A specific CpG oligodeoxynucleotide induces protective antiviral responses against grass carp reovirus in grass carp Ctenopharyngodon idella.

CpG oligodeoxynucleotides (ODNs) show strong immune stimulatory activity in vertebrate, however, they possess specific sequence feature among species. In this study, we screened out an optimal CpG ODN sequence for grass carp (Ctenopharyngodon idella), 1670A 5'-TCGAACGTTTTAACGTTTTAACGTT-3', from six published sequences and three sequences designed by authors based on grass carp head kidney mononuclear cells and CIK (C. idella kidney) cells proliferation. VP4 mRNA expression was strongly inhibited by CpG ODN 1670A in CIK cells with GCRV infection, showing its strong antiviral activity. The mechanism via toll-like receptor 9 (TLR9)-mediated signaling pathway was measured by real-time quantitative RT-PCR, and TLR21 did not play a role in the immune response to CpG ODN. The late up-regulation of CiRIG-I mRNA expression indicated that RIG-I-like receptors (RLRs) signaling pathway participated in the immune response to CpG ODN which is the first report on the interaction between CpG and RLRs. We also found that the efficient CpG ODN can activates interferon system. Infected with GCRV, type I interferon expression was reduced and type II interferon was induced by the efficient CpG ODN in CIK cells, especially IFNγ2, suggesting that IFNγ2 played an important role in response to the efficient CpG ODN. These results provide a theoretical basis and new development trend for further research on CpG and the application of CpG vaccine adjuvant in grass carp disease control.

In vitro immunocompetence of two compounds isolated from Polygala tenuifolia and development of resistance against grass carp reovirus (GCRV) and Dactylogyrus intermedius in respective host.

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1ß, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1ß, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L(-1)). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L(-1) and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections.

Characterizations of two grass carp Ctenopharyngodon idella HMGB2 genes and potential roles in innate immunity.

High-mobility group box 2 (HMGB2) protein is a chromatin-associated nonhistone protein, involved in transcriptional regulation and nucleic-acid-mediated innate immune responses in mammalian. However, the function of piscine HMGB2 in innate immune responses is still unknown. In the present study, two HMGB2 homologue genes (CiHMGB2a, CiHMGB2b) were identified and characterized in grass carp (Ctenopharyngodon idella). Both CiHMGB2a and CiHMGB2b genes encode proteins with 213 amino acids, sharing 71.4% identities and containing two basic HMG boxes and an acidic tail. The deduced protein sequences showed the most identities to HMGB2a (93%) and HMGB2b (86.4%) of zebrafish (Danio rerio), respectively. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that CiHMGB2a and CiHMGB2b were constitutively expressed in all the 15 tested tissues. Post grass carp reovirus (GCRV) infection, mRNA levels of CiHMGB2a and CiHMGB2b were strongly up-regulated in spleen and head kidney and mildly modulated in C. idella kidney (CIK) cells. Meanwhile, mRNA expressions of CiHMGB2a and CiHMGB2b were significantly regulated by viral pathogen associated molecular patterns (PAMPs) polyinosinic-polycytidylic potassium salt (poly(I:C)) and bacterial PAMPs lipopolysaccharide (LPS), peptidoglycan (PGN) challenge in CIK cells. In CiHMGB2a and CiHMGB2b over-expression cells, expressions of CiHMGB2a and CiHMGB2b facilitated each other; transcription levels of CiTRIF, CiMyD88, CiIPS-1 and CiMx1 were remarkably enhanced, whereas CiIFN-I was inhibited, compared with those in cells transfected with pCMV (control plasmid); after GCRV challenge, all those tested genes were up-regulated with divergent expression profiles. Antiviral activities of CiHMGB2a and CiHMGB2b were manifested by the delayed appearance of cytopathic effect (CPE) and inhibition of GCRV yield. All those results demonstrate that CiHMGB2a and CiHMGB2b not only mediate antiviral immune responses but also involve in responding to viral/bacterial PAMPs challenge, which provides novel insights into the essential role of HMGB2 in innate immunity.

Dot immunobinding assay method with chlorophyll removal for the detection of southern rice black-streaked dwarf virus.

Southern rice black-streaked dwarf virus (SRBSDV), a new virus from Fiji, has seriously damaged rice crops in southern China and northern Vietnam in recent years. This virus is difficult to diagnose in the early stages of infection, and is very destructive at the late stage. In the present study, a dot immunobinding assay (DIBA) that has a high sensitivity for diagnosing SRBSDV was developed. Two kinds of treatment for the DIBA were evaluated to determine the most effective one for removing chlorophyll interferences via rice extraction. The first included several reagents to remove chlorophyll, namely, the alkaline reagents like magnesium oxide and alumina oxide, the adsorbent reagents like activated carbon and bentonite, as well as the extraction agent acetone. The second and third treatments, which were used to remove chlorophyll in blot membrane-nitrocellulose and polyvinylidene fluoride (PVDF), included several organic solvents containing methanol, ethanol, acetone, ethyl acetate, and diethyl ether. The results showed that activated carbon and methanol yielded the best contrasting purple color for the infected samples by decreasing the chlorophyll content.

Identification and expression profiling analysis of grass carp Ctenopharyngodon idella LGP2 cDNA.

LGP2 (laboratory of genetics and physiology 2), a homologue of RIG-I (Retinoic acid inducible gene-I) and MDA5 (Melanoma differentiation associated gene 5) without the CARD (caspase activation and recruitment domain) required for signaling, plays a pivotal role in modulating signaling by RIG-I and MDA5 for interferon (IFN) synthesis. In this study, a novel LGP2 gene from grass carp Ctenopharyngodon idella (designated as CiLGP2) was isolated and characterized. The full-length cDNA of CiLGP2 was of 2920 bp with five instability motifs (ATTTA). The open reading frame was of 2043 bp and encoded a polypeptide of 680 amino acids, including five main overlapping structural domains: two DEXDc (DEAD/DEAH box helicase domain), one ResIII (conserved restriction domain of bacterial type III restriction enzyme), one HELICc (helicase superfamily c-terminal domain) and one RD (regulatory domain). There was one more alpha-helix in the RD, compared with that in human. The CiLGP2 mRNA was ubiquitous expression in the tested tissues, was high level in spleen, skin, heart and intestine tissues, and was up-regulated by grass carp reovirus (GCRV) injection by semi-quantitative RT-PCR (sqRT-PCR) assay. The CiLGP2 expression in spleen was significantly up-regulated at 12 h (14.5 folds, P < 0.05), reached the crest at 24 h (19.0 folds, P < 0.05), and then dropped a little at 48 h (10.4 folds) post-injection of GCRV and kept this level in the following test period (P < 0.05). In liver, the temporal expression of CiLGP2 mRNA was significantly increased at 24 h (3.8 times, P < 0.05), reached peak at 48 h (10.7 times, P < 0.05), and then decreased a little bit at 72 h (5.8 times, P < 0.05) and kept this high level by the end of the test (P < 0.05). These results collectively suggested that CiLGP2 was a novel member of RLR gene family, engaging in the early stage of antiviral innate immune defense in grass carp, and laid the foundation for the further mechanism research of LGP2 in fishes.

Role of neutralising antibody in passive immunity to bluetongue infection.

A group of sheep inoculated with serum obtained from sheep which had recovered from bluetongue virus type 3 infection were protected from challenge with the homologous virus type but not from heterologous challenge. Twin lambs which had received colostrum containing virus antibodies were shown to be only partially protected against homologous challenge. A monoclonal antibody directed against the type-determining protein of the virus was also shown to give partial protection against challenge. From this series of experiments it was concluded that antibody has a significant role in protection from bluetongue but that the outcome of challenge will depend on several interacting factors.

Comparison of paired whole milk and dried filter paper samples for anti-enterotoxin and anti-rotavirus activities.

Milk specimens, 75 from cows immunized against cholera toxin and 35 from a human population in which enterotoxigenic Escherichia coli and rotaviral infections are endemic, were collected as paired filter paper and frozen whole milk samples. Each pair was tested for antibody activity against heat-labile E. coli and Vibrio cholerae enterotoxins. Additionally, 12 of the 35 paired human milk samples stored as frozen whole milk and dried on filter paper were tested for anti-rotavirus immunoglobulin A. Anti-enterotoxin and anti-rotavirus immunoglobulin A titers in milk dried on filter paper compared favorably with those of their frozen whole milk pairs. Filter paper samples offered considerable advantages for field collection, transportation, and storage over frozen liquid samples.

Diarrhoea in dairy calves reduced by feeding colostrum from cows vaccinated with rotavirus.

Forty-two dairy calves remained with their dams for two days after birth, and then were removed to a calf rearing shed. Calves were allocated to three groups for the next 14 days, and received twice daily either whole milk, whole milk with a 10 per cent supplement of pooled normal bovine colostrum or whole milk with 10 per cent supplement of colostrum from cows vaccinated with rotavirus. A natural outbreak of diarrhoea occurred, affecting 28 of the 42 calves. Feeding immune colostrum delayed the onset of diarrhoea, and reduced its incidence, duration and severity. Live weight gains were consequently improved. The group fed normal colostrum had diarrhoea intermediate in severity between that of control calves and those fed immune colostrum. The aetiology of the diarrhoea was complex, with calves excreting rotavirus, enteropathogenic Escherichia coli and cryptosporidia.

[Persistence of transplacental antibodies against rotavirus in children less than 6 months of age]. / Persistencia de anticuerpos transplacentarios contra rotavirus en niños menores de seis meses de edad.

The persistence of antibodies against rotavirus was studied in the sera of 54 recently born infants up to six months of age; likewise, in the sera of their mothers. Serum positivities were found to be similar in both, showing 96% for those with antibodies and 94.4% for the latter. The percentage of infants with antibodies dropped gradually to the 4th month of age and since then rises in the titre of antibodies began to appear in four infants, which indicated there had been rotavirus infections. These studies lead to believe that in spite of the presence of serum antibodies, rotavirus multiply in the enteric tract and do not bring about serum antibodies, but perhaps, they stimulate the production of coproantibodies, which do not allow in the future the implantation of these viruses in the intestine.

Distribution of antibodies to rotavirus in serum and lacteal secretions of naturally infected swine and their suckling pigs.

Rotavirus antibodies were demonstrated in lacteal secretions and sera of 20 parturient sows and in sera of their newborn by an enzyme-linked immunosorbent assay blocking technique, using bovine rotavirus cell culture antigen and monospecific antibody to bovine rotavirus. Antibodies to rotavirus occur in the 3 immunoglobulin (Ig) classes IgM, IgA, and IgG in lacteal secretions. High and long-persisting antibody activity was mainly associated with the IgA class. The IgM and IgG decreased to undetectable concentrations in most sows during the 14-day investigation period. Serum antibodies of newborn pigs nursing their dams also decreased rapidly during this time. The heterologous enzyme-linked immunosorbent assay blocking technique was a reliable and rapid procedure for the demonstration of rotavirus antibodies.

Development of immunity to porcine rotavirus in piglets protected from disease by bovine colostrum.

Bovine colostrum with rotavirus-neutralizing activity was fed for 10 days to two groups of piglets, one of which was inoculated intranasally with a rotavirus of porcine origin. A third group, which did not receive colostrum, was also inoculated with the virus, and these piglets developed diarrhea, excreted rotavirus in the feces, and died 6 days after infection. In contrast, the infected piglets fed with bovine colostrum remained healthy, although they developed antibody to rotavirus. Twenty-seven days after the primary inoculation, piglets in the colostrum-fed groups were inoculated intranasally with virus. Those in the previously unexposed group became clinically ill and excreted rotavirus, whereas those which had experienced a previous subclinical infection (the colostrum-fed, virus-inoculated group) remained healthy. It was concluded that bovine colostrum protected piglets from the clinical effects of a porcine rotavirus and that these animals developed an immunity which prevented subsequent disease.

Detection of antirotavirus immunoglobulins A, G, and M in swine colostrum, milk, and feces by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to allow direct detection of class-specific antirotavirus antibodies. In colostrum and in milk, antirotavirus antibodies were found in the three immunoglobulin classes. Antirotavirus immunoglobulins G and M were predominant in colostrum, whereas antirotavirus immunoglobulin A was predominant in milk and feces.

Effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection.

Levels of antirotaviral secretory immunoglobulin A were measured by enzyme-linked immunosorbent assay in colostrum and milk samples collected daily for the first 5 days postpartum from 49 mothers breast-feeding their infants. The trypsin-inhibitory capacity of these lacteal secretion samples was assessed by their ability to inhibit the hydrolysis of alpha-N-benzoyl-DL-arginine-p-nitroanilide by trypsin. Stools passed by these breast-fed infants and by an additional 43 bottle-fed infants were pooled by individual and examined by electron microscopy for rotavirus. Stool trypsin levels were estimated with the gelatin hydrolysis test. Breast-fed infants were significantly less likely to become infected with rotavirus and showed significantly lower stool tryptic activity than did bottle-fed infants. Breast-fed infants who did not excrete rotavirus over the 5-day period received milk of significantly higher antirotaviral secretory immunoglobulin A or trypsin-inhibitory capacity or both than breast-fed infants who were infected with rotavirus. A case of probable maternal rotavirus infection during pregnancy, producing greatly elevated lacteal antirotaviral secretory immunoglobulin A levels lasting for 2 years, was detected. Results of this study suggest that both antibodies and trypsin inhibitors in human milk can be associated with the protection of neonates against rotavirus infection in the first 5 days of life.

Measurement of immunoglobulin A, G, and M class rotavirus antibodies in serum and mucosal secretions.

A solid-phase, enzyme-linked immunospecific assay for measurement of different immunoglobulin classes of human rotavirus antibodies is described. The antigen, which was adsorbed directly to polyvinyl microtiter plates, consisted of a clarified cell culture stock of the simian rotavirus SA 11. The assay was sensitive and reproducible and could readily be calibrated to determine concentrations of each class of antibody. The assay was applied to measurements of rotavirus antibodies in serum, colostrum, milk, and fecal samples. It particularly facilitates investigations of the role of immunoglobulin A antibodies in immunity to rotavirus infections.

Transfer of antirotaviral antibodies from mothers to their infants.

Levels of rotavirus-specific immunoglobulin G (IgG), IgA, IgM, and secretory immunoglobulin in maternal and cord serum, colostrum and milk, and infants' stools were measured by enzyme-linked immunosorbent assay in 92 mothers and their infants. Although antirotaviral IgG, IgA, and secretory immunoglobulin were present in most maternal sera, only IgG crossed the placenta. All samples of colostrum and milk tested contained antirotaviral secretory immunoglobulin and IgA except those of two women in whom IgA deficiency was subsequently described. Specific IgM and IgG were also detected in many colostral samples. Antirotaviral IgA was detected in many colostral samples. Antirotaviral IgA was detected in stools of breast-fed but not bottle-fed neonates. Apparently the human infant receives rotaviral antibodies both transplacentally and via maternal colostrum and milk.

[Antibodies to rotavirus in human milk (author's transl)]. / Antikörper gegen Rotavirus in der Muttermilch.

116 human milk specimens taken from 36 women during the first 10 days after delivery were investigated for antibodies against rotavirus by crossimmunoelectrophoresis. Antibodies were detected in 37 specimens from 24 women, the antibody titers proved to be lower than the serum antibody titers. Antibody concentrations in the colostral and transitory milk samples declined continuously during the 10 days after delivery. Women with high serum antibody titers against rotavirus possessed antibodies in their colostrum more frequently than women with low antibody titers. There was no difference between the antibody titers in the sera of the women and the corresponding cord blood.

Antiviral activity of colostrum and serum immunoglobulins A and G.

Enteric virus-specific IgA and IgG present in paired human sera and colostrums were measured by the enzyme-linked immunosorbent assay (ELISA). Virus-specific IgA was present in all colostrums, but virus-specific IgG could not be detected. The reverse was true when sera were assayed. Most of these colostrums also neutralized either polio virus or reovirus, as did IgA, which was separated from a pool of colostrums by exclusion chromatography. No correlation could be made between levels of neutralizing and ELISA antibody titers in colostrums.

Reovirus-like agent associated with fatal diarrhea in neonatal pigs.

Large numbers of a reovirus-like agent were visualized with electron microscopy in bacteria-free gut homogenates obtained from piglets with a fatal diarrhea resembling transmissible gastroenteritis. The syndrome, of vomiting, diarrhea, dehydration, and death, was reproduced in piglets artificially infected with these bacteria-free gut homogenates. Reovirus-like particles persisted in serial piglet passage and none was seen in uninfected, asymptomatic controls. Hyperimmune sera (made in recovered piglets) aggregated the reovirus-like particles, as judged by immunoelectron microscopy, and neutralized the infectious agent. The cytoplasm in enterocytes on infected intestinal epithelium fluoresced when this hyperimmune sera was used in an indirect fluorescent antibody test. Feeding cow colostrum or diets containing porcine gamma globulin protected infected piglets. No cytopathogenic effect was noted in infected tissue cultures, nor did this agent affect neonatal guinea pigs, hamsters, mice, and rats. The agent did not agglutinate human O or A erythrocytes.

Immune response to orally administered calf reovirus-like agent and coronavirus vaccine.

Twenty 6-to 7-hour-old gnotobiotic calves inoculated orally with attenuated calf diarrhea reovirus-like agent and challenge-inoculated with virulent virus 48-72 h post-inoculation (PI) remained clinically normal during the post-vaccination observation period; one developed mild diarrhea after challenge inoculation. Four non-vaccinated challenge control calves developed severe diarrhea. Twenty 6-to 7-hour-old gnotobiotic calves inoculated orally with attenuated calf diarrhea coronavirus and challenge inoculated with virulent virus 96 h later remained clinically normal during the post-vaccination and post-challenge observation period. Four non-vaccinated challenge control calves developed severe diarrhea and 2 of these died. Five-foot long isolated loops prepared in the lower ileum (Thiry-Vella loop) of newborn colostrum-deprived calves were inoculated with attenuated coronavirus. Daily loop washings were cultured for virus and tested for neutralizing antibody. Peak viral titers of 10(6.5) to 10(7) occurred 3-4 days PI and descended rapidly to 0 between 6 and 8 days PI. Neutralizing antibody was first detected in the washings 6-8 days PI and reached a titer of 128-256 7 to 9 days PI. Loop immunoglobulin separated by gel filtration and identified by immunodiffusion were primarily IgM and IgA. Initial resistance to virulent viral infection is thought to be due to an interference phenomenon, with later resistance due to local antibody.