Heterogeneity of Foot-and-mouth Disease Virus: Further Studies on Plaque Formation by Two Plaque-size Variants

Plaque production by a small-plaque (SP) and large-plaque (LP) variant of foot-and-mouth disease virus, type A, strain 119 (FMDV, A119), was influenced by a number of environmental factors. The SP variant produced plaques on cells of the IB-RS-2 cell line from swine kidney and to a lesser degree on primary cultures of swine kidney cells, but plaque formation was inhibited on primary cultures of bovine kidney (BK) cells unless diethylaminoethyl (DEAE) dextran was added to agar overlays. When DEAE dextran-treated agar overlay was used, the LP variant formed larger plaques on BK cells but not on IB-RS-2 cells. Concentrations of DEAE dextran from 0 to 100 µg/ml greatly enhanced the formation of SP virus plaques on BK cells but had little or no effect on the average size of plaques produced by the LP variant. Higher concentrations of polycation enlarged the plaques formed by both variants. Plaque sizes of the SP and LP variants increased as the concentration of agar or agarose in the overlays decreased. Reducing the concentration of agar to 0.75% facilitated the formation of SP virus plaques, but better plaque production occurred under agarose overlays.

The original parent virus consisted predominantly of virus particles that formed small plaques. The rate of neutralization of the parent virus by guinea pig antiserum prepared against the parent virus was faster than antiserum inactivation of a low-passage virus of the same serotype and strain.

     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

Studies on Trånsmissible Gastroenteritis of Swine: III. The Effect of Selective Inhibitors of Viral Replicåtion on a Cytopathogenic Virus from Transmissible Gastroenteritis

The effect on the plaque production of the Purdue strain of cytopathogenic virus from transmissible gastroenteritis of swine by 5-bromo-2'-deoxyuridine (BUDR), 5-iodo-2'-deoxyuridine (IUDR), actinomycin-D, puromycin, and amantadine-HCI (Symmetral) has been studied.

Amantadine-HCI reduced the plaque-forming units of virus per ml by approximately 98%. Puromycin prevented almost all virus reproduction while actinomycin-D caused approximately a 22% reduction. Both IUDR and BUDR produced approximately a 20% increase in plaque-forming units of virus per ml.

Swine testis cells stained with acridine orange early in the course of infection contained brick-red particles in the cytoplasm, indicative of a ribonucleic acid (RNA) type virus.

     

The Effect of Diethylaminoethyl Dextran and Agar Overlay pH on Plaque Formation by Two Plaque-size Variants of Foot-and-Mouth Disease Virus

The effect of diethylaminoethyl (DEAE) dextran and agar overlay medium pH on a small-plaque (SP) and large-plaque (LP) foot-and-mouth disease virus (FMDV), type A, strain 119 (A119) was studied. The SP virus was inhibited under normal agar overlay but the addition of 100, 1,000 and 2,000 µg DEAE dextran/ml of agar overlay permitted plaque development. By using untreated and DEAE dextran-treated agar overlay medium, plaque formation by the SP virus was enhanced when the pH of agar medium was raised to a more alkaline level before overlay. Plaques formed by the LP virus were relatively uninhibited under the regular overlay but were larger in the presence of 1,000 µg DEAE dextran/ml. The enhancement of LP virus plaques occurred at various pH levels and was also inversely related to the hydrogen ion concentration of agar overlays; regular and DEAE dextrantreated alkaline overlays produced larger plaques.     

Bovine Viral Diarrhea Virus and Escherichia Coli in Neonatal Calf Enteritis

This study was initiated to determine the etiologic and pathogenic significance of an American strain of bovine viral diarrhea (BVD) virus (strain NADL-MD) in enteritis of neonatal calves (calf scours).

Three colostrum-fed calves from dams exposed intravenously to BVD virus at 6, 16 and 25 days prepartum, respectively, had moderate diarrhea persisting until the eighth day of life. The BVD virus was isolated from all 3 calves and persisted up to 93 days in 1 calf, indicating either that BVD was transmitted in utero or via the dam's milk.

Three specific pathogen free (SPF) calves permitted dams' colostrum for the first 4 feedings and then given milk replacer were exposed orally on the day of birth to BVD virus. One calf died of neonatal enteritis 28 hours post-exposure and at necropsy the BVD virus was isolated from several of its organs. The remaining 2 calves had a mild diarrhea persisting to the eighth day of age.

Two calves permitted dams' colostrum ad lib. for 72 hours, and then weaned, were exposed orally to BVD virus. Both calves had a mild persistent diarrhea and BVD virus was isolated from their blood for 56 days post-exposure.

Of 13 SPF, colostrum-deprived calves exposed orally or intranasally at birth to the BVD virus, 4 had severe diarrhea and died of neonatal enteritis from 38 hours to 13 days postexposure. Isolations of BVD virus were made from several of the organs of the calves at necropsy. All of the 9 surviving calves had a moderate to severe diarrhea frequently persisting for 7 to 10 days, and BVD virus was isolated from the survivors up to 103 days postexposure.

Several strains of Escherichia coli were isolated from calves after the second day of life, but were neither pathogenic for mice, nor serologically related to strains of E. coli usually associated with outbreaks of calf scours. Four colostrum-deprived SPF calves were exposed orally at birth to a strain of E. coli isolated from the intestine of the calf with the most acute symptoms and fatal neonatal enteritis. None of the four calves receiving the E. coli had diarrhea. One calf, however, had respiratory distress and died on day 5.

Two SPF colostrum-deprived control calves had neither diarrhea nor respiratory distress.

The above findings support the conclusion that BVD virus should not be overlooked as a primary cause of the neonatal calf enteritis complex.

     

A Stable L-form of Haemophilus pleuropneumoniae

A stable L-form of Haemophilus pleuropneumoniae (Shope) was isolated from primary pig kidney cell tissue cultures which had been inoculated 28 days previously with glycine induced spheroplasts of this organism.

H. pleuropneumoniae was definitely cytopathic in primary pig kidney cell cultures, producing cell rounding, cytoplasmic vacuolation and nuclear enlargement with peripheral condensation of nuclear DNA. By contrast, the effect of spheroplasts was much less distinct, producing only loss of cytoplasmic granularity coincident with apparent loss of some cytoplasmic RNA, and slight nuclear enlargement.

Both the organism and its L-form were shown to be related by cultural methods, antibiotic sensitivity tests, immunofluorescence and immunodiffusion.

The L-form remained stable after 90 serial passages on agar and 45 in broth, each medium being capable of supporting the growth of both forms of the organism.

     

The relation between pathogenicity and plaque size of Getah virus Kanagawa strain in suckling mice

The relation among biological properties, particularly pathogenicity for suckling mice, and plaque size was studied in four virus strains: Getah virus strain Kanagawa; two strains obtained by plaque cloning of the Kanagawa strain, Getah Kanagawa SP (G-K-SP) strain which forms small plaques (SP) only and strain G-K-LP which forms large plaques (LP) only; and strain Haruna which forms SP only. There were no marked differences among the four strains in serological properties, growth curves and sensitivity to pH, trypsin and temperature. Strain G-K-LP showed higher pathogenicity for suckling mice than strain G-K-SP. However, the pathogenicity of strain Haruna, which forms SP only, was as high as that of strain G-K-LP. Some of the clones in SP of strain Kanagawa kill all mice in 5 to 6 days after inoculation while the others required 9 to 11 days or longer before causing death. The present study showed that the pathogenicity of Getah viruses shortly after being isolated from the field, such as the Kanagawa strain, is different between large and small plaques, and even among small plaques, at least in suckling mice, and that the pathogenicity has no relation to plaque size.     

Interactions of amino acids and hormones regulate the balance between growth and milk protein synthesis in lactating rats fed diets differing in protein content

Insulin-like growth factor-I (IGF-I), growth hormone (GH), and prolactin (PRL) play important roles in milk protein synthesis, and their plasma concentrations were reported to be affected by dietary protein intake. To investigate the relationship between circulating amino acid (AA) and concentrations of these hormones, 18 Wistar rats aged 14 wk were assigned to a low (LP; 9% protein), standard (SP; 21% protein), or high-protein (HP; 35% protein) diet from parturition through day 15 of lactation. Plasma, liver, pituitary gland, skeletal muscle, and mammary gland samples were collected at the end of treatment. Circulating and hepatic IGF-I concentrations increased linearly with elevated dietary protein concentrations (P < 0.0001). Rats receiving the HP diet had higher circulating GH (P < 0.01) and pituitary PRL concentrations (P < 0.0001) but lower pituitary GH concentration (P < 0.0001) relative to those in rats receiving the LP and SP diets. Pearson correlation test performed on composed data across treatments showed that several circulating AAs were correlated with circulating and tissue concentrations of IGF-I, GH, and PRL. Multiple linear regression analyses identified Leu, Gln, Ala, Gly, and Arg as the main AAs associated with hormone responses (R² = 0.37 ~ 0.80; P < 0.05). Rats fed the LP and HP diets had greater Igf1 and Ghr gene expression in skeletal muscle than those fed the SP diets (P < 0.01). However, LP treatment decreased Prlr mRNA abundance in mammary glands as compared with the SP and HP treatments (P < 0.05). The HP diets increased AA transporter expression (P < 0.01) but decreased mammalian target of rapamycin (P < 0.05) and 70 kDa ribosomal protein S6 kinase 1 (P < 0.01) phosphorylation in mammary glands as compared with the LP and SP diets. The results of the present study suggested that several circulating AAs mediated the effects of dietary protein supply on concentrations of IGF-I, GH, and PRL, which in turn altered the metabolism status in peripheral tissues including the lactating mammary glands.     

The Effect of Edema, Hydrocortisone Acetate, Concurrent Viral Infection and Immunization on the Clearance of Pasteurella hemolytica from the Bovine Lung

The influence of pulmonary edema, hydrocortisone, immunization against Pasteurella hemolytica and concurrent infection with parainfluenza-3 virus upon pulmonary clearance of aerosolized P. hemolytica was studied in 31 calves. Following the various treatments calves were challenged with an aerosol of P. hemolytica. One control calf was killed immediately after the aerosol and the numbers of bacteria in the lung taken as 100%. Two calves were killed four hours after challenge and the numbers of bacteria in the lungs were compared to the 100% of the control calf. The result was the percentage clearance of bacteria at four hours.

Pulmonary edema was induced by three different methods: by an aerosol of histamine, by intravenous injection of endotoxin and by intravenous injection of croton oil emulsion. The edema impaired the clearance of P. hemolytica, which was reflected in high numbers of P. hemolytica present in the lungs at four hours after challenge: 260% after histamine, 300% and 400% after endotoxin and 92% after croton oil.

Six days of treatment of four calves with high doses of hydrocortisone acetate produced inconsistent results: two calves treated with a higher daily dose (36 mg/kg) had normal clearance whereas two calves treated with a lower dose had pulmonary edema and displayed lowered clearance with 111% and 31% respectively of P. hemolytica retained in the lungs four hours after challenge.

Immunization of calves by three different methods, a subcutaneously injected bacterin of P. hemolytica (2 calves), single aerosol (2 calves) and four aerosols (4 calves) of live P. hemolytica was reflected in an accelerated pulmonary clearance of P. hemolytica (with a mean of 1.55% of bacteria retained at four hours).

Concurrent infection with parainfluenza-3 virus did not lower the clearance of P. hemolytica in the lungs of 12 calves over 15 days except on the first day following the exposure to parainfluenza-3 virus. These calves had hemagglutinating antibodies against P. hemolytica before exposure.

     

Alterations in the jejunum of young goats caused by feeding soy protein‐based diets

This study was designed to investigate whether soy protein or soy protein supplemented with indispensable amino acids (AA) change the protein expression pattern and utilization of pre‐cursors for RNA biosynthesis in jejunal mucosa in relation to casein and whether these changes affect mucosal cell growth. Kids were fed comparable diets based on cow`s milk, of which 50% of crude protein were replaced by either casein (CAS), soy protein (SP) or soy protein supplemented with indispensible AA (SPA) for 34 days (n = 4/group). Jejunal tissue was collected 5 h after adding a single dose of ¹⁵N‐RNA to the diet, in order to determine morphology, protein repertoire by two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry, and RNA biosynthesis by isotope ratio‐mass spectrometry. In mid‐jejunum, morphological alterations induced by partial replacement of casein with soy protein were accompanied by changes in mucosal proteins related to generation of the cytoskeleton and in pathways for mucosal RNA biosynthesis, resulting in a smaller re‐utilization of dietary RNA pre‐cursors and in an increased activity of enzymes involved in nucleic acid breakdown. Soy protein supplemented with indispensible aminoacids tended to revise mucosal growth retardation with no impact on salvage of dietary RNA pre‐cursors for mucosal RNA biosynthesis, but changes in cytoskeleton generation. Feeding soy protein with supplementation of indispensible AA does not ameliorate soy protein effects on mucosal morphology and RNA metabolism in the jejunum in a significant manner.     

Colony Formation in Culture by Bovine Granulopoietic Progenitor Cells

Calf bone marrow cells cultured in a semi-solid medium of 0.8% methyl cellulose produced colonies of granulocytic cells and macrophages by seven days. A prerequisite for colony growth was the presence of serum obtained from a calf three hours after intravenous injection of endotoxin. Three morphological types of colonies were seen but cell types within these types of colonies did not differ. Cultured cells were identified by morphological and cytochemical characteristics.

Optimum growth occurred when serum from endotoxin stimulated calves and fetal calf serum were present in a volumetric ratio of 7:3. Inhibition of colony growth occurred when endotoxin-stimulated serum was present at greater than optimum concentration. Normal calf serum, fetal calf serum, mouse L-cell conditioned medium and bovine urine did not stimulate significant colony growth when 8.0 x 10⁴ marrow cells were cultured.

There was a linear relationhip between the number of marrow cells in the cultures and the number of colonies produced. Colony forming efficiency ranged from 13 to 59 colonies per 10⁵ cells plated.

The behaviour of calf colony forming units in suspension culture was similar to that reported for mouse colony forming units.

     

Physicochemical Characterization of a Neonatal Calf Diarrhea Virus

Physicochemical characteristics of two isolates of a neonatal calf diarrhea virus were investigated. Neither isolate was sensitive to ether or chloroform, both were stable at pH 3.0, were relatively heat resistant, but were thermolabile when heated to 50°C for one hour in the presence of 1.0 M MgCl₂. Multiplication of virus was not inhibited by concentrations of 5-iodo-2'-deoxyuridine (IDUR) up to 500 µg/ml, which indicated that the nucleic acid was ribonucleic acid (RNA). Also, multiplication was not inhibited by concentrations of actinomycin D (AD) up to 0.5 µg/ml. Thermal denaturation studies demonstrated that the nucleic acid had a high melting temperature.

Resistance to lipid solvents, stability at an acid pH, relatively high thermostability, type of nucleic acid, plus previous reports from this laboratory on general morphology and cytopathogenicity suggest that the virus may belong to the diplornavirus (reovirus) group. However, thermolability in the presence of 1.0 M MgCl₂ is not consistent with characteristics of this group.

     

靶向env及LTR基因的siRNA抑制ALV-J 复制的研究

试验针对J亚群禽白血病病毒的env及LTR基因,根据它们的保守序列,分别设计合成了4~5对siRNA,并将其克隆至pSilencer 4.1,构建siRNA重组表达质粒。将该质粒转染DF-1细胞6 h后以10³ TCID₅₀接种ALV-J,利用Real-time RT-PCR在mRNA表达水平检测各重组质粒对病毒复制的影响。结果表明,与阴性对照相比,pSi4.1-env各重组质粒对env基因的抑制效果达到18.15%~63.43%,pSi4.1-LTR各重组质粒的抑制效果则达到19.37%~45.41%。     

A Comparison of Bluetongue Virus and EHD Virus: Electronmicroscopy and Serology

Bluetongue virus, BT₈, and the virus of epizootic hemorrhagic disease (EHD) of deer, NJ-55, were plaque purified and compared electronmicroscopically and serologically. The latter included a plaque reduction neutralization test, the agar gel precipitin test, and the complement fixation test. The viruses were indistinguishable morphologically, but antigenically different. A plaquing technique was described for EHD virus.     

Preliminary Observations on the Isolation of Escherichia coli Bacteriophages from the Intestinal Contents of Chickens

Eschericia coli bacteriophages were isolated from the intestines of chickens. These phages had different lytic patterns, and were propagated in nutrient broth containing 0.4 gm calcium chloride/litre. The agar layer technique was used to determine the routine test dilution (RTD) and plaque morphology. The phages differed in their 1) morphology, 2) RTD values, and their ability to lyse E. coli strains from various animals. All phages isolated lysed human K12 E. coli strains, whereas only two phages lysed the chicken E. coli strain. Phages isolated lysed E. coli from chicken, bovine, ovine, equine, and human, but not from porcine, canine and other avian species.     

Studies on Transmissible Gastroenteritis of Swine I. The Isolation and Identification of a Cytopathogenic Virus of Transmissible Gastroenteritis in Primary Swine Kidney Cell Cultures

Two virus isolates from transmissible gastroenteritis (TGE) of swine were adapted to grow in primary swine kidney cells. Growth of the virus was indicated by the resistance of the infected cells to the cytopathic effect of a virus diarrhea virus of cattle, and by the development of large round cells on the cell sheet.

Evidence that these virus isolates were TGE was obtained by the development of signs of the disease followed by death of exposed SPF pigs, or the resistance of the recovered pigs to further signs of disease when they were exposed to virulent TGE contained in virus bearing intestinal tissue.

The in vitro and in vivo serum neutralization tests, along with staining of infected cells by fluorescein conjugated TGE antiserum, gave further indication of the specific nature of the virus growing in the cell cultures.

     

Enhancement of infectious bovine rhinotracheitis virus replication with corticosterone

Infectious bovine rhinotrachetis virus (IBRV) progeny was increased ten to 12 fold in bovine turbinate (BT) cells treated with 10^?3 M corticosterone acetate (CCA) as demonstrated by plaque assay. Autoradiographic studies demonstrated an increased binding of ³H-corticosterone (³H-CS) in IBRV infected cells and the fractionation of labelled cells revealed 78–80% of the total hormone associated with the cytoplasmic components. Incorporation of ³H-uridine and ³H-valine precursors into cells treated with the hormone demonstrated up to 16-fold increase in RNA and protein synthesis which was inhibited by the addition of actinomycin D. The data suggest that increased rate of macromolecular synthesis in IBRV infected cells treated with the corticosteroid may result in the enhancement of virus production.     

Serological examination and egg production of progeny of fowl experimentally infected with Egg Drop Syndrome 1976 virus

Summary

Following EDS'76 virus (BC14 virus) infection of breeder chickens by the conjunctival route, vertical transmission occurred in the first week after infection. In the progeny which had been infected with EDS'76 virus by the vertical route, increasing haemagglutination inhibiting (HI) litres to BC14 virus and increasing numbers of birds with HI litres were observed from 3 weeks to 15 weeks of age. Sixty‐one per cent of the hens and 77 per cent of the cocks had ²log HI BC14 virus litres exceeding 4 at an age of 15 weeks.

Some birds which had been serologically negative throughout the rearing period, seroconverted between 25 and 28 weeks of age. This phenomenon occurred in hens as well as in cocks. Simulation of stress twice during the laying period by injection of corticosteroid hormone did not increase the number of birds serologically positive to EDS'76 virus.

EDS'76 was observed in the group of hens that was vertically infected, since egg production was significantly depressed between 28 and 34 weeks of age. Probably this was mainly the result of a production drop in the hens showing seroconversion at 27 or 28 weeks of age.

In the group of fowl vertically infected with EDS'76 virus, serologically positive birds appeared to be protected for the greater part to BC14 virus challenge at 50 weeks of age, while negative birds seemed to be fully susceptible. Chicks hatched from eggs collected in the third and fourth week after infection of the dams had maternal antibodies. Fertility and hatchability of apparently normally shelled eggs seemed not to be affected after BC14 virus infection of the dams. Intensive contact with contaminated faeces is probably an indispensable condition for lateral transmission of the virus.     

Effects of Tryptophan Analogs, Mitomycin c and Acridine Orange on the Production of Filtrable Hemolysin by Escherichia coli

Bacteriostatic doses of 5-methyltryptophan and of 7-azatryptophan exert a complete inhibition on the in vitro production of filtrable hemolysin by Escherichia coli. This inhibition is readily overcome by L-tryptophan, and does not seem to be specific but secondary to an interference with de novo protein synthesis and cell multiplication as is observed with chloramphenicol in sensitive strains. Although the influence of the two tryptophan analogs on hemolysin production and cell multiplication is similar, their mechanism of action at the molecular level appears to be different.

The addition of 50 µg/ml 7-azatryptophan which causes an unbalanced growth characterized by an arrest of the cellular division and an increase of cell size, blocks the production of an active hemolysin. Exposure to 4 µg/ml 5-methyltryptophan also prevents cell multiplication and hemolysin production but no sign of unbalanced growth is evident. Mitomycin C in concentrations sufficient to prevent increase in the number of viable units provokes an extreme elongation of E. coli cells and, apparently, does not stop the synthesis of hemolysin. In blocking the production of hemolysin the three inhibitors of protein synthesis used in this study were more effective than mitomycin C an agent known to affect deoxyribonucleic acid synthesis and to induce extrachromosomic genetic factors. Results of conjugation experiments also described here support the finding of other workers that the genetic factor that controls the production of the filtrable hemolysin in E. coli can be transmitted by conjugation. Acridine orange eliminated the hemolytic property from a large proportion of the population of a hemolytic strain which did not carry the R factor, but was little effective in the strains which had received both the R factor and the hemolytic character by conjugation.

     

Laboratory Studies on the Transmission of Western Equine Encephalitis Virus by Saskatchewan Mosquitoes I. Culex tarsalis

A Saskatchewan strain of the mosquito Culex tarsalis, transmitted a local strain of western equine encephalitis virus from chick to chick, between four and 44 days after an infective blood meal. At incubation temperatures of 69 and 75°F, 120 transmissions occurred out of a possible 141, and all but seven of these were by single infected mosquitoes. At 75°F virus titers in individual mosquitoes were more uniform and transmission was more efficient, than at 69°F, although infection rates were similar at both temperatures. The minimum concentration of virus required to infect 50% of C.tarsalis was 10^2.5 intracerebral three-week old mouse LD₅₀ per 0.03 ml of donor blood. These findings provide direct evidence that C. tarsalis of Saskatchewan is a highly efficient vector of western equine encephalitis virus.