Human Rotavirus Infection Research Articles

IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.

Cyclophilin A restricts rotavirus infection by enhancing type 1 interferon response in infected epithelial cells (68.1)

Abstract Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host protein synthesis, however, previous studies have revealed that RV infection can also lead to the upregulation of host proteins, which could play crucial roles in RV infection. Using proteomic methods, we found that Cyclophilin A (CYPA), a peptidyl-prolyl cis-trans isomerase (PPIase), was upregulated in MA104 cells infected by human rotavirus (HRV). Following infection, CYPA was recruited to the viroplasm where it interacted with HRV structural protein VP2, to inhibit viral protein expressions, thus producing a repressive effect on HRV replication, which was dependent on its PPIase activity. We further show that CYPA was required for host type-1 interferon (IFN-I) response to HRV infection, and this effect was independent on its PPIase activity but dependent on JNK signaling pathway. Furthermore, we found that the expression of CYPA in enterocytes correlates to the period when BALB/c mice become resistannt to RV infection-induced diarrhea. Together, our data identify CYPA as a new host restrictive factor that confers protection against rotavirus infection.

Lactobacillus acidophilus and Lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus

Probiotic lactic acid bacteria (LAB) have been shown to alleviate inflammation, enhance the immunogenicity of rotavirus vaccines, or reduce the severity of rotavirus diarrhoea. Although the mechanisms are not clear, the differential Th1/Th2/Th3-driving capacities and modulating effects on cytokine production of different LAB strains may be the key. Our goal was to delineate the influence of combining two probiotic strains of Lactobacillus acidophilus and Lactobacillus reuteri on the development of cytokine responses in neonatal gnotobiotic pigs infected with human rotavirus (HRV). We demonstrated that HRV alone, or HRV plus LAB, but not LAB alone, initiated serum cytokine responses, as indicated by significantly higher concentrations of IFN-α, IFN-γ, IL-12, and IL-10 at postinoculation day (PID) 2 in the HRV only and LAB+HRV+ pigs compared to LAB only and LAB-HRV- pigs. Peak cytokine responses coincided with the peak of HRV replication. LAB further enhanced the Th1 and Th2 cytokine responses to HRV infection as indicated by significantly higher concentrations of IL-12, IFN-γ, IL-4 and IL-10 in the LAB+HRV+ pigs compared to the LAB-HRV+ pigs. The LAB+HRV+ pigs maintained relatively constant concentrations of TGF-β compared to the HRV only group which had a significant increase at PID 2 and decrease at PID 7, suggesting a regulatory role of LAB in maintaining gut homeostasis. At PID 28, cytokine secreting cell (CSC) responses, measured by ELISpot, showed increased Th1 (IL-12, IFN-γ) CSC numbers in the LAB+HRV+ and LAB-HRV+ groups compared to LAB only and LAB-HRV- pigs, with significantly increased IL-12 CSCs in spleen and PBMCs and IFN-γ CSCs in spleen of the LAB+HRV+ group. Thus, HRV infection alone, but not LAB alone was effective in inducing cytokine responses but LAB significantly enhanced both Th1 and Th2 cytokines in HRV-infected pigs. LAB may also help to maintain immunological homeostasis during HRV infection by regulating TGF-β production.

Characterization of immune modulating functions of γδ T cell subsets in a gnotobiotic pig model of human rotavirus infection

We characterized immune modulating functions of porcine γδ T cell subsets in rotavirus infection using a gnotobiotic pig model of human rotavirus infection and sort-purified lymphocyte autologous co-cultures. We demonstrated that CD2+CD8- and CD2-CD8- γδ T cells have mainly pro-inflammatory function as evident by directly secreting IFN-γ or promoting CD4+ αβ T cell proliferation and IFN-γ production, whereas CD2+CD8+ γδ T cells mainly exert regulatory T cell function by expressing FoxP3, secreting IL-10 and TGF-β or increasing IL-10 and TGF-β production by CD4+ αβ T cells. γδ T cells responded to rotavirus infection by increasing TLR2, TLR3, TLR9 expression and IFN-γ and/or TGF-β production. The CD8- subsets likely differentiate into CD8+ subset by acquiring CD8 expression, explaining in part the apparently dual functions of CD2+CD8+ and CD2+CD8- subsets. Thus, both CD8+ and CD8- γδ T cell subsets can contribute to anti-rotavirus immunity and to the maintenance and restoration of intestinal and systemic homeostasis.

Rotavirus and biliary atresia

New knowledge on rotavirus infection in children and well established mouse models has renewed interest in whether rotavirus could cause biliary atresia, an idiopathic, obliterative infantile disease of bile ducts that is the primary indication for liver transplant in children. Studies in the rotavirus mouse model of biliary atresia indicate that infection of biliary epithelium is an inaugural event leading to biliary inflammation and obstruction, which is preceded by systemic spread of rotavirus, which also occurs during human rotavirus enteric infections. Viral factors, including rotavirus gene 4, are important for biliary infection and biliary atresia in mice. Specific host factors related to inflammatory processes (natural killer and T cells, interferon) are also critical, and a paucity of regulatory T cells in neonates may play a key role in pathogenesis in experimental biliary atresia. Rotavirus vaccination has substantially decreased rotavirus diarrheal disease worldwide and might enable demonstration of a cause-effect relationship between rotavirus infection and biliary atresia in humans. Rotavirus can be detected in the serum of mice and children and causes biliary atresia in neonatal mice. Approaches to re-examine whether rotavirus causes biliary atresia in children are discussed based on concepts from the mouse model of biliary atresia and rotavirus vaccination programs.

Evaluation of Hyperimmune Hen Egg Yolk Derived Anti-Human Rotavirus Antibodies (Anti-HRVIgY) against Rotavirus Infection

Oral delivery of specific IgY has been reported to be beneficial against rotavirus infection. However, the production of IgYs against globally prevalent human rotavirus (HRV) serotypes and their evaluation detailing the influence on the virological/histopathological consequences have not been reported to date. In the present study, anti-HRVIgY was generated in the eggs of specific pathogen free hens immunized with HRV serotypes G1 - G4 and G9 independently. Purified anti-HRVIgY preparations were tested to determine the ELISA and neutralizing antibody titers respectively in an indirect ELISA and cell culture based neutralization assay. Efficacy of pre and post infection treatment of anti-HRV-3IgY was assessed in an infant BALB/c mouse model of human rotavirus infection by monitoring percent diarrhea, severity and duration of diarrhea, intestinal viral load and histopathology. High (1:64000 - 1:512000) titered anti-HRVIgYs were obtained from the egg yolk of immunized hens with peak titer value (1:256000/1:512000) at 40 - 60 day of immunization. In-vitro, each of the anti-HRVIgY preparations showed the presence of multiserotypic neutralizing activity with high (1:1600 - ≥1:6400) homologous and low (≤1:50 - 1:800) heterologous titers. However, anti-HRV-3IgY neutralized all of the serotypes tested in the study indicating broader in-vitro neutralizing activity. In mice, post exposure treatment with anti-HRV-3IgY significantly reduced the extent of diarrhea and intestinal virus load and inhibited histopathological changes whereas pre exposure anti-HRV-3IgY treatment imparted immediate protection from development of rotavirus gastroenteritis. Thus, the anti-HRVIgY administered orally decreased morbidity and disease incidences in mice suggesting its potential implication in prophylactic and therapeutic usage in human to achieve reduction in rotavirus disease burden.

Detection of human G10 rotavirus strains with similarity to bovine and bovine-like equine strains from untypable samples

G10 rotaviruses, which are usually found in cattle, have also been reported in neonatal infections in recent years. During the rotavirus surveillances of children less than 4years of age between 2003 and 2006 in Kolkata, eastern India, 60 out of 1153 samples could not be typed. All 60 samples gave usual electropherotype pattern in polyacrylamide gel. Thirty-one out of these 60 G and P untypable rotavirus strains were successfully characterized during the study. Among 31 samples, G9P[4] (n=8), G12P[8] (n=8), G1P[8] (n=6), G10P[4] (n=6), and G2P[4] (n=3) genotypes were identified. In this study we report genetic analysis of the six G10 strains, which revealed close relations with Turkish (E29TR) bovine strains, as well as with bovine-like-equine strain (Erv2) from India. SimPlot of the VP7 gene segment suggested possible recombination event between the bovine and the bovine-like-equine rotaviruses in these human rotavirus infections.

Molecular characterization of group A rotaviruses detected in children with gastroenteritis in Ireland in 2006–2009

Community and hospital-acquired cases of human rotavirus are responsible for millions of gastroenteritis cases in children worldwide, chiefly in developing countries, and vaccines are now available. During surveillance activity for human rotavirus infections in Ireland, between 2006 and 2009, a total of 420 rotavirus strains were collected and analysed. Upon either PCR genotyping and sequence analysis, a variety of VP7 (G1-G4 and G9) and VP4 (P[4], P[6], P[8] and P[9]) genotypes were detected. Strains G1P[8] were found to be predominant throughout the period 2006-2008, with slight fluctuations seen in the very limited samples available in 2008-2009. Upon either PCR genotyping and sequence analysis of selected strains, the G1, G3 and G9 viruses were found to contain E1 (Wa-like) NSP4 and I1 VP6 genotypes, while the analysed G2 strains possessed E2 NSP4 and I2 VP6 genotypes, a genetic make-up which is highly conserved in the major human rotavirus genogroups Wa- and Kun-like, respectively. Upon sequence analysis of the most common VP4 genotype, P[8], at least two distinct lineages were identified, both unrelated to P[8] Irish rotaviruses circulating in previous years, and more closely related to recent European humans rotaviruses. Moreover, sequence analysis of the VP7 of G1 rotaviruses revealed the onset of a G1 variant, previously unseen in the Irish population.

Kefir Grain의 Polysaccharide에 의한 HRV S-2의 MA-104 세포 감염억제

The inhibitory effect of a polysaccharide from kefir on human rotavirus infection in MA-104 cells was investigated. The extracted polysaccharide was separated as fraction I in unbound materials and as fractions II, III, and IV in bound materials. Adding polysaccharide fractions II (4.8 mg/mL), III (5.3 mg/mL), and IV (1.4 mg/mL) inhibited the infection of MA-104 cells by human rotavirus. The 50% inhibitory concentrations (IC 50) were 0.075, 0.083, and 0.022 mg/mL, respectively. Based on these results, the kefir polysaccharide has anti-rotavirus activity. In conclusion, a polysaccharide from kefir had more than a 97% inhibition effect against human rotavirus infection in MA-104 cells.

Immune Responses to Rotavirus Infection and Vaccination and Associated Correlates of Protection

Group A rotavirus (RV) strains are a major cause of acute gastroenteritis (AGE) in infants and young children worldwide [1]. RV disease accounts for more than one-third of all diarrhea-related hospitalizations and 500,000–600,000 deaths per year [2–4]; most deaths occur in sub-Saharan Africa and Asia [3, 4]. Direct medical and indirect annual costs associated with RV disease are estimated to be €400 million in Europe [5–7] and to exceed US$ 1 billion in the United States [8]. RV strains form a genus of the Reoviridae family and possess a genome of 11 segments of double-stranded (ds) RNA, encoding 6 structural viral proteins (VPs) and 6 nonstructural proteins (NSPs). The infectious particle (ie, virion) consists of 3 layers: the inner layer (core) contains the viral genome, the viral RNA-dependent RNA polymerase (RdRp, VP1), the capping enzyme (VP3), and the scaffolding protein (VP2); the core is surrounded by a middle layer (VP6), and the outer layer consists of VP7 and VP4 [9]. RV infects mature enterocytes in the small intestine. Viral replication leads to increased intracellular Ca2+ level (effected by NSP4), increased Cl- secretion, and shut-off of host cell protein synthesis (effected by NSP3), resulting in acute osmotic and secretory diarrhea (described in [9]). Various RV genes have been implicated in the pathogenesis of AGE [10]. After RV infection, a viremic stage of, at present, unclear significance has been identified in humans and experimental animals [11–13]. The RV-encoded NSP1 blocks interferon (IFN) production by various pathways [14–17]. RV infection down-regulates the IFN- and pro-inflammatory cytokine–associated pathways in calves [18]. RV strains have a high genomic and antigenic diversity and are classified into at least 7 different groups (A–G), distinguished by different VP6. Most human RV infections are caused by group A RV strains, which are further subdivided into at least 2 subgroups (I, II), 23 G types (determined by VP7, a glycoprotein), and 31 P types (determined by VP4, a protease-sensitive protein) [9, 19–21]. RV strains with different G and P types cocirculate and change in geographical regions over time [22–25]. In temperate climate regions, most cocirculating RV strains are types G1–G4 and G9 (typically G1P1A[8], G2P1B[4], G3P1A[8], G4P1A[8], and G9P1A[8]), but other G types (G5, G8, G10, and G12), in combination with various P types, may be most prevalent in tropical areas [21, 23, 24]. Nonspecific (innate) and acquired virus-specific humoral and cellular immune responses are elicited by RV infection [26, 27] or RV vaccination [28–33]. Although currently licensed vaccines are highly efficacious in protecting children from severe RV AGE, the molecular mechanisms of protection are not fully understood. This article considers the immune responses to natural RV infection and RV vaccination in both experimental animals and humans as potential correlates of protection.

Analyses of clinical, pathological and virological features of human rotavirus strain, YO induced gastroenteritis in infant BALB/c mice

Experimental studies of human rotavirus infections in mice are limited and there is lack of information on the quantitative assessment of rotaviral replication and its relationship with histological changes. In the present study, consequences of human rotavirus strain, YO induced gastroenteritis in infant BALB/c mice were analyzed for the occurrence of clinical symptoms, histopathology and virological events. The infected animals developed diarrhea and dehydration and showed accumulation of vacuolated enterocytes with lodging of the rotavirus antigens and shortening of villi in the intestine over a period of 5 days. The ileum was identified as the most susceptible and supportive part of small intestine for perpetuation of rotavirus infection in mice. Rotaviral antigen/RNA in stool and RNA in intestine were detected throughout the clinical disease period. At 48–72 h post inoculation, diarrhea was at the peak (90–95%) in the infected animals with increased load of viral RNA and intense pathological lesions suggesting it as the critical time point in the course of infection. The rising titers of antirotavirus neutralizing antibodies ascertained the replication of human rotavirus strain, YO in mice. These data may contribute to the understanding of pathophysiological, immunological and virological characteristics of rotavirus infections in mice.

Innate immune responses to human rotavirus in the neonatal gnotobiotic piglet disease model

Intestinal and systemic dendritic cell (DC) frequencies, serum and small intestinal content cytokines and uptake/binding of human rotavirus (HRV) virus-like particles (VLP) were studied in HRV acutely infected or mock-inoculated neonatal gnotobiotic piglets. Intestinal, mesenteric lymph node (MLN) and splenic plasmacytoid DCs (pDCs), conventional DCs (cDCs) and macrophages/monocytes were assessed by flow cytometry. In infected pigs, serum and small intestinal content interferon-α (IFN-α) were highest, interleukin-12 (IL-12) was lower and IL-10, tumour necrosis factor-α and IL-6 were minimal. Compared with mock-inoculated piglets, frequencies of total intestinal DCs were higher; splenic and MLN DC frequencies were lower. Most intestinal pDCs, but few cDCs, were IFN-α(+) and intestinal macrophages/monocytes were negative for IFN-α. Serum IFN-α levels and IFN-α(+) intestinal pDCs were highly correlated, suggesting IFN-α production in vivo by intestinal pDCs (r=0·8; P<0·01). The intestinal pDCs and cDCs, but not intestinal macrophages/monocytes, of HRV-infected piglets showed significantly lower VLP uptake/binding compared with mock-inoculated piglets, suggesting higher activation of pDCs and cDCs in infected piglets. Both intestinal pDCs and cDCs were activated (IFN-α(+) and lower VLP binding) after HRV infection, suggesting their role in induction of HRV-specific immunity. Dose-effects of HRV on serum IFN-α and IFN-α(+) DCs were studied by infecting piglets with 100-fold higher HRV dose. A high dose increased parameters associated with inflammation (diarrhoea, intestinal pathology) but serum IFN-α and IFN-α(+) DCs were similar between both groups. The pDCs have both anti- and pro-inflammatory functions. Stimulation of the anti-inflammatory effects of pDCs after the high dose, without increasing their pro-inflammatory impacts, may be critical to reduce further immunopathology during HRV infection.

The Bovine Lactophorin C-Terminal Fragment and PAS6/7 Were Both Potent in the Inhibition of Human Rotavirus Replication in Cultured Epithelial Cells and the Prevention of Experimental Gastroenteritis

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-M(r) glycoprotein fraction (F1) from cow's milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 degrees C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.

The potential effects of Lactobacillus acidophilus on the prevention and treatment of neonatal mice infected with human rotavirus

Objective To investigate the potential effect of Lactobacillus acidophilus (L. acidophilus) on prevention and treatment of neonatal mice infected with human rotavirus (HRV). Methods Sixty 4-day-old kunming mice were randomly divided into control group. HRV infected group, L. acidophilus pretreated group (treated before HRV infection ) and L. acidophilus treated group(treated after HRV infection). The manifestation and pathological changes in small intestine of neonatal mice were observed. The HRV antigen in the feces and intestines were measured by ELISA and fluorescent-focus assay, respectively. Results The severity and duration of diarrhea as well as mortality in L. acidophilus pretreated group and treated group were lower than those in HRV infected group. The duration of HRV-antigens shedding following infection was considerably prolonged in HRV infected group compared to that in L. acidophilus pretreated group and treated group. Furthermore, decreased expression of HRV antigen and little pathological changes in intestinal mucosa were found in L. acidophilus pretreated group and treated group when compared with HRV infected group. Conclusion L. acidophilus may be used as an alternative approach for the prevention and treatment of neonatal mice infected with HRV. Key words: Rotavirus; Lactobacillus acidophilus ; Neonatal mice; Diarrhea; Intestinal mucosa

Effects of QWBZP on T-cell subsets and their cytokines in intestinal mucosa of HRV infection suckling mice

Aim of this study Qiwei Baizhu Powder (QWBZP) is a traditional herbal prescription that has been used traditionally for the treatment of infantile diarrhea, including the infantile diarrhea caused by Human Rotavirus (HRV). In this study, we investigated the pharmacological activity of QWBZP extract. Materials and methods NIH suckling mice with HRV induced diarrhea were used. Density of CD3 +, CD4 + and CD8 + T cells, mRNA expression of IL-2, IFN-γ, IL-4 and IL-10 in intestinal mucosa epithelial cells were assayed. Results QWBZP extract promoted the expressions of mRNA of IL-2, IL-4, IL-10 and IFN-γ in intestinal mucosa epithelial cells. Also, we found that the density of CD8 + cells in intestinal mucosa epithelial cells was significantly lower in QWBZP group than in Model group, while the density of CD8 + cells was significantly higher in QWBZP group than in Model group. Conclusion These data suggest that QWBZP extract may exhibit antiviral effects through modulating the densities of T-cell subsets and the expressions of their cytokines in small intestinal mucosa epithelial cells.

In Vitroandin VivoEvaluation of the Efficacy of Bovine Colostrum against Human Rotavirus Infection

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6-7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.

Genomic diversity among group A rotaviruses from diarrheic children, piglets, buffalo and cow calves of Madhya Pradesh

Diarrheal disease continues to be a global health problem, particularly among young ones in developing nations. Amongst several viral and non-viral agents associated with diarrhea, group A rotavirus has been recognized as the major etiological agent of childhood gastroenteritis in human infants as well as several animal species throughout the world. During this study, a total of 181 diarrheic stool samples collected from children, piglets, buffalo and cow calves of Madhya Pradesh, central India were analyzed by electrophoretic mobilities of the 11 segments of dsRNA by polyacrylamide gel electrophoresis (PAGE). This technique revealed prevalence of rotavirus among different species (human-26.09%, pig-25.71%, buffalo-23.61% and cattle-21.43%). Prevalence of existence of circulating 8 different electropherotypes of group A rotaviruses indicated high genomic diversity among rotaviruses in this geographical region. Majority of the electropherotypes from humans and animals were of long pattern (75%) than short electropherotypes (9.09%). Same electropherotype was found to exist either only in a single species or in more than one species implicating the possibility of cross species transmission of the rotavirus strains. As it was found that certain animal rotavirus strains had electropherotypic similarities to some human strains, speculation increased about whether animals play a role as a source of rotavirus infection in humans or vice-versa. There is a need for further detailed study on the molecular characterization of rotaviruses which would have important implication in vaccine evaluation program.

Rotavirus Induces a Biphasic Enterotoxic and Cytotoxic Response in Human‐Derived Intestinal Enterocytes, Which Is Inhibited by Human Immunoglobulins

The mechanisms of diarrhea due to rotavirus infection in humans are not fully understood; no specific therapy is available, but orally administered human serum immunoglobulins are effective in blocking stool output. We aimed to investigate the effect of rotavirus on ion transport and the role of NSP4 in human-derived enterocytes, and to test the efficacy of human serum immunoglobulin in a model of rotavirus infection. Soon after infection, rotavirus induces active chloride secretion in enterocytes. This effect is evident before viral replication leads to cell damage and correlates with NSP4 production. Inhibition of NSP4 prevents the early secretory phase but not cell damage. Incubation with human serum immunoglobulin blocks both ion secretion and cell damage. Rotavirus exerts an early NSP4-dependent ion secretion and subsequent tissue damage. The combined enterotoxic and cytotoxic effects may be responsible for the increased severity of diarrhea due to rotavirus infection, and both are counteracted by human serum immunoglobulin.

Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization

Rotavirus infections can be diagnosed in stool samples by serological and molecular methods. We developed a novel reverse transcriptase PCR (RT-PCR) method for the amplification of rotavirus RNA and a reverse hybridization assay on a strip to detect amplimers and identify the specific G and P genotypes present in human stool specimens. An additional aim was to permit specific identification of the rotavirus G1P[8] strain, used in the Rotarix vaccine. Novel broad-spectrum PCR primers were developed for both VP4 and VP7, permitting the amplification of a wide range of rotavirus genotypes. Primer sets comprise mixtures of defined primer sequences. For the identification of G and P genotypes, two reverse hybridization strip assays were developed. Both the VP4 and the VP7 strip contain universal probes for the detection of VP4 and VP7 sequences, irrespective of the G or P genotype. The VP4 strip contains type-specific probes for P[4], P[6], P[8], P[9], and P[10]. The VP7 strip contains type-specific probes for G1, G2, G3, G4, G5, G6, G8, and G9. In addition, probes to distinguish between wild-type G1 and G1 vaccine strain sequences were present. Testing by analysis of multiple reference strains confirmed that both RT-PCR methods allowed the detection of a broad spectrum of genotypes. RT-PCR for VP7 was more sensitive than RT-PCR for VP4, but all samples identified as positive for rotavirus antigen by an enzyme-linked immunosorbent assay (ELISA) were also positive for both VP4 and VP7. The high specificity of the reverse hybridization method was confirmed by sequence analysis as well as by type-specific PCR, and the vaccine strain could also be specifically identified. The reverse hybridization method permits accurate identification of mixed infections with different genotypes. Rotavirus genotypes for which no type-specific probes were present on the strip were adequately identified by the universal detection probes. The assay was formally validated by analyses of specificity, sensitivity, precision, accuracy, and robustness. In a panel of 149 ELISA-positive stool samples, comparison with conventional type-specific RT-PCR methods revealed the superiority of the novel method, mainly in cases of mixed rotavirus infections. This novel method permits highly accurate detection and identification of human rotavirus infections in stool samples. This validated assay could be useful for large-scale epidemiological and clinical trials.

Toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs

Toll-like receptors (TLR) play an important role in the recognition of microbes by host sentinel cells that leads to the subsequent innate and adaptive immune responses. In this study, we evaluated the patterns of TLR2-, TLR3- and TLR9-expressing antigen presenting cells (APCs) in spleen and blood of gnotobiotic (Gn) pigs after colonization with a mixture of two strains of lactic acid bacteria (LAB), Lactobacillus acidophilus and Lactobacillus reuteri or infection with the virulent human rotavirus (HRV) Wa strain. We also assessed the influence of LAB on TLR and serum innate cytokine responses induced by HRV. Distributions of subpopulations of APCs [CD14+/−SWC3+CD11R1- monocytes/macrophages and CD14+/−SWC3+CD11R1+ conventional dendritic cells (cDCs)] were described in our previous report (Zhang, W., Wen, K., Azevedo, M.S., Gonzalez, A.M., Saif, L.J., Li, G., Yousef, A.E., Yuan, L., 2008. Lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs. Vet. Immunol. Immunopathol. 121, pp. 222–231). We demonstrated that LAB induced strong TLR2-expressing APC responses in blood and spleen, HRV induced a TLR3 response in spleen, and TLR9 responses were induced by either HRV (in spleen) or LAB (in blood). LAB and HRV have an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of LAB. Overall, the frequencies of TLR-expressing CD14+ APCs were higher than CD14− APCs. LAB enhanced the IFN-γ and IL-4 responses in serum, but it had a suppressive effect on the TLR3- and TLR9-expressing CD14− APC responses in spleen and the serum IFN-α response induced by HRV. These results elucidated the systemic TLR2-, TLR3-, and TLR9-expressing monocyte/macrophage and cDC responses after HRV infection, LAB colonization, and the two combined. Our findings facilitate the understanding of the mechanism of LAB's adjuvant effect on rotavirus vaccines and the diverse innate and adaptive immune responses induced by commensal LAB colonization versus rotavirus infection and the interactions between them.