Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs.

The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV- from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n = 52) and conventional young pigs (n = 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative = 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to epitope D occurred using ELISA 2, indicating its greater specificity. The progression of seroresponses to the TGEV S protein epitopes A or D, as measured by the 2 ELISAs, was similar for both sets (A and B) of samples. Differentiation between TGEV and PRCV antibodies (based on seroresponses to epitope D) was consistently measured after the third week of inoculation.     

A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus

A competitive inhibition ELISA was developed to detect non-neutralizing antibodies to the peplomer protein of transmissible gastroenteritis virus (TGEV) in porcine sera using a monoclonal antibody as an indicator. It was demonstrated that field strains of the TGEV-related porcine respiratory coronavirus (PRCV) did not induce this antibody, whereas the Miller strain and field strains of TGEV did. The sensitivity of the competitive inhibition ELISA appeared to be similar to that of the virus neutralization (VN) test. The test enables differentiation of pigs which were previously infected with TGEV or PRCV and which cannot be distinguished by the classical anti-TGEV neutralization test. The present test is useful for selective serodiagnosis.     

Characterization and reactivity of monoclonal antibodies to the Miller strain of transmissible gastroenteritis virus of swine.

Hybridomas secreting monoclonal (MAB) to transmissible gastroenteritis virus (TGEV) were produced by fusion of SP2/0 myeloma cells and splenic lymphocytes of BALB/c mice immunized with the virulent cell-passaged Miller strain of TGEV. The MAB secreted by these hybridomas were partially characterized; 4 of them (MA4, MA5, MH11, MB2) had high-neutralization titer for TGEV. The remaining 7 (MC6, MD9, ME5, MG5, MF2, ME9, MG7) did not neutralize TGEV at 1:25 dilution. All 4 neutralizing and 2 of the nonneutralizing MAB reacted with the E2 protein of TGEV in a radioimmunoprecipitation assay. The remaining 5 MAB reacted with the E1 protein of TGEV. Reactivity of the MAB was tested in an indirect immunofluorescent assay with 3 cell culture-adapted strains of TGEV (Miller, Purdue, and Illinois) and 13 wild-type isolates of TGEV. Neutralizing MAB reacted with all 13 wild-type isolates and the 3 cell culture-adapted strains of TGEV. In contrast, nonneutralizing MAB that reacted with the Miller strain of TGEV varied in their reactivity with the wild-type TGEV isolates. Reactivity of neutralizing MAB was also tested, using plaque-reduction neutralization assays with Miller, Purdue, and Illinois strains and 5 wild-type isolates. All 4 neutralizing MAB neutralized the 8 virus isolates, but the neutralization titer was higher with the homologous virus than with the heterologous virus isolates. However, neutralization titers of the 4 neutralizing MAB were 4 to 16 times higher for the homologous Miller strain of TGEV than for the heterologous Illinois and Purdue strains, and were 4 to 1,000 times higher than for the wild-type isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

RT-PCR检测和区分猪传染性胃肠炎病毒和猪呼吸道冠状病毒的研究

根据猪传染性胃肠炎病毒（TGEV）和猪呼吸道冠状病毒（PRCV）的基因组核苷酸序列，在S基因（纤突蛋白基因）5′端保守区设计了一对引物P3／P4，该对引物在TGEV扩增跨幅约为2.4kb；而PRCV由于在此区域存在一约0.6kb碱基缺失，扩增跨幅约为1.8kb。用引物P3／P4对TGEV Miller株、Purdue株和PRCV AR310株分别进行RT－PCR，根据RT－PCR扩增片段大小可以直接区分TGEV和PRCV。用引物P3／P4与引物P1／P2作Nested-PCR,提高了该RT－PCR的特异性和敏感性，建立的RT－PCR可为临床上诊断TGEV及调查我国是否存在PRCV感染提供可靠的鉴别手段。     

贵州省猪传染性胃肠炎及呼吸冠状病毒病血清学调查

用酶联免疫吸附试验对采自贵州省内88个县(市)的2906份血清进行了猪传染性胃肠炎病毒及呼吸冠状病毒抗体检测,结果检出猪传染性胃肠炎病毒抗体阳性血清12份,总体阳性率为0.41%;猪呼吸冠状病毒抗体阳性血清44份,总体阳性率为1.51%;2818份血清两种抗体均呈阴性(阴性率为96.97%),32份血清检测无效或无结论(无效率为1.10%)。     

Characterization of two new monoclonal antibodies against Haemophilus paragallinarum serovar C hemagglutinating antigen

Two new monoclonal antibodies (MAbs), D6D8D5 and B3E6F9, both directed against Haemophilus paragallinarum serovar C hemagglutinating (HA) antigen, were produced, and characteristics of the MAbs were compared with those of the previously described MAb F2E6 in dot-blot and hemagglutination-inhibition (HI) tests using two representative H. paragallinarum strains each of serovars A, B, and C strains and 55 Japanese serovar C field isolates. MAb D6D8D5 and MAb F2E6 reacted with all serovar C strains and field isolates in the dot-blot test. However, MAb D6D8D5 showed various degrees of inhibition of the HA activity of field isolates. In the enzyme-linked immunosorbent assay-competition test, MAb D6D8D5 did not compete with MAb F2E6. MAb B3E6F9 reacted with strain S1, serovar C but not with strain Modesto, serovar C in both dot-blot and HI tests. Three out of 55 field isolates did not react with MAb B3E6F9. Neither MAb reacted with the serovar A and B strains.     

Antigenic analysis of feline coronaviruses with monoclonal antibodies (MAbs): preparation of MAbs which discriminate between FIPV strain 79-1146 and FECV strain 79-1683.

We prepared 31 monoclonal antibodies (MAbs) against either FIPV strain 79-1146 or FECV strain 79-1683, and tested them for reactivity with various coronaviruses by indirect fluorescent antibody assay (IFA). Sixteen MAbs which reacted with all of the 11 strains of feline coronaviruses, also reacted with canine coronavirus (CCV) and transmissible gastroenteritis virus (TGEV). In many of them, the polypeptide specificity was the recognition of transmembrane (E1) protein of the virus. We succeeded in obtaining MAbs which did not react with eight strains of FIPV Type I viruses (showing cell-associated growth) but reacted with FIPV Type II (79-1146, KU-1) and/or FECV Type II (79-1683) (showing non-cell associated growth). These MAbs also reacted with CCV or TGEV. These MAbs recognized peplomer (E2) glycoprotein, and many antigenic differences were found in this E2 protein. These results suggest that FIPV Type II and FECV Type II viruses are antigenically closer to TGEV or CCV than to FIPV Type I viruses. Furthermore, the MAb prepared in this study has enabled discrimination between FIPV strain 79-1146 and FECV strain 79-1683, which was thought to be impossible by the previous serological method.     

Serological studies of transmissible gastroenteritis in Great Britain, using a competitive ELISA.

A competitive ELISA which differentiates between transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was used to detect non-neutralising antibodies to the peplomer protein of TGEV in porcine sera. The test was shown to be TGEV specific, having a relative specificity of 100 per cent, and to have a relative sensitivity of 94.9 per cent when compared with the virus neutralisation test. The prevalence of TGEV in Great Britain is low; only 0.6 per cent of sows sampled in 1990 were seropositive to TGEV. Seroconversion to the virus neutralisation test occurred in a closed herd in 1984, with no apparent spread, but later testing by the ELISA did not detect any blocking antibodies. The possibility of the existence of a less contagious strain of PRCV is discussed. All British isolates of TGEV tested by the indirect fluorescent antibody test were recognised by the monoclonal antibody 1D.B12, the indicator antibody in the ELISA.     

Differentiation between transmissible gastroenteritis virus and porcine respiratory coronavirus using a cDNA probe.

A plasmid, pG3BS, containing a cDNA clone from the 5' coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.     

Field validation of a commercial blocking ELISA to differentiate antibody to transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus and to identify TGEV-infected swine herds.

A commercially available blocking ELISA was analyzed for its ability to identify antibodies to porcine coronaviruses (transmissible gastroenteritis virus [TGEV] or porcine respiratory coronavirus [PRCV]), to differentiate antibodies to TGEV and PRCV, and to identify TGEV-infected herds. Nine sera from uninfected pigs, 34 sera from 16 pigs experimentally infected with TGEV, and sera from 10 pigs experimentally infected with PRCV were evaluated using both the TGEV/PRCV blocking ELISA and a virus neutralization (VN) assay. The ELISA was not consistently effective in identifying pigs experimentally infected with TGEV until 21 days postinfection. Sera from 100 commercial swine herds (1,783 sera; median 15 per herd) were similarly evaluated using both tests. Thirty of these commercial herds had a clinical history of TGEV infection and a positive TGEV fluorescent antibody test recorded at necropsy within the last 35 months, while 70 herds had no history of clinical TGEV infection. The blocking ELISA and the VN showed good agreement (kappa 0.84) for the detection of porcine coronavirus antibody (TGEV or PRCV). The sensitivity (0.933) of the ELISA to identify TGEV-infected herds was good when considered on a herd basis. The ELISA was also highly specific (0.943) for the detection of TGEV-infected herds when the test results were evaluated on a herd basis. When sera from specific age groups were compared, the ELISA identified a greater proportion (0.83) of pigs in herds with TGEV antibody when suckling piglets were used. In repeatability experiments, the ELISA gave consistent results when the same sera were evaluated on different days (kappa 0.889) and when sera were evaluated before and after heating (kappa 0.888). The blocking ELISA was determined to be useful for herd monitoring programs and could be used alone without parallel use of the VN assay for the assessment of large swine populations for the detection of TGEV-infected herds.     

7种动物冠状病毒特异基因的克隆与序列分析

为了比较冠状病毒基因相关性,获得特异基因克隆制备冠状病毒基因芯片,根据发布的基因序列,每种病毒设计4~17对引物,利用火鸡冠状病毒(TCV)原毒和蔗糖密度梯度离心纯化浓缩的犬冠状病毒(CCV)、猫冠状病毒(FCV)、猫传染性腹膜炎病毒(FIPV)、猪传染性胃肠炎病毒(TGEV)、猪呼吸道冠状病毒(PRCV)、牛冠状病毒(BCV)细胞毒,提取总RNA并反转录和PCR扩增。回收PCR产物连接pGEM-T-easy载体并转化大肠杆菌TGI,经PCR鉴定后测序。将所有基因片段的核苷酸序列和推导的氨基酸序列,分别与GenBank有关病毒相关基因片段的核苷酸序列进行分析比较,确定它们的同源性。通过对不同冠状病毒不同基因片段的克隆和测序,发现同一群冠状病毒核苷酸序列间具有较高的同源性。     

Comparison of the antibody response to transmissible gastroenteritis virus and porcine respiratory coronavirus, using monoclonal antibodies to antigenic sites A and X of the S glycoprotein.

Pigs were inoculated with various strains of transmissible gastroenteritis virus (TGEV) or with porcine respiratory coronavirus (PRCV), and antigenic site-specific antibody responses were compared. A blocking-ELISA was used to study to what extent antibodies in convalescent sera interfered with the binding of monoclonal antibodies (MAB) 57.16 or 57.110 to the attenuated TGEV/Purdue virus. Monoclonal antibody 57.16 is directed against the A site on the peplomer, neutralizes virus, and recognizes TGEV and PRCV. Monoclonal antibody 57.110 is directed against the X site on the peplomer, but does not neutralize virus, and recognizes only TGEV. Antibodies directed against TGEV and PRCV could be detected in a blocking ELISA, using MAB 57.16 as a conjugate. Antibodies directed against both viruses were detectable as early as 1 week after inoculation. Antibody titers correlated well with those in a virus-neutralization test. Antibodies against TGEV could be detected in a blocking ELISA, using MAB 57.110 as a conjugate. Such antibodies were not induced by a PRCV infection. In the blocking ELISA, using MAB 57.110 as a conjugate, antibodies were detectable as early as 2 weeks after inoculation. There was a significant difference between antibody titers reached after infection with various TGEV strains, however. This difference is ascribed to a variation of the antigenic site defined by MAB 57.110 in TGEV strains. Conditions for a differential test for TGE serodiagnosis, and for serologic discrimination between TGEV- and PRCV-infected pigs, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycoplasma gallisepticum hemagglutinin V1hA, pyruvate dehydrogenase PdhA, lactate dehydrogenase, and elongation factor Tu share epitopes with Mycoplasma imitans homologues

Mycoplasma gallisepticum is a major pathogen of poultry. Mycoplasma imitans is genetically and antigenically closely related to M. gallisepticum, but so far, only a few proteins of M. imitans have been identified as sharing epitopes with M. gallisepticum. In this study, we identified three proteins of M. gallisepticum that share with M. imitans epitopes defined by monoclonal antibodies (MAbs). MAb 9D4 reacted with the 67-kD hemagglutinin V1hA (previously termed pMGA) of M. gallisepticum and with its continuously expressed 40-kD protein. This MAb also reacted with a 40-kD protein of M. imitans, but not with its putative V1hA. Two-dimensional (2D) immunoblots of M. gallisepticum strains showed that their 40-kD proteins reacting with MAb 9D4 are expressed as major forms with isoelectric points (pI) around 6, and also as less-abundant forms differing in pI. In M. imitans, major forms of 40-kD proteins recognized by MAb 9D4 had pI around 6, whereas minor forms had pI between 5.5 and 5.8. The N-terminal sequence of the M. gallisepticum 40-kD protein recognized by MAb 9D4 strongly indicates that this protein is pyruvate dehydrogenase E1, subunit alpha (PdhA protein, also termed AcoA). The position of elongation factor Tu (EF-Tu), detected by the reference MAb GB8, was very similar in the 2D proteome maps of M. gallisepticum and M. imitans (MW of about 45 kD; pI - 5.6). In both M. gallisepticum and M. imitans, MAb 7G1 reacted with proteins of about 36 kD with similar charges (major forms with pI of about 8). The position of this protein in the proteome map of M. gallisepticum and its N-terminal sequence strongly suggest that MAb 7G1 recognizes lactate (malate) dehydrogenase (Ldh or Mdh). Comparison of 2D proteomes of 10 M. gallisepticum strains indicated that positions of EF-Tu, PdhA, and Ldh proteins are rather consistent and can be used as reference points in further analyses of the M. gallisepticum proteome.     

Sows infected in pregnancy with porcine respiratory coronavirus show no evidence of protecting their sucking piglets against transmissible gastroenteritis

Eighteen litters of sucking piglets were challenged with one of two strains of transmissible gastroenteritis virus (TGEV). During pregnancy, their seronegative dams had been either inoculated intranasally with porcine respiratory coronavirus (PRCV), inoculated orally with TGEV or left untreated. On the basis of weight gain, clinical signs and survival, no differences in response to challenge was detected when piglets suckled by PRCV inoculated sows were compared with those suckled by uninoculated sows. Such a difference was evident when the litters of sows successfully pre-immunized with TGEV were compared with those of unicoculated or PRCV-inoculated sows. The possibility of transplacental transmission of PRCV was investigated in two litters born to sows that had been inoculated with this virus in late pregnancy. All sixteen live-born piglets were seronegative for the virus at birth and PRCV was not isolated from tissues taken from two stillborn piglets.     

Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus.

The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure. Four antigenic sites have been defined (A, B, C, and D). The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro. These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides. Both sites might be relevant in protection in vivo. PRCV does not have sites B and C, due to a genomic deletion. Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies. An epidemiological tree relating the TGEVs and PRCVs has been proposed. The estimated mutation fixation rate of 7 +/- 2 x 10(-4) substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses. In order to induce secretory immunity, different segments of the S gene have been expressed using a virulent forms of Salmonella typhimurium and adenovirus. These vectors, with a tropism for Peyer's patches may be ideal candidates in protection against TGEV.     

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.     

Evaluation of a panel of monoclonal antibodies in the subtyping of Haemophilus paragallinarum.

A panel of four monoclonal antibodies (MAbs) was evaluated, using a hemagglutination-inhibition test, for its ability to subtype 76 isolates of Haemophilus paragallinarum. The results of the MAb reactions were compared with the results of both the Page and Kume serotyping schemes (the serovars of the Page scheme correspond to the serogroups of the Kume scheme). One MAb (E5C12D10) was raised against a Page serovar A strain and the remaining MAbs (F2E6, D6D8D5, and B3E6F9) against a Page serovar C strain. Six different reaction patterns were found among the 76 isolates of H. paragallinarum. There was total correlation between the MAb reaction pattern and the Page scheme, and thus the Kume scheme, to the serogroup level. All 19 Page serovar A (= Kume serogroup A) strains reacted only with MAb E5C12D10, whereas all five Page serovar B (= Kume serogroup B) strains failed to react with any of the MAbs. All 52 remaining strains were Page serovar C (= Kume serogroup C), and all failed to react with MAb E5C12D10 but showed varying reaction patterns with the three other MAbs. Although the MAbs recognized four subdivisions within Kume serogroup C, these subdivisions differed from the four Kume C serovars. This panel of MAbs can be used to assign isolates of H. paragallinarum to either Page serovars or Kume serogroups. Although the subdivisions recognized by the MAbs within the Page serovar C strains do not correspond to the Kume serovars, they may be useful in epidemiological applications.     

Seroprevalence of economically important viral pathogens in swine populations of Trinidad and Tobago,West Indies

The objective of this study was to evaluate the seroprevalence and identify the strains of swine influenza virus (SwIV), as well as the seroprevalence of porcine parvovirus (PPV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine respiratory coronavirus (PRCV), porcine circovirus type 2 (PCV-2), and classical swine fever virus (CSFV) in pigs in Trinidad and Tobago (T&T). Blood samples (309) were randomly collected from pigs at farms throughout T&T. Serum samples were tested for the presence of antibodies to the aforementioned viruses using commercial ELISA kits, and the circulating strains of SwIV were identified by the hemagglutination inhibition test (HIT). Antibodies against SwIV were detected in 114 out of the 309 samples (37%). Out of a total of 26 farms, 14 tested positive for SwIV antibodies. HI testing revealed high titers against the A/sw/Minnesota/593/99 H3N2 strain and the pH1N1 2009 pandemic strain. Antibodies against PPV were detected in 87 out of the 309 samples (28%), with 11 out of 26 farms testing positive for PPV antibodies. Antibodies against PCV-2 were detected in 205 out of the 309 samples tested (66%), with 25 out of the 26 farms testing positive for PCV-2 antibodies. No antibodies were detected in any of the tested pigs to PRRSV, TGEV, PRCV, or CSFV.