An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

Rao, D. Narayana; Reddy, Harikrishna; Naidu, Jagadish R.; Raghavendra, A.; Radhika, N.S.; Karande, Anjali A.

doi:10.1016/j.jviromet.2015.08.007

Cited by 6 publications

(7 citation statements)

References 55 publications

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“…The non-polio enterovirus (NPEV) infections were associated with acute and persistent diarrhea (18–21% of diarrheal cases) ( Rao et al, 2013 ; Rao, Maiya & Babu, 2014 ) as well as increased frequency of bowel movements (approximately 29%) ( Rao et al, 2014 ). Another study also revealed the evidence of some enterovirus strains induced severe diarhea in mouse model ( Rao et al, 2015 ). In the case control study, the NPEV was more prevalent in patients with diarrhea (13.7–17%) than in healthy children (4.5–4.9%) ( Patil, Chitambar & Gopalkrishna, 2015 ; Rao et al, 2013 ).…”

Section: Discussionmentioning

confidence: 89%

Enterovirus infections in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, 2015–2018

Rojjanadumrongkul

Khamrin

Ukarapol

et al. 2020

PeerJ

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Background Infection with viruses especially rotavirus, norovirus, astrovirus, and adenovirus has been known to be a major cause of acute gastroenteritis in children under 5 years of age globally, particularly in developing countries. Also, some genotypes of enteroviruses (EVs) have been reported to be associated with gastroenteritis. This study is aimed to investigate the prevalence and genotype diversity of EV in children admitted to hospitals with acute gastroenteritis. Methods A total of 1,736 fecal specimens were collected from children hospitalized with diarrhea in Chiang Mai, Thailand from 2015 to 2018. All specimens were tested for the presence of EV by RT-PCR of the 5′ untranslated region. The genotypes of EV were further identified by nucleotide sequencing and phylogenetic analysis of the viral protein 1 (VP1) gene. Results EV was detected in 154 out of 1,736 specimens (8.9%) throughout the study period. The prevalence of EV detected in 2015, 2016, 2017, and 2018 was 7.2%, 9.0%, 11.2%, and 8.6%, respectively. EV was detected all year round with a high prevalence during rainy season in Thailand. Overall, 37 genotypes of EV were identified in this study. Among these, coxsackievirus (CV)-A24 and CV-B5 (7.5% each), and EV-C96 (6.8%) were the common genotypes detected. Conclusion This study demonstrates the prevalence, seasonal distribution, and genotype diversity of EV circulating in children hospitalized with acute gastroenteritis in Chiang Mai, Thailand during the period 2015 to 2018.

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Section: Discussionmentioning

confidence: 89%

Enterovirus infections in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, 2015–2018

Rojjanadumrongkul

Khamrin

Ukarapol

et al. 2020

PeerJ

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“…Turning next to the effects of regulating the synthesis of relocalized host cell proteins on infectious virus yields, Fig. 7 shows that while the siRNA-mediated downregulation of HuR, hnRNP D 45 , hnRNP I, and hnRNP K decreased progeny virus titers by almost 2 orders of magnitude, their overexpression resulted in only a 6-to 8-fold increase in the progeny virus titers, as determined in MA104 cells by an enzyme-linked immunoperoxidase focus assay (ELIFA) (44)(45)(46). In contrast, the downregulation of G3BP1, TIA1, and hnRNP C1 increased the progeny virus yields by 3, 2.4, and 2.5 orders of magnitude, respectively, while ectopic overexpression resulted in reductions of virus yields by 3.2, 1.6, and 2.0 orders of magnitude, respectively (Fig.…”

Section: Figmentioning

confidence: 99%

Cytoplasmic Relocalization and Colocalization with Viroplasms of Host Cell Proteins, and Their Role in Rotavirus Infection

Dhillon

Tandra

Chorghade

et al. 2018

J Virol

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Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), which are nucleated by two essential viral nonstructural proteins, NSP2 and NSP5. However, the precise composition of the VM, the intracellular localization of host proteins during virus infection, and their association with VMs or role in rotavirus growth remained largely unexplored. Mass spectrometry analyses revealed the presence of several host heterogeneous nuclear ribonucleoproteins (hnRNPs), AU-rich element-binding proteins (ARE-BPs), and cytoplasmic proteins from uninfected MA104 cell extracts in the pulldown (PD) complexes of the purified viroplasmic proteins NSP2 and NSP5. Immunoblot analyses of PD complexes from RNase-treated and untreated cell extracts, analyses of coimmunoprecipitation complexes using RNase-treated infected cell lysates, and direct binding assays using purified recombinant proteins further demonstrated that the interactions of the majority of the hnRNPs and ARE-BPs with viroplasmic proteins are RNA independent. Time course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells revealed a surprising sequestration of the majority of the relocalized host proteins in viroplasms. Analyses of ectopic overexpression and small interfering RNA (siRNA)-mediated downregulation of expression revealed that host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces the cytoplasmic relocalization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than is currently understood. Rotavirus replicates exclusively in the cytoplasm. Knowledge on the relocalization of nuclear proteins to the cytoplasm or the role(s) of host proteins in rotavirus infection is very limited. In this study, it is demonstrated that rotavirus infection induces the cytoplasmic relocalization of a large number of nuclear RNA-binding proteins (hnRNPs and AU-rich element-binding proteins). Except for a few, most nuclear hnRNPs and ARE-BPs, nuclear transport proteins, and some cytoplasmic proteins directly interact with the viroplasmic proteins NSP2 and NSP5 in an RNA-independent manner and become sequestered in the viroplasms of infected cells. The host proteins differentially affected viral gene expression and virus growth. This study demonstrates that rotavirus induces the relocalization and sequestration of a large number of host proteins in viroplasms, affecting host processes in both compartments and generating conditions conducive for virus growth in the cytoplasm of infected cells.

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“…ELIFA. Rotavirus titers in virus preparations and the effects of ectopic expression of cellular proteins and their downregulation of expression by siRNAs on the progeny virus yield were determined using an enzyme-linked immunoperoxidase focus assay (ELIFA) as previously described (11,136). HEK293T cells were infected with 400 focus-forming units (FFU) per well in 24-well plates, and the progeny virus titers in MA104 cells were determined as described by Dhillon et al (11).…”

Section: Fig 10mentioning

confidence: 99%

Rotavirus Induces Formation of Remodeled Stress Granules and P Bodies and Their Sequestration in Viroplasms To Promote Progeny Virus Production

Dhillon

Rao

2018

J Virol

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Rotavirus replicates in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), the composition and structure of which have yet to be understood. Based on the analysis of a few proteins, earlier studies reported that rotavirus infection inhibits stress granule (SG) formation and disrupts P bodies (PBs). However, the recent demonstration that rotavirus infection induces cytoplasmic relocalization and colocalization with VMs of several nuclear hnRNPs and AU-rich element-binding proteins (ARE-BPs), which are known components of SGs and PBs, suggested the possibility of rotavirus-induced remodeling of SGs and PBs, prompting us to analyze a large number of the SG and PB components to understand the status of SGs and PBs in rotavirus-infected cells. Here we demonstrate that rotavirus infection induces molecular triage by selective exclusion of a few proteins of SGs (G3BP1 and ZBP1) and PBs (DDX6, EDC4, and Pan3) and sequestration of the remodeled/atypical cellular organelles, containing the majority of their components, in the VM. The punctate SG and PB structures are seen at about 4 h postinfection (hpi), coinciding with the appearance of small VMs, many of which fuse to form mature large VMs with progression of infection. By use of small interfering RNA (siRNA)-mediated knockdown and/or ectopic overexpression, the majority of the SG and PB components, except for ADAR1, were observed to inhibit viral protein expression and virus growth. In conclusion, this study demonstrates that VMs are highly complex supramolecular structures and that rotavirus employs a novel strategy of sequestration in the VM and harnessing of the remodeled cellular RNA recycling bins to promote its growth.IMPORTANCE Rotavirus is known to replicate in specialized virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), but the composition and structure of VMs are not yet understood. Here we demonstrate that rotavirus interferes with normal SG and PB assembly but promotes formation of atypical SG-PB structures by selective exclusion of a few components and employs a novel strategy of sequestration of the remodeled SG-PB granules in the VMs to promote virus growth by modulating their negative influence on virus infection. Rotavirus VMs appear to be complex supramolecular structures formed by the union of the triad of viral replication complexes and remodeled SGs and PBs, as well as other host factors, and designed to promote productive virus infection. These observations have implications for the planning of future research with the aim of understanding the structure of the VM, the mechanism of morphogenesis of the virus, and the detailed roles of host proteins in rotavirus biology.

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An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

Cited by 6 publications

References 55 publications

Enterovirus infections in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, 2015–2018

Enterovirus infections in pediatric patients hospitalized with acute gastroenteritis in Chiang Mai, Thailand, 2015–2018

Cytoplasmic Relocalization and Colocalization with Viroplasms of Host Cell Proteins, and Their Role in Rotavirus Infection

Rotavirus Induces Formation of Remodeled Stress Granules and P Bodies and Their Sequestration in Viroplasms To Promote Progeny Virus Production

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