Genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine.

Modified live vaccines (MLVs) based on genotype 1 strains, particularly C-strain, have been used to prevent and control classical swine fever virus (CSFV) worldwide. Nevertheless, a shift in the predominant CSFV strains circulating in the field from genotype 1 or 3 to genotype 2 is seen. Genotype 2 is genetically distant from the vaccine strains and was recently reported during outbreaks after vaccine failure; this has raised concerns that vaccination has influenced viral evolution. In Korea in 2016, there was an unexpected CSF outbreak in a MLV-vaccinated commercial pig herd. The causative CSFV strain was genetically distinct from previously isolated Korean strains but similar to recent Chinese strains exhibiting enhanced capacity to escape neutralization; this suggests the need for global cooperative research on the evolution of CSFV. We analysed global E2 sequences, using bioinformatics tools, revealing the evolutionary pathways of CSFV. Classical swine fever virus genotypes 1 and 2 experienced different degrees and patterns of evolutionary growth. Whereas genotype 1 stayed relatively conserved over time, the genetic diversity of genotype 2 has progressively expanded, with few fluctuations. It was determined that genotype 2 evolved under lower immune pressures and at a higher evolutionary rate than genotype 1. Further, several selected codons, under diversifying selection in genotype 1 but under purifying selection in genotype 2, correspond to antigenic determinants, which could lead to evasion of vaccine-induced immunity. Our findings provide evidence that evolutionary changes in CSFV are the result of the disproportionate usage of the CSF MLVs in endemic areas; this underscores the need to develop mitigation strategies to minimize the substantial risk associated with the emergence of vaccine-escaping mutants.

Hematological adverse effects and pharmacokinetics of ribavirin in pigs following intramuscular administration.

Ribavirin (RBV) is a synthetic guanosine analog that is used as a drug against various viral diseases in humans. The in vitro antiviral effects of ribavirin against porcine viruses were demonstrated in several studies. The purposes of this study were to evaluate the adverse effects and pharmacokinetics of ribavirin following its intramuscular (IM) injection in pigs. Ribavirin was formulated as a double-oil emulsion (RBV-DOE) and gel (RBV-Gel), which were injected into the pigs as single-dose IM injections. After injection of RBV, all of the pigs were monitored. The collected serum and whole blood samples were analyzed by liquid chromatography-tandem mass spectrometry and complete blood count analysis, respectively. All of the ribavirin-treated pigs showed significant decreases in body weight compared to the control groups. Severe clinical signs including dyspnea, anorexia, weakness, and depression were present in ribavirin-treated pigs until 5 days postinjection (dpi). The ribavirin-treated groups showed significant decrease in the number of red blood cells and hemoglobin concentration until 8 dpi. The mean half-life of the RBV-DOE and RBV-Gel was 27.949 ± 2.783 h and 37.374 ± 3.502 h, respectively. The mean peak serum concentration (Cmax ) and area under the serum concentration-time curve from time zero to infinity (AUCinf ) of RBV-DOE were 8340.000 ± 2562.577 ng/mL and 16 0095.430 ± 61 253.400 h·ng/mL, respectively. The Cmax and AUCinf of RBV-Gel were 15 300.000 ± 3764.306 ng/mL and 207526.260 ± 63656.390 h·ng/mL, respectively. The results of this study provided the index of side effect and pharmacokinetics of ribavirin in pigs, which should be considered before clinical application.

Immunohistochemical application of an antibody specific for human CD1a to the diagnosis of canine mast cell tumour.

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.

Cellular characterization of multidrug resistance P-glycoprotein, alpha fetoprotein, and neovascular endothelium-associated antigens in canine hepatocellular carcinoma and cirrhotic liver.

P-glycoprotein (P-gp), which is encoded by the multidrug resistance gene (MDR-1); alpha fetoprotein (AFP); and vascular endothelium-associated antigens are well-known markers for human and canine hepatic diseases. We obtained liver tissues from 5 dogs with hepatocellular carcinoma (HCC) and 12 dogs with cirrhosis, and we performed histopathologic and immunohistochemical evaluations using anti-P-gp, anti-AFP, anti-CD31, and anti-CD34 antibodies. P-gp was expressed at higher levels in HCC than in cirrhotic livers ( P < .01), and was most commonly localized in biliary canaliculi and small ductuli. AFP was localized mainly in the cytoplasm in HCC ( P < .01) and in a few cases of cirrhosis. In both HCC and cirrhosis, the AFP-positive cells were morphologically similar to normal hepatocytes and showed an even cytoplasmic distribution of AFP. The endothelial markers CD31 and CD34 were used to investigate vascular distribution. CD31 was expressed strongly in the portal area and parenchyma in HCC, but it was rarely observed in the parenchyma in cirrhosis. CD34 expression could not be detected in both HCC and cirrhosis. This study constitutes the first comprehensive study of P-gp, AFP, and endothelial markers in canine HCC and cirrhosis. The importance of these markers in HCC and cirrhosis in dogs was demonstrated and provides a more accurate basis for a definitive diagnosis of HCC and cirrhosis in dogs.

A simple and rapid chromatographic strip test for detection of antibody to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a major economic problem for swine industries worldwide despite several disease-reduction strategies such as age-segregated early weaning and all-in-all-out pig movement. Routine diagnosis of PRRSV is carried out by the combined use of an antibody-detecting enzyme-linked immunosorbent assay (ELISA), immunofluorescence, reverse transcription-polymerase chain reaction, and virus isolation. These assays require specialized laboratory equipment in addition to multistep sample handling and sample preparation. The objective of this study was to evaluate a simple pen-side assay (BioSign PRRSV) for rapid detection of PRRSV antibody based on a lateral flow chromatographic strip immunoassay system. This assay uses Escherichia coli-expressed viral nucleocapsid protein antigen for detecting antibodies against PRRSV in swine sera. In this report, the authors describe the evaluation of this assay using sera from both clinical samples and experimentally infected piglets. The results were compared with those of a standard, commercially available antibody ELISA (HerdChek PRRS ELISA) and an indirect immunofluorescence assay using the same serum samples. The BioSign PRRSV assay was capable of detecting antibodies in sera known to contain antibodies to PRRSV, resulting in 93.2% sensitivity for samples from experimentally infected pigs and 98.7% sensitivity for clinical serum samples. For sera that did not contain antibodies to PRRSV, the specificity was found to be 98.5% and 99.2% for clinical and experimental serum samples, respectively.

Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population.

Hepatitis E virus (HEV) is a very important public health concern in many developing countries where epidemics of hepatitis E are common. Sporadic cases of clinical hepatitis E not only occur in these countries but also occur uncommonly in patients with no known epidemiological exposure to HEV in industrialized countries. The source of infection in industrialized countries is unknown but it has been suggested that animals might serve as a reservoir for HEV in both settings. We recently identified and characterized an HEV strain (swine HEV) that infects large numbers of pigs in the United States. To assess the potential of pigs to serve as a global reservoir of HEV, we measured the prevalence of HEV antibodies in pigs in two countries where hepatitis E is endemic and two countries where it is not. Swine herds in all four countries contained many pigs that were seropositive for IgG anti-HEV, although the percentage of seropositive pigs varied greatly from herd to herd. A very limited number of pig handlers in the two endemic countries were also tested and most of them were found to be seropositive for HEV. The results from this study suggest that hepatitis E is enzootic in pigs regardless of whether HEV is endemic in the respective human population. J. Med. Virol. 59:297-302, 1999. Published 1999 Wiley-Liss, Inc.

The VP4 and VP7 of bovine rotavirus VMRI are antigenically and genetically closely related to P-type 5, G-type 6 strains.

We have previously reported the isolation of a bovine rotavirus, designated VMRI, with a super-short electropherotype. We have characterized this strain further as it has shown antigenic differences with the prototype G6 strain NCDV-Lincoln. In this communication, we report the antigenic and molecular characterization and the nucleotide sequence of the VP4 and VP7 genes of this strain. Virus neutralization tests indicated 2- to 13-fold differences in the titers between NCDV-Lincoln, B641 and VMRI strains. Northern blot hybridization results indicated a degree of heterogeneity in the VP4 gene of these strains which can be detected under conditions of high stringency. The VP4 and VP7 genes of the VMRI strain were cloned and sequenced and compared with the published sequences of other bovine rotavirus strains. The VP4 gene of VMRI had a high degree of homology with that of UK and B641 strains but differed significantly from that of both NCDV-Lincoln and B223 strains. Sequence analysis of the VP7 gene of VMRI and other strains indicated a high degree of conservation and the amino acid identity between the different strains was 96%. Sequence information regarding these strains and field isolates will assist in the generation of effective vaccination strategies for control of neonatal calf diarrhea.

Evaluation of rotavirus infection and diarrhea in Iowa commercial pigs based on an epidemiologic study of a population represented by diagnostic laboratory cases.

Group A, B, and C rotaviruses were identified in 9% (96/1,048) of pig fecal specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 1987 and 1988. Six of the rotaviruses were group B, 5 were group C, and the remaining 89% were group A. Of the rotavirus cases with more than 1 serotype, 5 were multiple group A serotypes, 1 involved a group A and B serotype, and 1 included 2 group C serotypes. A retrospective epidemiologic evaluation of pig diarrhea in herds of origin was done using data obtained from the accession records of the rotavirus and 88 matched nonrotavirus pig diarrhea control cases. Herds from which rotavirus cases were derived experienced lower morbidity, mortality, and case fatality rates than matched control herds. The incidence of diarrhea decreased rapidly among all pigs from birth to 3 weeks of age. The peak incidence for piglet diarrhea occurred in February, and a moderate rise occurred in August-September. Definitive evidence for transmissible gastroenteritis virus was found in 12% of nonrotavirus cases but none of the rotavirus cases in which it was sought. Other pathogenic microorganisms were identified less frequently and inconsistently.

Prevalence of P types among porcine rotaviruses using subgenomic VP4 gene probes.

Nucleic acid probes were developed to differentiate VP4 (P) types among porcine rotaviruses. These probes were then used to determine the relative prevalence of P types 6 (Gottfried-like) and 7 (OSU-like) in cultivated rotaviruses and field specimens. The variable regions between bases 205-551 of the VP4 gene of rotavirus strains OSU and Gottfried were amplified by the polymerase chain reaction and radiolabeled with 32P by random primer extension. Radiolabeled probes were tested in a dot blot hybridization assay. The subgenomic probes prepared from VP4 gene detected as little as 5 ng oF rotavirus RNA and were specific for differentiating the two porcine rotavirus P types. The probes were used to determine the P type of several reference rotavirus strains and recently cultivated porcine rotavirus strains. Eight of the 10 cultivated, previously untyped, rotaviruses isolates tested were of P type 7 (OSU-like). Two rotavirus strains neither reacted with OSU nor with the Gottfried probe, therefore, their P type could not be determined. Seventeen out of the 26 rotavirus (65.4%) field samples tested had a P type 6 (Gottfried-like) whereas 5 out of 26 (19.2%) had a P type 7 (OSU-like). Four of the 26 samples (15.4%) reacted neither with the OSU nor with the Gottfried probe and possibly represent previously unrecognized P types in swine. Data in this study suggests that (1) rotaviruses with P type 7 are most common among the cultivated rotaviruses, (2) rotaviruses with P type 6 are the most abundant type of rotavirus in natural infections and (3) rotaviruses with additional P types are also associated with diarrheic swine.

Immunogens of rotaviruses.

Rotaviruses cause gastroenteritis in neonates of many animal species including cattle, swine, horses, dogs, cats, chickens and turkeys. Rotavirions are nonenveloped, are about 75 nm in diameter, have a double capsid, and contain 11 double-stranded RNA segments as their genome. Several antigenically distinct groups of rotaviruses have been identified and have been alphabetically designated as A through G. Group A rotaviruses were the first group of rotaviruses isolated and are the most commonly detected rotaviruses in diarrheic animals. Group A rotaviruses have two surface proteins, VP4 and VP7, both of which are important in serotype determination and in inducing neutralizing antibodies and protective immunity. Multiple serotypes of group A rotavirus based on glycoprotein VP7 (designated as G types) and based on VP4 (P types) have been identified. The immune response to rotaviruses is essentially serotype specific, however, cross-reactive or heterotypic epitopes have also been identified. Currently acceptable methods for immunogen quantitation include the induction of neutralizing antibody in host or laboratory animals. The in vivo efficacy of vaccines against rotavirus-associated gastroenteritis remains the standard method against which in vitro methods must be compared. Several animal models have been developed which could potentially be used in evaluating the efficacy of candidate vaccines. Monoclonal antibodies to rotavirus immunogens are also currently available and serve as valuable reagents for in vitro quantitation of rotaviral immunogens.

Isolation of a bovine rotavirus with a "super-short" RNA electrophoretic pattern from a calf with diarrhea.

A rotavirus with a "super-short" RNA electropherotype was isolated from a calf with diarrhea and was designated VMRI strain. Segments 10 and 11 of this rotavirus migrated more slowly than did those of bovine rotavirus strains NCDV, B641, and B223. The electrophoretic pattern of the VMRI strain was similar to that reported for rotaviruses with super-short RNA electropherotypes from humans and rabbits. Northern (RNA) blot hybridization indicated that gene 11 of the VMRI strain was altered and migrated between gene segments 9 and 10. The subgroup of the VMRI strain was shown to be subgroup I. The VMRI strain of bovine rotavirus was neutralized by antisera containing polyclonal antibodies to rotavirus serotype 6 (bovine rotavirus serotype I) strains NCDV and B641 and by ascitic fluid containing monoclonal antibodies directed to VP7 of serotype 6 rotavirus. The VMRI strain was not neutralized by either polyclonal or monoclonal antibodies to strain B223 (bovine rotavirus serotype II). Collective data on the neutralization of the VMRI strain with monoclonal antibodies and polyclonal antibodies suggest that this virus is a member of the NCDV group (serotype 6) of rotaviruses (bovine rotavirus serotype I).

High-resolution chromatography of nucleic acids on the Gen-Pak FAX column.

High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules. HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved. Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species. Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules. Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules. Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products. Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column. HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase. Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species. In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride. Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid. Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.

Isolation of two new serotypes of porcine rotavirus from pigs with diarrhea.

Two group A rotaviruses (ISU-64 and ISU-65) with subgroup I antigen were isolated from pigs with diarrhea and were shown by two-way neutralization tests to be two new serotypes of porcine rotavirus.