Generation of recombinant rotaviruses encoding a split NanoLuc peptide tag.

Recombinant viruses expressing fluorescent or luminescent reporter proteins are used to quantitate and visualize viral replication and transmission. Here, we used a split NanoLuc luciferase (NLuc) system comprising large LgBiT and small HiBiT peptide fragments to generate stable reporter rotaviruses (RVs). Reporter RVs expressing NSP1-HiBiT fusion protein were generated by placing an 11 amino acid HiBiT peptide tag at the C-terminus of the intact simian RV NSP1 open reading frame or truncated human RV NSP1 open reading frame. Virus-infected cell lysates exhibited NLuc activity that paralleled virus replication. The antiviral activity of neutralizing antibodies and antiviral reagents against the recombinant HiBiT reporter viruses were monitored by measuring reductions in NLuc expression. These findings demonstrate that the HiBiT reporter RV systems are powerful tools for studying the viral life cycle and pathogenesis, and a robust platform for developing novel antiviral drugs.

Antiviral activities of resveratrol against rotavirus in vitro and in vivo.

BACKGROUND: Rotavirus (RV) is the primary causative agent for viral gastroenteritis among infants and young children worldwide. Currently, no clinically approved and effective antiviral drug for the treatment of RV infection is available. PURPOSE: We investigated the potential anti-RV activity of resveratrol and underlying mechanisms by which resveratrol acted against RV. METHODS: The anti-RV activity of resveratrol in vitro was evaluated using plaque reduction assays. The effects of resveratrol on yield of virion progeny, viral polyprotein expression and genomic RNA synthesis were respectively investigated using enzyme-linked immunosorbent assays, western blotting and qRT-PCR assays. Further, we also measured the antiviral effect of resveratrol by evaluation of antigen clearance and assessment of changes in proinflammatory cytokines/chemokines in RV-infected neonatal mouse model. RESULTS: Our results indicated that 20 µM of resveratrol significantly inhibited RV replication in Caco-2 cell line by suppressing RV RNA synthesis, protein expression, viroplasm plaque formation, progeny virion production, and RV-induced cytopathy independent of the different strains and cell lines of RV that we used. Analysis of the effect of time post-addition of resveratrol indicated that its application inhibited early processes in the RV replication cycle. Further study of the underlying mechanism of anti-RV activity indicated that resveratrol inhibited RV replication by suppressing expression of heat-shock protein 90 (HSP90) mRNA and protein, and that the effect occurred in a dose-dependent manner. Overexpression of HSP90 was found to have attenuated the inhibitory effect of resveratrol on RV replication. Interestingly, the application of resveratrol were found to down-regulate the level of inhibition of RV-mediated MEK1/2 and ERK phosphorylation. Using a RV-infected suckling mice model, we found that application of resveratrol significantly lessened the severity of diarrhea, decreased viral titers, and relieved associated symptoms. Levels of mRNA expression of interleukin-2, interleukin-10, tumor necrosis factor-α, interferon-γ, macrophage inflammatory protein 1, and monocyte chemotactic protein-1 were all found to have been sharply reduced in intestinal tissue from mice which had been treated with resveratrol (10 or 20 mg/kg) after RV infection (p < 0.05). CONCLUSION: These findings implied that resveratrol exhibits antiviral activity and could be a promising treatment for rotavirus infection.

Vitamin D Alleviates Rotavirus Infection through a Microrna-155-5p Mediated Regulation of the TBK1/IRF3 Signaling Pathway In Vivo and In Vitro.

(1) Background: Vitamin D (VD) plays a vital role in anti-viral innate immunity. However, the role of VD in anti-rotavirus and its mechanism is still unclear. The present study was performed to investigate whether VD alleviates rotavirus (RV) infection through a microRNA-155-5p (miR-155-5p)-mediated regulation of TANK-binding kinase 1 (TBK1)/interferon regulatory factors 3 (IRF3) signaling pathway in vivo and in vitro. (2) Methods: The efficacy of VD treatment was evaluated in DLY pig and IPEC-J2. Dual-luciferase reporter activity assay was performed to verify the role of miR-155-5p in 1α,25-dihydroxy-VD3 (1,25D3) mediating the regulation of the TBK1/IRF3 signaling pathway. (3) Results: A 5000 IU·kg-1 dietary VD3 supplementation attenuated RV-induced the decrease of the villus height and crypt depth (p < 0.05), and up-regulated TBK1, IRF3, and IFN-ß mRNA expressions in the jejunum (p < 0.05). Incubation with 1,25D3 significantly decreased the RV mRNA expression and the RV antigen concentration, and increased the TBK1 mRNA and protein levels, and the phosphoprotein IRF3 (p-IRF3) level (p < 0.05). The expression of miR-155-5p was up-regulated in response to an RV infection in vivo and in vitro (p < 0.05). 1,25D3 significantly repressed the up-regulation of miR-155-5p in vivo and in vitro (p < 0.05). Overexpression of miR-155-5p remarkably suppressed the mRNA and protein levels of TBK1 and p-IRF3 (p < 0.01), while the inhibition of miR-155-5p had an opposite effect. Luciferase activity assays confirmed that miR-155-5p regulated RV replication by directly targeting TBK1, and miR-155-5p suppressed the TBK1 protein level (p < 0.01). (4) Conclusions: These results indicate that miR-155-5p is involved in 1,25D3 mediating the regulation of the TBK1/IRF3 signaling pathway by directly targeting TBK1.

Lentinan administration relieves gut barrier dysfunction induced by rotavirus in a weaned piglet model.

Rotavirus (RV) is a pathogen that induces severe diarrhea in infants and young animals. Shiitake mushroom is a traditional food, which can improve physiological function, including gut health. Lentinan (LNT) is the main functional component of Shiitake mushroom. This study aimed to verify whether LNT administration could improve intestinal barrier function, thereby decreasing RV-induced diarrhea in a porcine model. According to initial weight and origin, a total of 28 weaned piglets were randomly fed 2 diets containing 0 or 84 mg kg-1 LNT for 19 d (n = 14). On day 15, RV was orally infused to half of the pigs in each group. RV-induced diarrhea (P < 0.05), the positive rate of RV non-structural protein 4 (NSP4), impaired intestinal morphology, antioxidant capacity and microbiota (P < 0.05), and increased apoptosis of jejunal epithelial cells (P < 0.05) were assessed in the piglets. Dietary LNT supplementation was found to improve intestinal morphology, permeability, antioxidant capacity and microbiota (P < 0.05). Supplementation also further alleviated the effects of RV infection on diarrhea, intestinal morphology, permeability, antioxidant capacity, microbiota and apoptosis of jejunal epithelial cells in piglets (P < 0.05). Thus, these results suggest that LNT administration relieved RV-induced diarrhea in piglets, which could be due to the increase in antioxidant capacity, reduction in apoptosis and improvement of the microbiota-increased gut barrier.

Mechanism of Anti-rotavirus Synergistic Activity by Epigallocatechin Gallate and a Proanthocyanidin-Containing Nutraceutical.

Epigallocatechin gallate (EGCG) of green tea and the nutraceutical CystiCran®-40 (containing 40% proanthocyanidins) of the cranberry plant have been associated with antiviral activity. The purpose of this work was to determine the mechanism of antiviral synergy between each compound. Coliphage T4II (phage T4) and the rotavirus strain SA-11(RTV) were used as model virus systems. Individual and combined flavonoids structural and molecular weight analyses were performed by NMR and HPCL/MS, respectively. A suboptimal concentration of EGCG or C-40 alone or in combination reduced phage infectivity by ≤10%. Similarly, EGCG (30 µg/ml) and C-40 (25 µg/ml), respectively, reduced RTV titers by 3 and 13%. However, RTV titers were reduced by 32% (p < .05) with both flavonoids used in combination. RTV was not recognized in host cells by electron microscopy 24-h post-inoculation. NMR and HPLC/MS findings revealed significant structural and potential changes in molecular weight of the flavonoids in complex.

Medicinal plants and natural molecules with in vitro and in vivo activity against rotavirus: A systematic review.

BACKGROUND: Rotaviruses can cause life-threatening health disorders, such as severe dehydrating gastroenteritis and diarrhea in children. Vaccination is the main preventive strategy to reduce rotavirus diarrhea and the severity of episodes, but vaccines are not fully effective and new episodes may occur, even in vaccinated children. The WHO recommends oral rehydration therapy and zinc supplementation for rotavirus-induced diarrhea management. There is little preclinical evidence to support the use of phytotherapeutics in the management of rotaviral infections. PURPOSE: We aim to review the use of medicinal plants and natural molecules in the management of rotavirus infections in experimental studies. METHODS: Articles, published in the English language between 1991 and 2016, were retrieved from PubMed, Scopus and Web of Science using relevant keywords. The scientific literature mainly focusing on plant natural products with therapeutic efficacies against experimental models of rotavirus, were identified and tabulated. In addition, an assessment of the reliability of animal experiments was determined under ``Risk of Bias'' criteria. CHAPTERS: After an initial search and a revision of the inclusion criteria, 41 reports satisfied the objectives of the study. 36 articles were found concerning the anti-rotaviral potential in rotavirus infected cell lines. Among the active secondary metabolites screened for rotavirus inhibition, the polyphenols of flavonoid structure had acquired the highest number of studies in our survey, compared to phenolic acids, stilbenoids, tannins, pectins, terpenoids and flavonoid glycosides. Also, many phytochemicals reduced the efficacy of viral capsid proteins foremost to their elimination and improved the tendency of host-cell inhibiting virus absorption or by prevention of viral replication. Furthermore, five in vivo studies reported that herbs, as well its components, reduced the duration and severity of diarrhea in mice and piglets. The anti-rotavirus efficacy were highlighted based on improvements in reduction on liquid stool, fecal virus shedding, small intestinal histology, levels of inflammation related cytokines and signaling receptors. However, the quality of the experiments in animal studies contained certain types of bias in terms of how they were conducted and reported. CONCLUSION: We identified and summarized studies on medicinal plants and natural molecules having anti-rotavirus activity in order to further future developments of cures for rotavirus gastroenteritis.

Anti-VP6 VHH: An Experimental Treatment for Rotavirus A-Associated Disease.

Species A Rotaviruses (RVA) remain a leading cause of mortality in children under 5 years of age. Current treatment options are limited. We assessed the efficacy of two VP6-specific llama-derived heavy chain antibody fragments (VHH) -2KD1 and 3B2- as an oral prophylactic and therapeutic treatment against RVA-induced diarrhea in a neonatal mouse model inoculated with virulent murine RVA (ECw, G16P[16]I7). Joint therapeutic administration of 2KD1+3B2 (200 µg/dose) successfully reduced diarrhea duration, RVA infection severity and virus shedding in feces. While the same dose of 2KD1 or 3B2 (200 µg) significantly reduced duration of RVA-induced diarrhea, 2KD1 was more effective in diminishing the severity of intestinal infection and RVA shedding in feces, perhaps because 2KD1 presented higher binding affinity for RVA particles than 3B2. Neither prophylactic nor therapeutic administration of the VHH interfered with the host's humoral immune response against RVA. When 2KD1 (200 µg) was administered after diarrhea development, it also significantly reduced RVA intestinal infection and fecal shedding. Host antibody responses against the oral VHH treatment were not detected, nor did viral escape mutants. Our findings show that oral administration of anti-VP6 VHH constitute, not only an effective prophylactic treatment against RVA-associated diarrhea, but also a safe therapeutic tool against RVA infection, even once diarrhea is present. Anti-VP6 VHH could be used complementary to ongoing vaccination, especially in populations that have shown lower immunization efficacy. These VHH could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Effect of 25-hydroxyvitamin D3 on rotavirus replication and gene expressions of RIG-I signalling molecule in porcine rotavirus-infected IPEC-J2 cells.

The study evaluated whether a 25-hydroxyvitamin D3 (25D3) supplementation decreases the replication of rotavirus by the retinoic acid-inducible gene I (RIG-I) signalling pathway in a porcine small intestinal epithelial cell line (IPEC-J2). The results show that IPEC-J2 cells express high baseline levels of 1α-hydroxylase (CYP27B1), which converts inactive 25D3 to the active 1,25-dihydroxyvitamin D3 (1,25D3). Porcine rotavirus (PRV) infection alone resulted in a significant increase in CYP27B1 mRNA, which augmented the production of active vitamin D. Physiological concentrations of 25D3 were found to decrease PRV replication in IPEC-J2 cells. RIG-I plays an important role in the recognition of double-stranded RNA virus by host cells. Upon recognition, RIG-I triggers a series of signalling molecules such as interferon-ß (IFN-ß) promoter stimulator 1 (IPS-1) leading to the expression of type I interferons (IFN-ß). Active 25D3 that was generated by PRV-infected IPEC-J2 cells led to an increased expression of toll-like receptors 3 (TLR3), RIG-I, IPS-1, IFN-ß and IFN-stimulated genes 15 (ISG15) with important innate immune functions. Inhibiting CYP27B1 also failed to increase RIG-I, IPS-1, IFN-ß and ISG15 mRNA expression. These observations suggest that 25D3 can directly inhibit PRV in IPEC-J2 cells, which requires this active form of vitamin D. The anti-rotavirus effect of 25D3 is mediated at least in part by RIG-I signalling pathways in IPEC-J2 cells.

An evaluation of the inhibitory effects against rotavirus infection of edible plant extracts.

BACKGROUND: Rotaviruses are the single most important cause of severe diarrhea in young children worldwide. The developments of specific, potent and accessible antiviral treatments that restrain rotavirus infection remain important to control rotavirus disease. METHODS: 150 plant extracts with nutritional applications were screened in vitro on MA-104 cells for their antiviral activity against rhesus rotavirus (RRV). One extract (Aspalathus linearis (Burm.f.) R.Dahlgren) was also tested for its effect on the loss of transepithelial resistance (TER) of Caco-2 cells caused by simian rotavirus (SA-11) infection. RESULTS: Aqueous extracts of Nelumbo nucifera Gaertn. fruit, Urtica dioica L. root, Aspalathus linearis (Burm.f.) R.Dahlgren leaves, Glycyrrhiza glabra L. root and Olea europaea L. leaves were found to have strong significant antiviral activity with a 50% inhibitory concentration (IC50) < 300 µg/ml. The pure compound 18ß-glycyrrhetinic acid from Glycyrrhiza glabra was found to have the strongest antiviral activity (IC50 46 µM), followed by luteolin and vitexin from Aspalathus linearis (IC50 respectively 116 µM and 129 µM) and apigenin-7-O-glucoside from Melissa officinalis (IC50 150 µM). A combination of Glycyrrhiza glabra L. + Nelumbo nucifera Gaertn. and Urtica dioica L. + Nelumbo nucifera Gaertn. showed synergy in their anti-viral activities. Aspalathus linearis (Burm.f.) R.Dahlgren showed no positive effect on the maintenance of the TER. CONCLUSIONS: These results indicate that nutritional intervention with extracts of Nelumbo nucifera Gaertn., Aspalathus linearis (Burm.f.) R.Dahlgren, Urtica dioica L., Glycyrrhiza glabra L. and Olea europaea L. might be useful in the treatment of diarrhea caused by rotavirus infection.

Inhibition of rotavirus infection in cultured cells by N-acetyl-cysteine, PPARγ agonists and NSAIDs.

Although the current rotavirus vaccines have shown good tolerance and significant efficacy, it would be useful to develop alternative or complementary strategies aimed at preventing or treating acute diarrhoeal disease caused by this viral agent. A variety of antiviral strategies other than vaccines have been assayed for rotavirus infection management. The recently demonstrated sensitivity of rotavirus infectivity to thiol/disulfide reagents prompted assays for screening drugs that potentially affect cellular redox reactions. MA104 or Caco-2 cells were inoculated with the rotavirus strains RRV, Wa, Wi or M69 and then incubated with different concentrations of drugs belonging to a selected group of 60 drugs that are currently used in humans for purposes other than rotavirus infection treatment. Eighteen of these drugs were able to inhibit rotavirus infectivity to different extents. A more systematic evaluation was performed with drugs that could be used in children such as N-acetylcysteine and ascorbic acid, in addition to ibuprofen, pioglitazone and rosiglitazone, all of which affecting cellular pathways potentially needed by the rotavirus infection process. Evidence is provided here that rotavirus infectivity is significantly inhibited by NAC in different cell-culture systems. These findings suggest that NAC has the potential to be used as a therapeutic tool for treatment and prevention of rotavirus disease in children.

Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja saponaria Molina.

Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide with approximately 300,000 pre-adolescent deaths each year. Quillaja saponins are a natural aqueous extract obtained from the Chilean soapbark tree. The extract is approved for use in humans by the FDA for use in beverages as a food addictive. We have demonstrated that Quillaja extracts have strong antiviral activities in vitro against six different viruses. In this study, we evaluated the in vivo antiviral activity of these extracts against rhesus rotavirus (RRV) using a mouse model. We established that at a dosage of 0.015 mg/mouse of saponin extract, RRV induced diarrhea can be significantly reduced from 79% to 11% when mice are exposed to 500 plaque-forming-units (PFU) for each of five consecutive days. Additionally, while a reduction of RRV induced diarrhea depended both on the concentration of virus introduced and on the amount of Quillaja extract given to each mouse, the severity and interval of diarrhea under a variety of conditions tested, in all the treated mice were greatly reduced when compared to those that did not receive the Quillaja extracts. Mechanistically, there is strong evidence that the Quillaja extracts are able to "block" rotavirus infection by inhibiting virus-host attachment through disruption of cellular membrane proteins and/or virus receptors. We believe that Quillaja extracts have promise as antivirals to reduce rotavirus infection and the severity of the disease in humans.

In vitro and in vivo evaluation 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.

An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections.

Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50 of 0.125 microgram/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50 of 251.39 micrograms/ ml. The combination of TF1 + TF2A + TF2B + TF3 was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50 was 34.7 micrograms/ml.

[Effects of qiwei baizhu san in inhibiting replication of human rotavirus in vitro].

Qiwei Baizhu San was found to have an inhibitory effect on human rotavirus (HRV)at monolayer of MA104 Cells. A 50% reduction in plaque number, a 10(1.86)TCID50 decrease in viral replication index and around 60% inhibition in viral RNA synthesis were observed at concentration of 100 mg/ml. Furthermore, the cytotoxicity of the decoction was low, while its promoting effect on growth and proliferation of the culture cells was observed at the concentrations of 12.5-50 mg/ml. The decoction was also found to have effects in prolonging the survival time of HRV infected cells and promoting the regeneration of the infected cells.

Use of the piglet to study the role of growth factors in neonatal intestinal development.

Milk growth factors are thought to contribute to postnatal gastrointestinal growth. The roles of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) in the neonatal piglet intestine were investigated. In the first study, piglets were infected with rotavirus on d 4 postpartum and received formula containing 0, 500 or 1000 micrograms/l of EGF for 8 days. A non-infected control group received no EGF. Infected piglets developed severe diarrhea and gained 60% less weight than controls. Rotaviral infection caused a 37% decrease in villus height and 40% decreases in intestinal enzyme activities compared to control. Oral EGF increased villus height and lactase activity in a linear dose-response fashion. Our results suggest that supplementation of formulas with high physiological levels of EGF may aid in small intestinal recovery. A second study investigated absorption of orally administered IGF-I. Newborn piglets were fitted with catheters via the umbilical artery and vein. Piglets were given formula containing 25 microCi of [125I]-IGF-I and blood samples were drawn for 24O min. Total radioactivity, protein bound counts, and counts immunoprecipitable with an antibody to IGF-I were determined in plasma. Radioactivity was detected in portal and arterial plasma within 15 min and rose throughout the study, however, protein bound counts were stable at 20-30% of total counts between 30 and 180 min postgavage. Approximately 10% of the counts were immunoprecipitable by a polyclonal antibody to IGF-I, suggesting that up to 10% of orally administered IGF-I may be absorbed intact.

Kinetics of viral replication in experimental rotavirus infection: effects of high dietary fat.

Epidemiological studies suggest that dietary fat can affect the frequency and severity of diarrhoeal illnesses in childhood. To study the effects of dietary fat on the kinetics of rotavirus antigen, 3-week-old mice of rotavirus-seronegative mothers were fed isocaloric diets with 40% of the total calories as fat [either butter (high saturated fat), olive oil (high monounsaturated fat), or corn oil (high polyunsaturated fat)] with one group on low fat (10% of calories) standard diet as controls. Seven-day-old mice from the first litter were killed and their stomach contents (milk) analyzed for total fatty acids. Seven-day-old mice from the second litter were inoculated with EDIM rotavirus, and some were killed at 48 h and others at 120 h postinoculation. The stool was removed from the distal colon of each mouse and examined for evidence of diarrhoea. The small intestine was removed, the contents washed, and the intestine divided into equal thirds. All stool and tissue samples were made to 10% (wt/vol), homogenised, and tested for rotavirus antigen by ELISA. We found that (a) dietary fat markedly altered the fatty acid profile of milk, (b) high saturated dietary fat (butter) delayed the onset of diarrhoea, reduced the excretion of rotavirus antigen in stool, and reduced mucosal antigen in the lower third of the small intestine at 120 h compared to 48 h postinoculation, (c) high polyunsaturated dietary fat initially increased the frequency of diarrhoea and viral antigen in the middle and lower segment at 120 h, followed by a reduction in rotavirus excretion in stool.(ABSTRACT TRUNCATED AT 250 WORDS)

Rotavirus replication in colostrum-fed and colostrum-deprived pigs.

A porcine rotavirus isolate was titrated in neonatal colostrum-fed and colostrum-deprived pigs. The stock rotavirus suspension had a titer of 10(-6.5)/ml and was in its fifteenth cell culture passage in MA-104 cells. Fourteen colostrum-fed pigs were orally inoculated with dilutions of the stock virus suspension ranging from undiluted to 10(-5). These pigs did not develop notable clinical signs during the 7-day experimental trial and no pathologic changes were found in intestine, liver, lung, kidney, spleen, or brain. However, rotavirus was detected in feces of the colostrum-fed pigs, using virus isolation and electron microscopic techniques. Rotavirus was also isolated from lung, brain, or spleen of 4 of 12 of these pigs. Sixteen colostrum-deprived pigs were orally inoculated with dilutions of the stock virus suspension ranging from 10(-1) to 10(-8). Diarrhea developed in 10 of 12 pigs that were given up to the 10(-6) dilution. Seven of these 12 pigs died because of the severity of diarrhea. Pigs that died of rotavirus-induced diarrhea had severe villus loss in the jejunum and ileum. Villi of the small intestine of colostrum-deprived pigs that survived the severe diarrhea were within normal limits at the end of the 7-day trial. The colostrum-deprived pigs that were inoculated with a dilution less than 10(-6) and survived past 96 hours underwent seroconversion. Rotavirus was detected by virus isolation and electron microscopy in the feces of all colostrum-deprived pigs that survived beyond 18.5 hours after inoculation. Virus was isolated from lungs, brain, or spleen of 12 of 16 colostrum-deprived pigs.

Isolation of bovine colostral lymphocytes: in vitro blastogenic responsiveness to concanavalin A and bovine rotavirus.

In order to study the in vitro proliferative activity of bovine colostral lymphocytes, the lacteal secretions from four Holstein heifer cows were collected at days 1 and 2 after calving for lymphocyte isolation. A density gradient centrifugation using a Ficoll-diatrizoate solution (sp gr 1.084 g/ml) allowed to isolate a cell population which consisted mainly of lymphocytes. The cell cultures incubated with Con A showed a significant in vitro lymphoblastic transformation, although the mitogenic response was lower than that which was seen with autologous blood lymphocytes. Two of the cows immunized with an adjuvanted-rotavirus vaccine during pregnancy also demonstrated a significant in vitro blastogenic blood and colostrum activity to bovine rotavirus, the higher response being observed with colostral lymphocytes. The immunocompetent cells found in bovine colostrum, with immunological activity to specific antigens, might have a role in passive protection of the neonate.

Effects of environmental variables and soil characteristics on virus survival in soil.

Because of the increasing emphasis placed upon land application as a means of wastewater disposal, it is important to evaluate the influences of different factors upon virus survival in soil. The objective of this study was to measure the effects of various environmental variables on virus persistence. Test samples of soil were placed in vials, and the soil was wetted with suspensions of virus in either distilled water, unchlorinated secondary sewage effluent, or mixtures of effluent and water. The viruses used were coxsackieviruses A9 and B3, echovirus 1, poliovirus 2, rotavirus SA11, and bacteriophages T2 and MS2. The rate of virus inactivation was evaluated statistically with regard to conditions under which the vials were incubated and to the soil characteristics. The factors that were found to influence virus survival were temperature, soil moisture content, presence of aerobic microorganisms, degree of virus adsorption to the soil, soil levels of resin-extractable phosphorus, exchangeable aluminium, and soil pH. Overall, temperature and virus adsorption to soil appeared to be the most important factors affecting virus survival.