteen samples (88.2%, 15/17) were sequenced and identified as classical HAstV, with the following genotypes detected: HAstV-1 (60.0%, 9/15), HAstV-3 (20.0%, 3/15), and HAstV-4 (20.0%, 3/15). Non-classical HAstV sequencing was performed on 12 (93.2%, 12/13) positive specimens, characterized as HAstV-1 (50%, 6/12), HAstV-3 (25%, 3/12), HAstV-4 (16.7%, 2/12), and HAstV-MLB-3 (8.3%, 1/12). A probable recombinant strain was identified, classified as HAstV-4 based on the ORF2 region and HAstV-1 based on the ORF1b region. This study provides updated epidemiological data on HAstV in the Brazilian Amazon and highlights the genetic diversity of both classical and non-classical genotypes. Notably, it reports the first detection - and the second complete genome repository - of the rare MLB-3 genotype in the Americas.