Human astrovirus MLB replication in vitro: persistence in extraintestinal cell lines

MLB astroviruses were identified 10 years ago in feces from children with gastroenteritis of unknown etiology and have been unexpectedly detected in severe cases of meningitis/encephalitis, febrile illness of unknown etiology, and respiratory syndromes. The aim of this study was to establish a cell culture system supporting MLB astrovirus replication. We used two clinical strains to infect several cell lines, an MLB1 strain from a gastroenteritis case, and an MLB2 strain associated with a neurologic infection. Efforts to propagate the viruses in the Caco-2 cell line were unsuccessful. In contrast, we identified two human nonintestinal cell lines, Huh-7 and A549, permissive for both genotypes. After serial passages in the Huh-7.5 cell line, the adapted strains were able to establish persistent infections in the Huh-7.5, Huh-7AI, and A549 cell lines, with high viral loads (up to 10 log10 genome copies/ml) detected by quantitative reverse transcription-PCR (RT-qPCR) in the culture supernatant. Immunofluorescence assays demonstrated infection in about 10% of cells in persistently infected cultures. Electron microscopy revealed particles of 32 to 33 nm in diameter after negative staining of cell supernatants and capsid arrays in ultrathin sections with a particularly high production in Huh-7.5 cells. Interferon (IFN) expression by infected cells and effect of exogenous IFN varied depending on the type of infection and the cell line. The availability of a cell culture system to propagate MLB astroviruses represents a key step to better understand their replicative cycle, as well as a source of viruses to conduct a wide variety of basic virologic studie

A Spanish case-control study in <5 year-old children reveals the lack of association between MLB and VA astrovirus and diarrhea

Novel human astroviruses (HAstV) were discovered 10 years ago and have been associated with fatal cases of central nervous system infections. Their role in gastroenteritis is controversial, as they have been identified in symptomatic and asymptomatic subjects. The aim of the study was to investigate novel HAstV in a gastroenteritis case-control study including a pediatric population in Spain over a one-year period. We included stool samples from patients with gastroenteritis and negative results for viruses screened by routine diagnostics, and stool samples of control subjects who sought for a routine medical consultation. All samples were screened by real-time RT-PCR assays for novel HAstV. An additional screening for rotavirus, norovirus GI, GII, sapovirus, classic HAstV and adenovirus was also performed for the control group. Overall, 23/363 stool samples from case patients (6.3%) and 8/199 stool samples from control patients (4%) were positive for ≥1 novel HAstV. MLB1 was predominant (64.5% of positives). Seasonality was observed for the case group (p = 0.015), but not the control group (p = 0.95). No difference was observed in the prevalence of novel HAstV between the case and control groups (OR 1.78, 95% CI 0.68-5.45; p = 0.30). Nevertheless, MLB genome copy numbers/ml of fecal suspension was significantly higher in the control group than in the case group (p = 0.008). In our study, we identified a lack of association between novel HAstV and gastroenteritis in the studied population, which could indicate a potential role of reservoir for children, especially given the higher viral load observed in the asymptomatic group for some of them

Characterization of emerging novel human astrovirus: form bedside to bench

[eng] 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.[spa] 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

Infections respiratoires virales chez le patient transplanté pulmonaire: revue et analyse critique des études cliniques

Les patients transplantés pulmonaires sont à haut risque de complications liées aux infections respiratoires. Ce travail est une revue de la littérature dont le but est d'identifier un lien de causalité entre les infections respiratoires virales et les complications de la transplantation. Trente-quatre études cliniques portant sur les virus influenza, virus respiratoire syncytial, parainfluenza, metapneumovirus, rhinovirus, enterovirus, coronavirus, bocavirus et adenovirus ont été retenues. L'incidence des infections respiratoires est de 1.4% à 60%. La présence d'un virus est cinq fois plus fréquente en présence de symptômes (OR=4.97, CI=2.11-11.68). En revanche, sur la base des données disponibles, nous ne pouvons retenir d'association entre les infections respiratoires virales et le rejet de greffe aigu (OR=1.35, CI=0.41-4.43). Nous retrouvons une incidence de 18% versus 11.6% de syndrome de bronchiolite oblitérante (BOS) chez les patients avec et sans infection respiratoire virale respectivement. Cependant, le nombre limité de BOS ne permet pas de confirmer d'associatio

Novel human astroviruses: novel human diseases ?

Astroviruses are small, non-enveloped, single-stranded positive RNA viruses that belong to the Astroviridae family. While classical human astroviruses (HAstV) are a well-recognized cause of acute non-bacterial diarrhea among young children worldwide, novel astroviruses, named HAstV-MLB and HAstV-VA/HMO, have been identified recently in humans by molecular assays. They are phylogenetically more related to animal astroviruses than to classical human astroviruses, thus suggesting cross-species transmission. Serological studies demonstrated a surprisingly high seroprevalence in certain populations and highlighted a high infection rate in the early years of life. Although their pathogenic role has not yet been clearly determined, novel astrovirus RNA sequences have been identified in different biological specimens of symptomatic patients, including the feces, plasma, cerebrospinal fluid, and brain biopsies. Thus, there is evidence that they could contribute not only to digestive tract infection, but also to unexpected clinical syndromes, notably encephalitis and meningitis. Severe infections affect mainly immunocompromised patients. These findings indicate that novel astroviruses should be considered in the differential diagnosis of immunocompromised patients with meningitis or encephalitis of unknown origin

Epidemiology of classic and novel human astrovirus: gastroenteritis and beyond

Since they were identified in 1975, human astroviruses have been considered one of the most important agents of viral acute gastroenteritis in children. However, highly divergent astroviruses infecting humans have been recently discovered and associated with extra-intestinal infections. The report of cases of fatal meningitis and encephalitis, especially in immunocompromised individuals, has broadened their disease spectrum. Although zoonotic transmission among animal and human astroviruses has not been clearly recognized, the genetic similarity between some human and animal viruses makes it likely to occur. This review provides an update on the epidemiology of both classic and novel human astroviruses, and a comprehensive view on confirmed or potential association between astrovirus and human disease

A case report of CT-diagnosed renal infarct secondary to syphilitic aortitis

BACKGROUND: Even though reported cases of syphilis have been increasing, cases of tertiary syphilis remain extremely rare. The majority of our knowledge with regard to complications of syphilis such as aortitis was acquired before the advent of relatively modern technologies such as CT, MRI and PET. This case report presents a rare case of syphilitic aortitis associated with a renal infarct caused by a peripheral arterial embolism diagnosed by CT. CASE PRESENTATION: We present a young man with sudden abdominal pain and flank tenderness without fever. Blood tests showed acute kidney failure. Computed tomography showed a right renal infarct and a non-circular thickening of the descending thoracic aortic wall with intra-luminal thrombus. Serology confirmed the diagnosis of syphilis. Treatment with anticoagulant and penicillin resulted in a good outcome. Follow-up PET-MRI showed resolution of the thrombus with a metabolically inactive atheromatous plaque. CONCLUSION: Technologies, such as CT, PET-CT and PET-MRI, that were not present during the pre-antibiotic era, can provide new insights into rare presentations of tertiary syphilis such as aortitis. These imaging modalities show promise for early radiological diagnosis of aortitis in syphilis and may be useful for determining the response to treatment in specific cases

Human Astrovirus MLB replication in vitro: Persistence in extraintestinal cell lines

MLB astroviruses were identified 10 years ago in feces from children with gastroenteritis of unknown etiology and have been unexpectedly detected in severe cases of meningitis/encephalitis, febrile illness of unknown etiology, and respiratory syndromes. The aim of this study was to establish a cell culture system supporting MLB astrovirus replication. We used two clinical strains to infect several cell lines, an MLB1 strain from a gastroenteritis case, and an MLB2 strain associated with a neurologic infection. Efforts to propagate the viruses in the Caco-2 cell line were unsuccessful. In contrast, we identified two human nonintestinal cell lines, Huh-7 and A549, permissive for both genotypes. After serial passages in the Huh-7.5 cell line, the adapted strains were able to establish persistent infections in the Huh-7.5, Huh-7AI, and A549 cell lines, with high viral loads (up to 10 log10 genome copies/ml) detected by quantitative reverse transcription-PCR (RT-qPCR) in the culture supernatant. Immunofluorescence assays demonstrated infection in about 10% of cells in persistently infected cultures. Electron microscopy revealed particles of 32 to 33 nm in diameter after negative staining of cell supernatants and capsid arrays in ultrathin sections with a particularly high production in Huh-7.5 cells. Interferon (IFN) expression by infected cells and effect of exogenous IFN varied depending on the type of infection and the cell line. The availability of a cell culture system to propagate MLB astroviruses represents a key step to better understand their replicative cycle, as well as a source of viruses to conduct a wide variety of basic virologic studies.IMPORTANCE MLB astroviruses are emerging viruses infecting humans. More studies are required to determine their exact epidemiology, but several reports have already identified them as the cause of unexpected clinical diseases, including severe neurologic diseases. Our study provides the first description of a cell culture system for the propagation of MLB astroviruses, enabling the study of their replicative cycle. Moreover, we demonstrated the unknown capacity of MLB astrovirus to establish persistent infections in cell culture. Whether these persistent infections are also established in vivo remains unknown, but the clinical consequences would be of high interest if persistence was confirmed in vivo Finally, our analysis of IFN expression provides some trails to understand the mechanism by which MLB astroviruses can cause persistent infections in the assayed cultures

Factors related to outcome of early and delayed prosthetic joint infections

Comment on : Ascione T, Pagliano P, Mariconda M, Rotondo R, Balato G, Toro A, Barletta V, Conte M, Esposito S. Factors related to outcome of early and delayed prosthetic joint infections. J Infect. 2015 Jan;70(1):30-6. doi: 10.1016/j.jinf.2014.07.008. Epub 2014 Jul 28. PMID: 25077990