Characterization of emerging novel human astrovirus: form bedside to bench

Abstract

Novel human astrovirus (HAstV) are enteric virus belonging to the Astroviridae family and were discovered 10 years ago by high-throughput sequencing. They are divided in two different clades, the HAstV-MLB including 3 genotypes (MLB1-3) and HAstV-VA including 5 genotypes (VA1-5). While their role during gastroenteritis is debated, they have been reported as the sole agent identified in many cases of severe central nervous system infection, mainly in immunocompromised patients. This suggests that these emerging and highly divergent viruses can be associated with serious clinical manifestations, requiring additional basic and epidemiological studies to better understand their pathogenesis, prevalence and clinical correlation. We implemented several cell culture systems allowing the propagation of two distinct genotypes of novel HAstV from clinical stool samples, namely HAstV-MLB1 and HAstV-MLB2. Both strains could efficiently replicate in human HuH-7 hepatoma and A549 respiratory cell lines. In addition, both strains could establish a persistent infection over several cell passages in both cell lines, and HAstV-MLB1 could also establish a persistent infection in HuH-7.5 cells. In the latter, electron microscopy revealed a high production of capsid arrays and significant intracellular reorganization. Immunofluorescence assays revealed only a low proportion (5-10%) of infected cells. We explored the innate immune response to HAstV-MLB infection and observed that IFN expression was either abolished or delayed and diminished, depending on the cell line, during acute infection. During persistent infection, IFN expression was abolished in all cases, while when co-stimulated with poly I:C, IFN expression remained inhibited in a cell and genotype-dependent manner. Addition of exogenous IFN led to the cure (IFN-β) and relative inhibition (IFN-λ) of HAstV-MLB infection in HuH-7 cell line, while there was no effect in A549 infected cell line. At the epidemiological level, using a sensitive and specific real-time RT-PCR assay, we found that novel HAstVs could be identified in 6-10% of cases of undiagnosed gastroenteritis in Spanish pediatric children of < 5 years old. Together with a Japanese study, our prevalence is the highest observed to date. Children under 2 years old had a higher prevalence rate, compared to older ones. HAstV-MLB1 and HAstV-VA1 accounted for 31% and 26% of all novel HAstV observed in our cohort, while no HAstV-MLB3 were detected. Nevertheless, we found a high rate of co-infection with other enteric viruses (66%), precluding to assess a firm association between the presence of novel HAstV and the occurrence of gastroenteritis in such cases. We could not identify differences in the mean Cq values between mono- and co-infection episodes. We then performed a case-control study to assess the role of novel HAstV in gastroenteritis. We found a prevalence of 6.3% and 4% in symptomatic and asymptomatic children, respectively, without statistical difference between groups. However, we found that asymptomatic children had statistically significant higher HAstV-MLB viral load (median 6.52 log10 genomes/ml, IQR 4.52-6.84) compared to symptomatic children (median 2.35 log10 genomes/ml, IQR 2.13-3.76). Similarly, in symptomatic patients, we observed a higher viral load when novel HAstVs were found in coproculture-positive (median 5.19 log10 genomes/ml, IQR 4.24-6.22) compared to coproculture-negative (median 2.31 log10 genomes/ml, IQR 2.11-3.32) stool samples. These findings suggest that novel HAstV are not associated with gastroenteritis, but could modulate the gut microbiome and may confer protection to invading pathogens, although the mechanism remains to be elucidated.

Los astrovirus humanos (HAstV) no clásicos son virus entéricos emergentes que pertenecen a la familia de los Astroviridae, la cual incluye virus asociados a gastroenteritis principalmente en la población pediátrica. Se descubrieron por primera vez hace 10 años mediante secuenciación masiva, y hoy se dividen en dos grupos filogenéticos distintos: los HAstV-MLB (MLB1-3), y los HAstV-VA (VA1-5). Su asociación con gastroenteritis no está del todo confirmada, y también han sido identificados en casos de meningoencefalitis en pacientes inmunodeprimidos. En nuestro trabajo hemos implementado varios sistemas de cultivo celular permisivos para la replicación de dos genotipos de HAstV-MLB, HAstV-MLB1 y HAstV-MLB2, utilizando muestras clínicas. Ambos genotipos pueden replicar en las líneas celulares humanas HuH-7 y A549, de hepatoma y tejido respiratorio, respectivamente. Además, ambos pueden establecer una infección persistente en el cultivo, detectándose señal positiva por inmunofluorescencia en 5-10% de las células. La microscopía electrónica identifica una gran cantidad de cápsides víricas dentro de las células infectadas, y una importante reorganización intracelular. En los cultivos persistentemente infectados no se detecta inducción de la respuesta interferón (IFN), y la capacidad de los virus para bloquear la expresión de IFN inducida por poliI:C es distinta para cada tipo celular. La sensibilidad frente a un tratamiento exógeno tanto con IFN-β como con IFN-λ, es efectivo en las células HuH-7 pero nulo en las células A549. En nuestros estudios epidemiológicos en niños menores de 5 años con gastroenteritis no diagnosticada, se detectaron HAstV no clásicos en 6-10% de los casos. MLB1 y HAstV-VA1 representaron el 31% y el 26% de todos ellos, respectivamente. Se detectó co-infección con algún otro virus entérico en 66% de las muestras positivas, y no se observaron diferencias en los valores Cq entre casos de mono- y co-infección. En un estudio de casos y controles, su prevalencia fue similar en ambos grupos (6.3% versus 4%, respectivamente). No obstante, se observó que el promedio de carga vírica en los casos asintomáticos fue significativamente superior que en los niños enfermos, y en pacientes sintomáticos, se observó una carga viral mayor en aquellas heces que eran positivas para coprocultivo en comparación con las negativas.