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Human Species D Adenoviruses Isolated from Diarrheal Feces Show Low Infection Rates in Primary Nasal Epithelial Cells.

ABSTRACT: The importance of adenovirus (Ad) research is significantly increasing with respect to virotherapy for vaccine development, tumor, and gene therapy. Due to the different species and subtypes of this virus, the characterization of the biological significance of especially rare Ad is necessary. Previously, rare Ad types 70, 73, and 74 were originally isolated from fecal samples of immunocompromised patients and they represent recombinants of other Ad types. Here we investigated transduction experiments of these reporter gene tagged Ad types in primary cells exemplified by subject-derived primary nasal epithelial cells (NAEPCs). To analyze the transduction rates, we performed flow cytometry, quantitative polymerase chain reaction (PCR), and cytokine analyses 25 h post-infection. We found that, in contrast to Ad type 5 (as a positive control), the transduction rates of NAEPCs with Ad types 70, 73, and 74 were interestingly low. The major Ad receptor (coxsackievirus-adenovirus receptor and CD46) expression levels showed no significant change after infection with Ad types 70, 73 and 74. Moreover, Interleukin 6 (IL-6) was not released after in vitro Ad transduction. Due to the high risk of developing life-threatening complications in immunocompromised patients by these human species D Ads, even more attention needs to be investigated into the development of diagnostic and therapeutic concepts to prevent and treat those opportunistic infections in susceptible patients.

SUBMITTER: Aydin M

PROVIDER: S-EPMC8307086 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that is faster and requires fewer resources. Here, we describe two LAMP assays for the detection of human adenoviruses in the feces of children with acute intestinal infections. We designed сolorimetric LAMP (c-LAMP) and real-time LAMP (f-LAMP) with fluorescent probes to detect the DNA of the adenovirus F human adenovirus 40/41 (hAdV40/41) hexon gene. The detection limit of both developed methods was 103 copies/mL, which is comparable to the sensitivity of PCR. The specificities of both c-LAMP and f-LAMP were high, with no false-positive results for clinical samples that do not contain adenovirus F, when testing other viruses and microorganisms. Comparative tests of PCR and LAMP on clinical samples from patients with acute gastroenteritis were carried out. For all samples with a PCR threshold cycle (CT) of up to 36, the PCR and LAMP results completely coincided; however, at low viral loads, the diagnostic sensitivity of LAMP, especially c-LAMP with colorimetric detection, was inferior to that of PCR. The combination of LAMP with modern methods of nucleic acid extraction, both in manual and automatic modes, can reduce the time for a complete study, including extraction of nucleic acid material and amplification, to 60 min. IMPORTANCE In April 2022, several cases of acute hepatitis of unknown origin were reported in children from 12 countries. In many cases, enteric adenovirus or SARS-CoV-2 and adenovirus coinfection were detected. It is known that human adenoviruses can cause different infections of varying severity, from asymptomatic to severe cases with lethal outcomes. There is a need to increase the diagnostic capabilities of clinical laboratories to identify such an underestimated pathogen as adenovirus. Although PCR remains the gold standard for pathogen detection, this method requires specialized equipment and has a long turnaround time to process samples. Previously, LAMP assays for the detection of human adenovirus have been based on measuring the turbidity, the fluorescence of intercalated dyes, or electrophoretic separation. Herein, we present LAMP-based assays with colorimetric or fluorescent detection and perform a detailed assessment of their sensitivity, specificity, and diagnostic performance.

Project description:Human adenovirus species F (HAdV-F) type 40 and 41 are commonly associated with acute diarrheal disease (ADD) across the world. Despite being the largest state in southeastern Brazil and having the second largest number of inhabitants, there is no information in the State of Minas Gerais regarding the role of HAdV-F in the etiology of ADD. This study was performed to determine the prevalence, to verify the epidemiological aspects of infection, and to characterize the strains of human adenoviruses (HAdV) detected. A total of 377 diarrheal fecal samples were obtained between January 2007 and August 2011 from inpatient and outpatient children of age ranging from 0 to 12 years. All samples were previously tested for rotavirus, norovirus, and astrovirus, and 314 of 377 were negative. The viral DNA was extracted, amplified using the polymerase chain reaction and the HAdV-positive samples were sequenced and phylogenetically analyzed. Statistical analyses were performed using the Chi-square test (p<0.05), considering two conditions: the total of samples tested (377) and the total of negative samples for the remaining viruses tested (314). The overall prevalence of HAdV was 12.47% (47/377); and in 76.60% (36/47) of the positive samples, this virus was the only infectious agent detected. The phylogenetic analysis of partial sequences of 32 positive samples revealed that they all clustered with the HAdV-F type 41. The statistical analysis showed that there was no correlation between the onset of the HAdV infection and the origin of the samples (inpatients or outpatients) in the two conditions tested: the total of samples tested (p=0.598) and the total of negative samples for the remaining viruses tested (p=0.614). There was a significant association in the occurrence of infection in children aged 0-12 months for the condition 1 (p=0.030) as well as condition 2 (p=0.019). The occurrence of infections due to HAdV did not coincide with a pattern of seasonal distribution. These data indicate the significant involvement of HAdV-F type 41 in the etiology of ADD in Minas Gerais, which demonstrates the importance of other viral agents in the development of the disease after the introduction of rotavirus vaccine immunization.

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Human Species D Adenoviruses Isolated from Diarrheal Feces Show Low Infection Rates in Primary Nasal Epithelial Cells.

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