Cause Of Severe Diarrheal Disease Research Articles

Cryptosporidium infection of human small intestinal epithelial cells induces type III interferon and impairs infectivity of Rotavirus

ABSTRACT Cryptosporidiosis is a major cause of severe diarrheal disease in infants from resource poor settings. The majority of infections are caused by the human-specific pathogen C. hominis and absence of in vitro growth platforms has limited our understanding of host-pathogen interactions and development of effective treatments. To address this problem, we developed a stem cell-derived culture system for C. hominis using human enterocytes differentiated under air-liquid interface (ALI) conditions. Human ALI cultures supported robust growth and complete development of C. hominis in vitro including all life cycle stages. Cryptosporidium infection induced a strong interferon response from enterocytes, possibly driven, in part, by an endogenous dsRNA virus in the parasite. Prior infection with Cryptosporidium induced type III IFN secretion and consequently blunted infection with Rotavirus, including live attenuated vaccine strains. The development of hALI provides a platform for further studies on human-specific pathogens, including clinically important coinfections that may alter vaccine efficacy.

Model-based estimation of the impact on rotavirus disease of RV3-BB vaccine administered in a neonatal or infant schedule

ABSTRACT Rotavirus infection is a common cause of severe diarrheal disease and a major cause of deaths and hospitalizations among young children. Incidence of rotavirus has declined globally with increasing vaccine coverage. However, it remains a significant cause of morbidity and mortality in low-income countries where vaccine access is limited and efficacy is lower. The oral human neonatal vaccine RV3-BB can be safely administered earlier than other vaccines, and recent trials in Indonesia have demonstrated high efficacy. In this study, we use a stochastic individual-based model of rotavirus transmission and disease to estimate the anticipated population-level impact of RV3-BB following delivery according to either an infant (2, 4, 6 months) and neonatal (0, 2, 4 months) schedule. Using our model, which incorporated an age- and household-structured population and estimates of vaccine efficacy derived from trial data, we found both delivery schedules to be effective at reducing infection and disease. We estimated 95–96% reductions in infection and disease in children under 12 months of age when vaccine coverage is 85%. We also estimate high levels of indirect protection from vaccination, including 78% reductions in infection in adults over 17 years of age. Even for lower vaccine coverage of 55%, we estimate reductions of 84% in infection and disease in children under 12 months of age. While open questions remain about the drivers of observed lower efficacy in low-income settings, our model suggests RV3-BB could be effective at reducing infection and preventing disease in young infants at the population level.

Lambda Red Recombineering in Shigella flexneri.

Shigellosis remains a major cause of severe diarrheal disease and death throughout the world. Vaccine development against shigellosis has been hampered by an incomplete understanding of the molecular mechanisms by which Shigella spp. causes disease and difficulties in manipulating Shigella spp. genomes. While homologous recombination protocols for the construction of precise gene deletions exist, construction of mutants in S. flexneri has not become commonplace. We describe the steps for construction of gene deletions using λ-red recombination using tools that we have developed in our laboratory.

The Biology of the Intestinal Intracellular Parasite Cryptosporidium

Cryptosporidium emerged as a leading global cause of severe diarrheal disease in children. The parasite occupies a unique intracellular niche at the brush border of intestinal epithelial cells, where it undergoes a complex sexual life cycle. How this life cycle unfolds and how host and parasite interact remain largely to be discovered. A series of technical advances now offer genetic and immunological tools for mechanistic investigation of the parasite. Here we introduce the pathogen and disease and highlight important questions to tackle onward. We invite scientists to consider this versatile parasite model to probe the biology and immunology of the intestine.

Articles of Significant Interest in This Issue

Rotaviruses are a leading cause of severe diarrheal disease in young children throughout the world. Using reverse genetics technologies, Philip and Patton (e00959-20) found that the rotavirus RNA genome is remarkably flexible and can accommodate additional coding sequence sufficient to allow expression of separate foreign proteins. Among the recombinant rotaviruses that were engineered are those that produce fluorescent reporters, enabling the study of rotavirus biology and pathogenesis by live cell imaging. Given the capacity of rotavirus to encode foreign proteins, the potential exists for developing rotavirus vaccines as vector systems against other enteric and mucosal diseases.

Evaluation of the safety profile of rotavirus vaccines: a pharmacovigilance analysis on American and European data

Rotaviruses (RVs) are the most common cause of severe diarrheal disease. To date two rotavirus oral vaccines are licensed: Rotarix and Rotateq. Our aim was to contribute to the post-marketing evaluation of these vaccines safety profile. We collected all RV vaccines-related reports of Adverse Events Following Immunization (AEFI) in US Vaccine Adverse Events Reporting System (VAERS) and VigiBase between January 2007 and December 2017. A disproportionality analysis using Reporting Odds Ratio (ROR) was performed. A total of 17,750 reports in VAERS and 6,358 in VigiBase were retrieved. In VAERS, 86.2% of the reports concerned RotaTeq, whereas in VigiBase 67.7% of them involved Rotarix. Across the databases, diarrhea (1,672 events in VAERS, 1,961 in VigiBase) and vomiting (1,746 in VAERS, 1,508 in VigiBase) were the most reported AEFIs. Noteworthy, the RV vaccines-intussusception pair showed a ROR greater than 20 in both databases. Some new potential safety signals emerged such as fontanelle bulging, hypotonic-hyporesponsive episode, livedo reticularis, and opisthotonus. Overall, our data show that most of the reported AEFIs are listed in the Summary of Product Characteristics (SPCs). However, there remains the need to investigate the potential safety signals arose from this analysis, in order to complete the description of the AEFIs.

Genetic ablation of purine salvage in Cryptosporidium parvum reveals nucleotide uptake from the host cell

The apicomplexan parasite Cryptosporidium is a leading global cause of severe diarrheal disease and an important contributor to early-childhood mortality. Waterborne outbreaks occur frequently, even in countries with advanced water treatment capabilities, and there is currently no fully effective treatment. Nucleotide pathways are attractive targets for antimicrobial development, and several laboratories are designing inhibitors of these enzymes as potential treatment for Cryptosporidium infections. Here we take advantage of newly available molecular genetics for Cryptosporidium parvum to investigate nucleotide biosynthesis by directed gene ablation. Surprisingly, we found that the parasite tolerates the loss of classical targets including dihydrofolate reductase-thymidylate synthase (DHFR-TS) and inosine monophosphate dehydrogenase (IMPDH). We show that thymidine kinase provides a route to thymidine monophosphate in the absence of DHFR-TS. In contrast, only a single pathway has been identified for C. parvum purine nucleotide salvage. Nonetheless, multiple enzymes in the purine pathway, as well as the adenosine transporter, can be ablated. The resulting mutants are viable under normal conditions but are hypersensitive to inhibition of purine nucleotide synthesis in their host cell. Cryptosporidium might use as-yet undiscovered purine transporters and salvage enzymes; however, genetic and pharmacological experiments led us to conclude that Cryptosporidium imports purine nucleotides from the host cell. The potential for ATP uptake from the host has significant impact on our understanding of parasite energy metabolism given that Cryptosporidium lacks oxidative phosphorylation and glycolytic enzymes are not constitutively expressed throughout the parasite life cycle.

A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability

SummaryDespite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using “air-liquid interface” (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

Colostrum from cows immunized with a veterinary vaccine against bovine rotavirus displays enhanced in vitro anti-human rotavirus activity

Human rotaviruses represent a major cause of severe diarrheal disease in infants and young children. The limited impact of oral vaccines on global estimates of rotavirus mortality and the suboptimal use of oral rehydration justify the need for alternative prophylactic and therapeutic strategies, especially for immunocompromised hosts. The protective effects of colostrum—the first milk produced during the initial 24 to 48 h after parturition—are well documented in the literature. In particular, the ingestion of hyperimmune bovine colostrum has been proposed as an alternative preventive approach against human rotavirus gastroenteritis. Although the immunization of pregnant cows with human rotavirus boosts the release of specific immunoglobulin G in bovine colostrum, it raises regulatory and safety issues. In this study, we demonstrated that the conventional bovine rotavirus vaccine is sufficient to enhance the anti-human rotavirus protective efficacy of bovine colostrum, thus providing a conservative approach to produce hyperimmune bovine colostrum, making it exploitable as a functional food.

Forward Genetics in <I>Cryptosporidium</I> Enabled by Complete in Vitro Development in Stem Cell-Derived Intestinal Epithelium

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile culture methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using “air-liquid interface” (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable complete development of the parasite life cycle in vitro. ALI cultures supported parasite expansion >100-fold and led to the production of viable oocysts that were transmissible in vitro and to mice. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating conventional meiosis and Mendelian segregation. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

Prevalence and genotypes of group A rotavirus among outpatient children under five years old with diarrhea in Beijing, China, 2011\u20132016

BackgroundRotavirus is a leading cause of severe diarrheal disease, and one of the common causes of death in children aged under five years old. The dominant epidemic strains may change in different years in the same area. In order to provide evidence for rotavirus epidemic control and inform vaccine development, we analyzed epidemiological patterns and genetic characteristics of rotavirus in Beijing during 2011–2016.MethodsStool specimens of outpatient children under five years old were collected from three children’s hospitals on a weekly basis. Group A rotavirus antigens were detected using enzyme-linked immunosorbent assay (ELISA) kit. The partial VP4 genes and VP7 genes of rotavirus were both amplified and sequenced. Genotyping and phylogenetic analyses were performed. Logistic regression and Chi-square tests were performed to determine differences across age groups, districts and years in rotavirus prevalence and genotype distribution.ResultsA total of 3668 stool specimens from children with acute diarrhea identified through hospital-based surveillance were collected from 2011 to 2016 in Beijing. A total of 762 (20.8%) specimens tested positive for rotavirus. The rotavirus-positive rate was highest among the 1–2 years old age group (29.0%, 310/1070). November, December and January were the highest rotavirus-positive rate months each year. G9 was the most common G genotype (64.4%, 461/716), and P [8] was the most common P genotype (87.0%, 623/716) among the 716 rotavirus-positive specimens. G9P [8], G3P [8] and G2P [4] were the most common strains. The rotavirus-positive rates of samples in 2012 and 2013 were higher than that in 2011, and the dominant genotype changed from G3P [8] to G9P [8] in 2012 and 2013. VP7 gene sequences of G9 strains in this study clustered into two main lineages. Most of the G9 strains exhibited the highest nucleotide similarity (99.1%~ 100.0%) to the strain found in Japan (MI1128). VP4 gene sequences of P [8] strains were almost P[8]b.ConclusionsRotavirus accounted for more than one fifth of childhood diarrhea in Beijing during the study period. Targeted measures such as immunization with effective rotavirus vaccines should be carried out to reduce the morbidity and mortality due to rotavirus.

Oral Administration of a Seed-based Bivalent Rotavirus Vaccine Containing VP6 and NSP4 Induces Specific Immune Responses in Mice.

Rotavirus is the leading cause of severe diarrheal disease among newborns. Plant-based rotavirus vaccines have been developed in recent years and have been proven to be effective in animal models. In the present study, we report a bivalent vaccine candidate expressing rotavirus subunits VP6 and NSP4 fused with the adjuvant subunit B of E. coli heat-labile enterotoxin (LTB) in maize seeds. The RT-PCR and Western blot results showed that VP6 and LTB-NSP4 antigens were expressed and accumulated in maize seeds. The expression levels were as high as 0.35 and 0.20% of the total soluble protein for VP6 and LTB-NSP4, respectively. Oral administration of transgenic maize seeds successfully stimulated systemic and mucosal responses, with high titers of serum IgG and mucosal IgA antibodies, even after long-term storage. This study is the first to use maize seeds as efficient generators for the development of a bivalent vaccine against rotavirus.

Molecular surveillance of rotavirus strains circulating in Yaoundé, Cameroon, September 2007–December 2012

Rotavirus is the most common cause of severe diarrheal disease in children under 5years of age worldwide. The World Health Organization (WHO) estimated that 453,000 rotavirus-attributable deaths occur annually. Through the WHO, the Rotavirus Sentinel Surveillance Program was established in Cameroon in September 2007 with the Mother and Child Center (MCC) in Yaoundé playing the role of sentinel site and national laboratory for this program. The objectives of this surveillance were to assess the rotavirus disease burden and collect baseline information on rotavirus strains circulating in Cameroon. Diarrheal stool samples were collected in a pediatric hospital from children under 5, using the WHO case definition for rotavirus diarrhea. Antigen detection of rotavirus was performed by using an enzyme immunoassay (EIA). The genotypic characterization was performed using multiplexed semi-nested reverse transcription-polymerase chain reaction (RT-PCR) assays. Between September 2007 and December 2012, 2444 stool samples were received at the MCC laboratory for rotavirus antigen detection, of which 999 (41%) were EIA positive. Among EIA positive samples 898 were genotyped. Genotype prevalence varied each year. Genotype G9P[8] was the dominant type during 2007 (32%) and 2008 (24%), genotype G3P[6] predominated in 2010 (36%) and 2011 (25%), and G1P[8] was predominant in 2012 (44%). The findings showed that the rotavirus disease burden is high and there is a broad range of rotavirus strains circulating in Yaoundé. These data will help measure the impact of vaccination in the future.

Predicted structure and domain organization of rotavirus capping enzyme and innate immune antagonist VP3.

Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5'-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2'-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2'-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2'-5' oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3. Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.

Rotavirus gastroenteritis in Latin America: A hospital-based study in children under 3 years of age

Rotavirus is the leading cause of severe diarrheal disease and dehydration in infants in both developed and developing countries. Vaccines have recently been developed, but detailed epidemiological information, which is needed for decisions about how and where to introduce vaccination, was lacking for many Latin American countries. The primary objective of this study was to measure the incidence and disease burden of rotavirus in young children presenting to Latin American hospitals with gastroenteritis. In addition it allowed to setting up the methodology to further conduct a large phase III trial with a rotavirus vaccine in the region. This was a prospective, multi-center surveillance study of gastroenteritis in children <3years old presenting to hospitals in 11 Latin American countries. Questionnaires and stool samples were collected from 6521 of 8031 enrolled cases (73% inpatients). Among these, 3122 (49%) were rotavirus positive. Of the rotavirus-positive cases, 12% were <6months, 48% <1year and 87% <2years old; 23% received antibiotics before diagnosis. Median hospital stay was 2days, 78% received intravenous rehydration. Overall strain distribution was G1 (59%), G2 (1%), G3 (12%), G4 (20%), G9 (6%), G12 (1%), untypable (7%) with large local variations. The direct economic impact on families was considerable: 48% of caregivers lost time from paid work and 69% of families were financially affected by their child's illness. This study confirms the high disease burden of rotavirus gastroenteritis among children in Latin America, which might be reduced by the use of effective vaccines.

Review of Rotavirus Studies in Africa: 1976–2006

Rotavirus is a common cause of severe diarrheal disease in children worldwide. Ninety percent of the associated deaths occur in sub-Saharan Africa and South East Asia. Our aim was to review the prevalence of rotavirus infection in Africa over the past 30 years. Eligible studies were identified from 3 Medline searches. Only studies including children <5 years of age that included >50 children and had an observational period >3 months were included. The data were analyzed during 3 periods (1976-1985, 1986-1995, and 1996-2006), as a summary (1976-2006), and by different study settings (ie, hospital, outpatient department, and combined). The initial search identified 206 studies from 27 countries during 1976-2006. The refined search yielded 101 studies, of which 58 (57%) were hospital based, 25 (25%) were outpatient studies, and 18 (18%) were combined. Rotavirus was detected in 25% (interquartile range, 16%-32%) of stool samples. Rotavirus was the most common agent identified in 73% of studies in which multiple diarrheal agents were determined. Rotavirus is an important cause of severe diarrheal disease in children <5 years of age in Africa. Clinical trials in South Africa and Malawi have shown that severe rotavirus disease is a vaccine-preventable entity in Africa.

Burden and Epidemiology of Rotavirus Diarrhea in Selected African Countries: Preliminary Results from the African Rotavirus Surveillance Network

Severe rotavirus diarrhea in children <5 years of age is a major public health problem; however, limited regional and country specific data on rotavirus disease burden are available from sub-Saharan Africa. In June 2006, the World Health Organization Regional Office for Africa initiated rotavirus surveillance in selected African countries. With use of standardized methodology developed by the World Health Organization, children <5 years of age who were hospitalized with severe diarrhea were enrolled, and stool specimens were collected for detection of rotavirus strains with use of a commercial enzyme immunoassay. Rotavirus strains were further characterized for G and P types with use of a reverse-transcriptase polymerase chain reaction. From June 2006 through December 2008, rotavirus surveillance was established at 14 sites in 11 African countries. Of 5461 stool samples collected from children enrolled in 8 countries with 1 or 2 complete years of data, 2200 (40%) were positive for rotavirus. Ninety percent of all rotavirus hospitalizations occurred among children aged 3-12 months. Predominant types included G1P[8] (21%), G2P[4] (7%), and P [8] (29%); however, unusual types were also detected, including G8P[6] (5%), G8P[8] (1%), G12P[6] (1%), and G12P[6] (1%). A high percentage of mixed rotavirus infections was also detected. These preliminary results indicate that rotavirus is a major cause of severe diarrheal disease in African children.

Porcine rotavirus strain Gottfried-based human rotavirus candidate vaccines: Construction and characterization

Rotavirus gastroenteritis remains the leading cause of severe diarrheal disease in infants and young children worldwide, and thus, a safe and effective rotavirus vaccine is urgently needed in both developing and developed countries. Various candidate rotavirus vaccines that were developed by us and others have been or are being evaluated in different populations in various parts of the world. We have recently confirmed that a porcine rotavirus Gottfried strain bears a P (VP4) serotype (P2B[6]) closely related to human rotavirus P serotype 2A[6] which is of epidemiologic importance in some regions of the world. Based on the modified Jennerian approach to immunization, we have constructed 11 Gottfried-based single VP7 or VP4 gene substitution reassortant vaccine candidates which could provide: (i) an attenuation phenotype of a porcine rotavirus in humans; and (ii) antigenic coverage for G serotypes 1–6 and 8–10 and P serotype 1A[8], 1B[4] and 2A[6]. In addition, following immunization of guinea pigs with Gottfried VP4, we found low but consistent levels of neutralizing antibodies to VP4 with P1A[8] or P1B[4] specificity, both of which are of global epidemiologic importance. Thus, porcine-based VP7 reassortant rotavirus vaccines may provide an advantage over rhesus- or bovine-based VP7 reassortant vaccines since the VP4s of the latter vaccines do not evoke antibodies capable of neutralizing the viruses bearing P1A[8], P1B[4] or P2A[6] VP4.

Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb

Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb’s directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.

Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb.

Rotaviruses are the leading cause of severe diarrheal disease in young children. Intestinal mucosal IgA responses play a critical role in protective immunity against rotavirus reinfection. Rotaviruses consist of three concentric capsid layers surrounding a genome of 11 segments of double-stranded RNA. The outer layer proteins, VP4 and VP7, which are responsible for viral attachment and entry, are targets for protective neutralizing antibodies. However, IgA mAb's directed against the intermediate capsid protein VP6, which do not neutralize the virus, have also been shown to protect mice from rotavirus infection and clear chronic infection in SCID mice. We investigated whether the anti-VP6 IgA (7D9) mAb could inhibit rotavirus replication inside epithelial cells and found that 7D9 acted at an early stage of infection to neutralize rotavirus following antibody lipofection. Using electron cryomicroscopy, we determined the three-dimensional structure of the virus-antibody complex. The attachment of 7D9 IgA to VP6 introduces a conformational change in the VP6 trimer, rendering the particle transcriptionally incompetent and preventing the elongation of initiated transcripts. Based on these observations, we suggest that anti-VP6 IgA antibodies confers protection in vivo by inhibiting viral transcription at the start of the intracellular phase of the viral replication cycle.