Generation and Characterization of Monoclonal Antibody Against Porcine Epidemic Diarrhea Virus Nonstructural Protein 13.

Porcine epidemic diarrhea virus (PEDV) is an enteric swine coronavirus. Recent PEDV eruption in East Asian and North American countries made it notorious and caused huge economic losses to the porcine industry. Nonstructural protein 13 (nsp13) is a nucleic acid helicase/NTPase that plays a critical role in viral gene transcription and viral replication. To investigate the function of nsp13 in the context of PEDV infection, here, PEDV nsp13 gene was amplified and cloned into pET28a/pET30a/pGEX-6P-1 expression vectors. The recombinant his-tagged nsp13 and GST-tagged nsp13 were expressed. Purified his-tagged nsp13 from pET28a-nsp13 vectors was chosen for immunization. Three monoclonal antibodies (mAbs) named 5A9, 5C7, and 5G7 were identified from the hybridoma cells, and were characterized by Western blot analysis and immunofluorescent assay, which demonstrated high specificity of these three mAbs with pCAGGS-HA-nsp13. All three mAbs belong to IgG1+ kappa subclass. However, only mAb 5A9 could effectively and specifically recognize PEDV expressing nsp13. Furthermore, the generated antibody against nsp13 could be applied to investigate nsp13 function during PEDV replication.

Inhibition of porcine transmissible gastroenteritis virus infection in porcine kidney cells using short hairpin RNAs targeting the membrane gene.

The membrane (M) protein is the most abundant component of the porcine transmissible gastroenteritis virus (TGEV) particle. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, three plasmids (pRNAT-1, pRNAT-2, and pRNAT-3) expressing short hairpin RNAs were designed to target three different coding regions of the M gene of TGEV. The plasmids were constructed and transiently transfected into a porcine kidney cells, PK-15, to determine whether these constructs inhibited TGEV production. The analysis of cytopathic effects demonstrated that pRNAT-2 and pRNAT-3 could protect PK-15 cells against pathological changes specifically and efficiently. Additionally, indirect immunofluorescence and 50% tissue culture infectious dose (TCID50) assays showed that pRNAT-2 and pRNAT-3 inhibited the multiplication of the virus at the protein level effectively. Quantitative real-time PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the three plasmids were reduced by 13, 68, and 70%, respectively. This is the first report showing that RNAi targeting of the M gene. Our results could promote studies of the specific function of viral genes associated with TGEV infection and might provide a theoretical basis for potential therapeutic applications.

Effective inhibition of porcine transmissible gastroenteritis virus replication in ST cells by shRNAs targeting RNA-dependent RNA polymerase gene.

Transmissible gastroenteritis virus (TGEV) is identified as one of the most important pathogenic agents during swine enteric infection, leading to high mortality in neonatal pigs and severe annual economic loss in swine-producing areas. Up to date, various vaccines developed against TGEV still need to be improved. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, two shRNA-expressing plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) targeting the RNA-dependent RNA polymerase (RdRp) gene of TGEV were constructed and transfected into swine testicular (ST) cells. The cytopathic effect (CPE) and MTS assays demonstrated that both shRNAs were capable of protecting cells against TGEV invasion with very high specificity and efficiency. A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the two plasmids were reduced by 95.2% and up to 100%, respectively. Our results suggest that RNAi might be a promising new strategy against TGEV infection.

Diagnosis of neonatal pig diarrhea.

To be effective, swine practitioners should develop a unit health program. Development should involve unit managers, owners, and employees involved in day-to-day operations. Emphasis on training personnel and management to reduce disease and collection of accurate records is necessary. Routine diagnostics are needed to solve disease problems. Communication with laboratory personnel to ascertain what samples are needed for diagnosis of particular problems cannot be overemphasized. General diagnosis of disease problems outlined by Vinson can be similarly followed within the specifics of diarrheal problems within units. 1. Observe symptoms exhibited by pigs, i.e., huddling, fecal material around perineum, extreme thirst, etc. 2. Evaluate the degree of morbidity and potential production losses. 3. Analyze possible specific causes of symptoms, i.e., environmental cleanliness, affected litter distribution, age of affected neonates, and other populations affected. 4. Examine live animals, i.e., obtain serum samples from a random population, take rectal temperatures of affected neonates, and evaluate fecal pH. 5. Necropsy dead or dying pigs [that] appear to represent the problem. 6. Submit live pigs or appropriate tissues from necropsied pigs to a diagnostic laboratory. 7. Re-evaluate environmental conditions that may be contributing to the problem (remember, unit employees are a part of the pigs' environment). 8. Evaluate management procedures contributing to the disease problem, i.e., lack of adherence to all-in all-out, rapid turn-around decreasing cleaning time etc. Following this format and communicating with diagnosticians should provide for positive results for the producers both entities serve.

Treatment and prophylaxis of some infectious diseases of swine and cattle using of natural alpha-interferons.

On the basis of experimental studies which proved stimulating action on absorbing and bactericidal function of blood phagocytes and antibody genesis the methods of therapy and prophylaxis of wide spread infections in modern live-stock (transmissive gastroenteritis of pigs, parainfluenza of calves, colibacteriosis of swine and cattle) were created. It was established, that optimal one-time therapeutical dose of homologous alpha-IFN for intramuscular administration to new-born pigs was 2000-4000 IU per head, prophylactic dose was 1000-2000 IU. Therapeutical doses of alpha-IFN for calves did not exceed 4000-6000 IU, prophylactic--4000 IU. As a rule, it is necessary to introduce the preparation three times with 48 h intervals. With the account of these data the large-scale commissional trials of these preparations were carried out.

Oral treatment of transmissible gastroenteritis with natural human interferon alpha: a field study.

During a natural outbreak of transmissible gastroenteritis (TGE), groups of piglets were treated orally for 4 consecutive days with placebo or 1.0, 10.0 or 20.0 international units (IU) natural human interferon alpha (nHuIFN alpha). Piglets that were 1-12 days of age and given 1.0, 10.0 or 20.0 IU nHuIFN alpha had significantly (P < 0.01) greater survival rates than placebo-treated piglets; survival rates were the greater for the highest level of nHuIFN alpha treatment. In contrast, beneficial effects of nHuIFN alpha were not observed in piglets farrowed during the disease outbreak and given nHuIFN alpha within hours of birth. Oral nHuIFN alpha therapy modulates the natural course of high morbidity and mortality commonly seen with TGE.

[Changed health problems in a changing pig-farming concern in The Netherlands until 1980]. / Veranderende gezondheidsproblematiek in een veranderende varkenshouderij in Nederland tot 1980.

In 1905 Poels published 'Disease of swine in the Netherlands'. This book dealt predominantly with swine fever, erysipelas, tuberculosis and 'pneumonia'. Between 1920 and 1940 others reported on streptococci, lobular haemmorrhagic pneumonia, bordetellosis, Aujeszky's disease and postweaning diarrhoea. After the second world war, particularly after 1960, the Dutch pig-farming industry developed at a tremendous rate. As a consequence, the health problems changed. Certain diseases became less important: tuberculosis, erysipelas, Leptospira tarrasovi, enteroviruses. Yet other diseases including postweaning diarrhoea, atrophic rhinitis and Aujeszky's disease became problems of increasing importance. At the end of the seventies the knowledge of E. coli toxin types was substantial. On the other hand, information concerning the pathogenesis and pathophysiology was very limited. Bordetella bronchiseptica was still considered to be the most important agent in AR, zootechnical factors being predisposing. However, one was aware of missing links. Aujeszky's disease was obscure until the late fifties. Until 1972 only occasional reports were made. In that year, an epidemic occurred in the Gelderse Vallei. Another epidemic occurred in 1974 in the provinces of Brabant and Limburg. By 1980 proper vaccines were available and Aujeszky's disease was not yet a political problem.

Fluid therapy trials in neonatal piglets infected with transmissible gastroenteritis virus.

The main purpose of this study was to evaluate the effectiveness of an oral fluid therapy alone or combined with parenteral administration of a 5% dextrose solution to attenuate the clinical signs and the pathophysiological consequences of transmissible gastroenteritis in neonatal piglets. Eighteen two day old conventional piglets were infected with transmissible gastroenteritis virus while six others were used as controls (Group 1). At the onset of diarrhea, infected piglets were divided into three groups of six (Groups 2, 3 and 4). Piglets in group 2 were not treated and were fed a milk replacer ad libitum. Piglets in group 3 were removed from the milk replacer and placed on an oral glucose-glycine-electrolyte solution ad libitum. Those in group 4 were placed on oral fluid therapy and received a 5% dextrose solution intraperitoneally at the rate of 25 mL/kg of body weight once a day. Blood samples were collected in heparin within minutes after the infected piglets became comatose and from the controls at four or five days of age. The following variables were measured: packed red cell volume, blood pH, total plasma protein and bicarbonate, blood urea nitrogen, and plasma glucose, creatinine, chloride, inorganic phosphorus, sodium, potassium, magnesium and calcium. Vomiting and diarrhea appeared 12 to 24 hours postinoculation in the infected piglets. There was a sudden and rapid progression into a comatose and moribund state one or two days later whether the infected piglets were treated or not.(ABSTRACT TRUNCATED AT 250 WORDS)

[Isolation and use of a farm-specific hyperimmune serum against transmissible gastroenteritis]. / Poluchavane i prilagane na obornospetsifichen khiperimunen serum sreshtu transmisiven gastroenterit.

A specific stationary hyperimmune serum was obtained in a pigbreeding complex with a transmissive gastroenteritis infection via trifold (at a fourteen-day interval) i/m injection of sows with 5 cm3 each of undiluted virus (10(6) TCCPE50 per cm3). Tested were hyperimmunization programmes with pigs in the final fattening period. It was found that the use of undiluted virus led to the equation of sows in terms of their immunologic state and to the essential rise of the titer of humoral antibodies. In order to obtain high titer immune serum against transmissive gastroenteritis it is sufficient to proceed with the i/m injection of pigs or adult swine in the final stage of fattening at rising amounts (from 3 to 10 cm3) of attenuated virus of sufficiently high titer (10(6) TCCPE50 per cu. cm). The hyperimmune serum produced a very good prophylactic effect with newborn pigs on the same farm - the twofold oral administration of 5 to 10 cm3 on the day of birth and a couple of days later led to a drop of both morbidity and mortality rate as well as to the improvement of body development.

Observations on clinical aspects of transmissible gastroenteritis of pigs in Norfolk and Suffolk, 1980-81.

Outbreaks of transmissible gastroenteritis in 56 breeding units, six maiden gilt sites and 64 fattening units were investigated during the period December 1980 to October 1981. The disease invariably occurred in classical virulent form, resulting in very high mortality in baby piglets. Reproductive sequelae were sometimes evident. Individual outbreaks continued for longer than in previous years. There was a high rate of recrudescene, particularly in large breeding units, and infection also persisted in many fattening units. The value of control measures and supportive therapy is discussed.

[Transmissible Gastroenteritis in Swine (author's transl)]. / Transmissible gastroenteritis bij varkens

Transmissible gastroenteritis or TGE is a virus diarrhoea which occurs in pigs of all ages and is associated with high mortality rates in the young piglets. Growth of virus in the columnar epithelium of the small intestine causes atrophy of the intestinal villi, malabsorption, watery diarrhoea and dehydration. Faecal excretion of virus usually continues up to fourteen days after infection but chronic carriers have been found to occur. TGE is self-limiting on the majority of pig-breeding farms but the virus may persist in particular conditions and an enzootic form of the disease will appear in this case. In typical outbreaks, the diagnosis can usually be based on clinical symptoms. When the disease runs an enzootic course, a clinical diagnosis will be out of the question. TGE should be differentiated from colibacillosis and from another virus diarrhoea, the aetiology of which is not precisely known. A rapid and correct diagnosis may be established by direct fluorescent antibody studies of frozen sections of the small intestine in infected piglets. When sows have been spontaneously infected, their offspring will be protected by lactogenic immunity. The presence of TGE antibodies of IgA class in the milk is required to ensure complete immunity of the piglets lasting for weeks on end. Intramuscular inoculation of a commercially available vaccine in sows will only stimulate the production of antibodies of the IgG class in the milk. These antibodies will merely afford short-lived immunity. The vaccine cannot prevent symptoms of disease from appearing in piglets following infection with virulent TGE virus but it does reduce mortality

[Economic problems in the control of transmissible gastroenteritis of swine]. / Ekonomická problematika tlumení virové gastroenteritidy prasat

The losses ascribable to transmissible gastroenteritis of pigs were financially determined at eight sites with 133-1269 large animal units, as converted to pig stocks. The overall losses were divided as follows: mortality and confiscation - 42.38%, weight gain reduction - 30.50%, extra costs of the enterprise - 13.11%, emergency slaughters 7.79%, and losses in progenies 6.22%. The coefficient of loss per one diseased animal was 42.63 to 114.65 crowns. Furthermore, the level of the assumed losses due to mortality and morbidity prevented by veterinary measures was also calculated. The calculation of the effectiveness of these measures showed that 3.14--60.01 crowns are saved per 1 crown spent. The total number of losses, loss coefficient per one diseased animal, the level of prevented loss, and the money saved per 1 crown were calculated from the over-all societal viewpoint and from the viewpoint of the agricultural enterprise after the detraction of the indemnities from the State Insurance Company.