Epidemiology, disease and control of infections in ruminants by herpesviruses--an overview

There are at least 16 recognised herpesviruses that naturally infect cattle, sheep, goats and various species of deer and antelopes. Six of the viruses are recognised as distinct alphaherpesviruses and 9 as gammaherpesviruses. Buffalo herpesvirus (BflHV) and ovine herpesvirus-1 (OvHV-1) remain officially unclassified. The prevalence of ruminant herpesviruses varies from worldwide to geographically restricted in distribution. Viruses in both subfamilies Alphaherpesvirinae and Gammaherpesvirinae cause mild to moderate and severe disease in respective natural or secondary ruminant hosts. Accordingly, the economic and ecological impact of the viruses is also variable. The molecular characteristics of some members have been investigated in detail. This has led to the identification of virulence-associated genes and construction of deletion mutants and recombinant viruses. Some of the latter have been developed as commercial vaccines. This paper aims to give an overview of the epidemiology and pathogenesis of infection by these viruses, immuno-prophylaxis and mechanisms of recovery from infection. Since there are 128 ruminant species in the family Bovidae, it is likely that some herpesviruses remain undiscovered. We conclude that currently known ruminant alphaherpesviruses occur only in their natural hosts and do not cross stably into other ruminant species. By contrast, gammaherpesviruses have a much broader host range as evidenced by the fact that antibodies reactive to alcelaphine herpesvirus type 1 have been detected in 4 subfamilies in the family Bovidae, namely Alcelaphinae, Hippotraginae, Ovibovinae and Caprinae. New gammaherpesviruses within these subfamilies are likely to be discovered in the future.     

Production and characterization of monoclonal antibodies to bovid herpesvirus-4

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.     

Preparation and partial characterization of monoclonal antibodies against the protective protein antigen of Erysipelothrix rhusiopathiae

Thirteen mouse monoclonal antibodies (Mabs) against the protective protein antigen (P64) of Erysipelothrix rhusiopathiae were prepared and partially characterized. The titres of the Mabs varied from 200 to 1,638,400 as determined by enzyme-linked immunosorbent assay (ELISA). Of the 13 Mabs 10, two and one belonged to the IgG2a, IgG1 and IgM subclasses, respectively. All Mabs reacted strongly with the 64 kDa protein and weakly with the 43 kDa protein upon Western blotting of the alkaline extract (AE) of E. rhusiopathiae. The protective activity (PD50/ml) of the 13 Mabs against E. rhusiopathiae infection in mice varied from < 50 to > 50,000. These Mabs were classified into three groups, highly protective Mabs, moderately protective Mabs and Mabs which did not possess protective activity, based on the protective index (ratio of the PD50/ml to the antibody titre). These results suggest that the 64 kDa protein is an effective protective antigen, which is easily cleaved into many small proteins, including the 43 kDa protein, and possesses at least two epitopes related to its protective activity and at least one epitope which is not related to protection of mice against E. rhusiopathiae infection.     

Ruminant alphaherpesviruses related to bovine herpesvirus 1

Herpesviruses have mainly co-evolved with their hosts for millions of years. Consequently, different related host species may have been infected by various genetically related herpesviruses. Illustrating this concept, several ruminant alphaherpesviruses have been shown to form a cluster of viruses closely related to bovine herpesvirus 1 (BoHV-1): namely bovine herpesvirus 5, bubaline herpesvirus 1, caprine herpesvirus 1, cervid herpesviruses 1 and 2 and elk herpesvirus 1. These viruses share common antigenic properties and the serological relationships between them can be considered as a threat to BoHV-1 eradication programmes. BoHV-1 is a herpesvirus responsible for infectious bovine rhinotracheitis, which is a disease of major economic concern. In this article, the genetic properties of these ruminant alphaherpesviruses are reviewed on a comparative basis and the issue of interspecific recombination is assessed. The pathogenesis of these infections is described with emphasis on the host range and crossing of the host species barrier. Indeed, the non bovine ruminant species susceptible to these ruminant alphaherpesviruses may be potential BoHV-1 reservoirs. The differential diagnosis of these related infections is also discussed. In addition, available epidemiological data are used to assess the potential of cross-infection in ruminant populations. A better knowledge of these ruminant alphaherpesvirus infections is essential to successfully control infectious bovine rhinotracheitis.     

Development of an ELISA to detect antibodies to a protective lipopolysaccharide fraction of Leptospira borgpetersenii serovar hardjo in cattle.

Monoclonal antibodies (Mabs) were produced against Leptospira borgpetersenii serovar hardjo-type Bovis antigens. A panel of 28 Mabs were characterised. Only the nine Mabs toward a lipopolysaccharide (LPS) fraction of 18, 24 kDa bands and a 26-28 kDa smear showed agglutinating, leptospiricidal and growth-inhibition activities, and passively protected hamsters against renal infection with hardjo. They also reacted strongly in the CH-ELISA, captured killed whole hardjo leptospires, gave good fluorescence in indirect FAT against smears of hardjo culture and exhibited no cross reactivity with strains in heterologous serogroups. On the basis of optimal activity in a range of tests, one IgG class Mab (designated 25) was selected for use in an antibody-capture ELISA system for the detection of bovine anti-hardjo antibodies. The system gave a wide separation of absorbance values between positive and negative sera at a 1:10 dilution. The antibodies detected by this assay are believed to be protective anti-LPS IgG.     

Study of bovine herpesvirus type 1 strains with monoclonal antibodies.

Fourteen strains of bovine herpesvirus type 1 (BHV-1, IBRV) representing all three groups of BHV-1 (BHV-1.1, BHV-1.2, BHV-1.3) were studied by ELISA using 106 monoclonal antibodies (Mabs) produced against BHV-1. On the basis of the ELISA, the Mabs could be divided into three groups. The first group (40 Mabs, 38%) reacted with all strains, the second group (43 Mabs, 41%) with the respiratory and genital strains (BHV-1.1 and BHV-1.2) while the third group (23 Mabs, 22%) only with the respiratory strains. Only 5 out of the antibodies neutralized respiratory and genital strains, and none of them neutralized the encephalitogenic strains (1.3). Three Mabs selected from each of the 3 groups, and the above five neutralizing strains were studied by Western blot. Antibodies of groups 1 and 3, and two neutralizing antibodies bound to a 90k protein (gpIII), whereas members of group 2 and 3 neutralizing antibodies reacted with a 74k and a 130k protein (both gpl). The results indicate that reactivity with monoclonal antibodies is as suitable for the classification of BHV-1 strains as is restriction endonuclease (RE) analysis but it cannot distinguish between subgroups within the groups.     

A new approach for the diagnosis of hog cholera

Pestiviruses were isolated from seven cases of suspect hog cholera. Using peroxidase conjugates of monoclonal antibodies (Mabs) six isolates were identified as hog cholera viruses (HCV), while one isolate was of ruminant origin, possibly bovine viral diarrhea virus. In parallel attempts were made to develop an ELISA for the detection of HCV-specific antibodies in pig sera. The Mab HCTC26 coated to polystyrol plates efficiently captured the major viral glycoprotein gp53 from crude antigen suspensions prepared from infected cells. The immobilized gp53 served as diagnostic antigen. Five pigs experimentally infected with the HCV strain Glentorf were sequentially bled and the development of antibodies was monitored by neutralization tests and the ELISA. Results showed that both tests detected antibodies simultaneously after infection. Titres measured by ELISA were slightly higher than those registered by neutralization.     

Neutralizing and Complement-fixing Monoclonal Antibodies as an Aid to the Diagnosis of Equine Herpesvirus-1 Infection

One complement-fixing (C-MAb) and three complement-dependent neutralizing monoclonal antibodies (N-MAbs) were raised against Hisar-90-7 equine herpesvirus-1 (EHV-1) strain. The target antigen of the C-MAb (2A5) and two of the N-MAbs (1H6, 9C4) was identified as a 140 kDa polypeptide in Western blotting. The target antigen of N-MAb (9C6) could not be identified. Purified polypeptides of five EHV-1 strains isolated from different regions and at different times gave intense bands at 140 kDa when reacted with N-MAb (1H6) in Western blots. In sandwich ELISA, all four MAbs captured the viral antigen from clinical materials, giving a reliable and rapid diagnosis of EHV-1 infection in equines.     

Monoclonal precipitating antibodies to porcine immunoglobulin M

Fusion of splenic immunocytes from a porcine IgM-immunized BALB/c mouse with SP2/0 mouse myeloma cells resulted in 231 primary hybrids. Culture fluids of the primary hybrids were screened for antibody production by enzyme-linked immunosorbent assay (ELISA) against porcine IgM and by radial immunodiffusion versus porcine serum. Culture fluids of 10 of the primary hybrids were positive in IgM-ELISA and radial immunodiffusion. Six of these primary hybrids (1A11, 1D10, 2D7, 2E2, 3B11, and 5C9) were cloned, and ascitic fluids were produced using cloned primary hybrids. The monoclonal antibodies (Mabs) in ascitic fluids were characterized as to their reactivity with porcine immunoglobulin isotypes. All six Mabs had mouse IgG1, K isotype and were mu-chain specific as they formed single precipitin lines against porcine serum and porcine IgM and no lines against porcine IgG, IgA, and fetal porcine serum in immunodiffusion and immunoelectrophoresis. In indirect ELISA, all Mabs reacted with porcine serum, porcine IgM, and mu-chains but did not react with porcine IgG, IgA, or light chains. All six Mabs were species-specific and recognized either of two antigenic regions of mu-chain. These Mabs have been successfully used to detect IgM-containing cells in tissue sections, to detect IgM in serum, and to quantitate surface membrane IgM-bearing cells in peripheral blood.     

Identification of Actinobacillus pleuropneumoniae strains of serotypes 7 and 4 using monoclonal antibodies: demonstration of common LPS O-chain epitopes with Actinobacillus lignieresii

Monoclonal antibodies (Mabs) against Actinobacillus pleuropneumoniae serotype 7 were produced and characterized. Three Mabs directed against surface polysaccharides were selected. One of the Mabs was directed against a capsular polysaccharide epitope (CPS) of A. pleuropneumoniae serotype 7 whereas two other Mabs reacted with different epitopes of the LPS O-chain. One of the latter reacted with the reference strain of serotype 7 and the other one with serotypes 7 and 4. These three Mabs were used to test, by Dot-ELISA, 508 field strains of A. pleuropneumoniae. None of the strains belonging to other serotypes different from serotypes 4 and 7 were positive with the Mabs. Used in combination, the CPS and one of the LPS O-chain directed Mabs were shown to be suitable for serotyping since they detected 100% of serotype 7 strains. In this study, we confirm for the first time that A. pleuropneumoniae serotype 4 is present in North America. Finally, both O-chain specific Mabs also reacted with the O-chain of Actinobacillus lignieresii. The cross-reactivity between the two species was confirmed using sera from pigs experimentally infected with A. pleuropneumoniae serotype 7 and A. lignieresii, using immunoblotting and ELISA. This is the first report of a specific cross-reactivity between the LPS of these bacterial species.     

Acute and latent infection by bovine herpesvirus type 5 in experimentally infected goats

The ability of alphaherpesviruses to infect different ruminant species may have important implications for control/eradication efforts. Serological data indicate that goats may be naturally infected with bovine herpesviruses. To investigate the susceptibility of goats to bovine herpesvirus-5 (BoHV-5), 3-4-month-old kids were inoculated intranasally with each of three Brazilian BoHV-5 isolates (G1, n=8; G2, n=5; G3, n=5). The acute infection was characterized by virus shedding in nasal secretions for up to 14 days (titers up to 10(5.97)TCID(50)/mL), mild respiratory signs and conjunctivitis. All animals seroconverted to BoHV-5, developing virus neutralizing (VN) titers from 4 to 32 to the homologous viruses. At day 60 post inoculation (pi), two animals from each group were euthanized for tissue collection and the remaining goats were submitted to dexamethasone administration (0.4 mg kg(-1) for 5 days). Dexamethasone treatment resulted in virus reactivation in 9 out of 12 animals, as ascertained by virus shedding and/or by increase in VN titers. Virus shedding was detected in 8/12 animals and lasted from 1 to 9 days. Latent viral DNA was detected by PCR in the olfactory bulb and/or trigeminal ganglia of 6/6 goats euthanized at day 60 pi and in 12/12 animals euthanized 40 days post-dexamethasone. These results show that young goats are susceptible to BoHV-5 and may shed virus upon reactivation of latent infection. Thus, it is reasonable to expect that goats raised in close contact with cattle in areas where BoHV-5 is endemic may be infected and therefore should be considered potential reservoirs of the virus.     

Characterization of a 39kDa capsular protein of avian Pasteurella multocida using monoclonal antibodies

The role of a 39kDa protein of avian Pasteurella multocida in pathogenesis of fowl cholera was investigated using monoclonal antibodies (Mabs). Mabs were prepared by immunization of BALB/c mice with a crude capsular extract (CCE) of P. multocida strain P-1059 (serovar A:3). Totally eight hybridomas producing Mab were obtained. Immunoblot analysis of the hybridomas revealed that all the Mabs recognized a 39kDa protein of CCE. Treatment of CCE antigen with proteinase K or periodic acid indicated that the epitope recognized was proteinaceous. The Mabs reacted with a major 39kDa protein of CCE from encapsulated strains but not with any protein of non-capsulated strains indicating that a direct correlation between encapsulation and the 39kDa protein. Immunoelectron microscopy on strain P-1059 and the non-capsulated derivative P-1059B (serovar -:3) reacting with the Mabs and gold-labeled anti-mouse IgG indicated that the protein is associated with the capsule. The Mabs significantly inhibited the adherence of encapsulated P. multocida strains to chicken embryo fibroblast cells, but only slightly that of non-capsulated strains. Mice passively immunized with the Mabs were protected from lethal challenge with virulent strains P-1059 and X-73 (serovar A:1). Thus the capsular 39kDa protein was determined to be an adherence factor and a cross-protective antigen of avian P. multocida type A strains.     

Diarrheal condition in dogs associated with viruses antigenically related to feline herpesvirus

Viruses with properties consistent with herpesvirus were isolated from dogs with diarrhea. The viruses were shown to be antigenically related to feline herpesvirus-1 (FHV-1) by virus neutralization tests. It was also observed that a canine herpesvirus (CHV) prototype, D004, and two field isolates from fatal CHV infections in 2-week-old and 6-week-old puppies were neutralized at a low level by antiserum to FHV-1. Reciprocal neutralization tests with CHV antiserum against FHV-1 were negative. These results indicated that viruses related to FHV-1 can infect the dog and that there appears to be uni-directional virus neutralization of CHV by FHV-1 antibody.     

Cross-reactivity of monoclonal antibodies to bovine immunoglobulins with immunoglobulins of other species.

Monoclonal antibodies (Mabs) to bovine immunoglobulin heavy chain of the four major isotypes gamma 1, gamma 2, alpha, mu and the light chains (combined kappa and lambda) were produced and found to cross-react in enzyme-linked immunoassay (ELISA) with immunoglobulins of some other animal species despite the discrete specificity associated with an antibody derived from a single clone. This cross-reactivity, particularly amongst ruminants, could be utilized in serological testing for the diagnosis of disease in these species. For example, Mabs produced against bovine immunoglobulin light chain cross-react with bison immunoglobulin light chain and were used successfully in serological testing as the secondary detection antibody in an indirect ELISA for the diagnosis of Brucella abortus in bison herds in north-western Canada.     

Bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in Algerian dromedary camels (<Emphasis Type="Italic">Camelus dromaderius</Emphasis>)

This study was performed to investigate the presence of bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in dromedary camels (Camelus dromaderius) kept in mixed herds with sheep and goats in Algeria, since the prevalence of BHV-1, BVDV, and BLV infections among dromedary camels in Algeria is unknown. Totally, 111 camel sera were collected from two provinces (Laghouat and Ghardaia) in Algeria. The sera were analyzed for BHV-1 specific antibodies, BVDV specific antibodies and BVDV antigen using the ELISA, and BLV nucleic acid using PCR. The seropositivity rate was 9.0% for BVDV-specific antibody, although 41.4% of camels tested were positive for BVDV antigen. Moreover, there was no evidence of BHV-1 and BLV infections. The results indicated that camels might represent an important source for BVDV infection in all ruminants, including cattle, sheep, and goats bred in mixed herds in Algeria, since they had a higher BVDV prevalence rates. Therefore, the prevention and control measures for BVDV infection should put in place in camel populations to limit the spread of BVDV infection to ruminant populations in Algeria.     

猪戊型肝炎病毒Ⅳ型衣壳蛋白单克隆抗体的制备与鉴定

为获得猪戊型肝炎病毒(Hepatitis E virus,HEV)Ⅳ型衣壳蛋白单克隆抗体,将猪HEV衣壳蛋白的C端267(408—675)个氨基酸基因序列克隆入原核表达载体pET-28a(+),构建重组质粒pET-28a-ORF2-C,转化E.coli Rosetta(BL21)感受态细胞进行诱导表达,SDS-PAGE和Western blot鉴定,纯化后免疫小鼠。取免疫小鼠的脾脏与鼠骨髓瘤细胞SP2/0融合制备单克隆抗体。通过间接ELISA和竞争ELISA方法筛选并鉴定单抗。结果表明蛋白得到正确、高效表达,获得3株识别不同的抗原表位区的单克隆抗体,分别命名为Mab-1E4(IgG1)、Mab-2C7(IgG1)和Mab-2G9(IgG2b),其中1E4和2G9能阻断临床阳性猪血清,提示该2株单克隆抗体识别的抗原表位是猪HEVⅣ型衣壳蛋白上重要的抗原表位区,而单抗Mab-2C7不能阻断。本研究为猪HEVⅣ型的诊断及研究提供重要工具。     

腺胃病变型鸡传染性支气管炎病毒变异株的比较研究

腺胃病变型鸡传染性支气管炎病毒（ＩＢＶ）变异株Ｈ９５提纯样品经ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ,证明Ｈ９５株单抗ＤＥ７和Ｍ４１株单抗６ＤＨ８均能识别Ｈ９５株５４０００蛋白多肽。采用单抗、多抗介导间接ＥＬＩＳＡ试验表明,Ｈ９５毒株能与抗ＩＢＶＭ４１Ｎ蛋白单抗６ＤＨ８和ＩＢＶＭ蛋白单抗ＭＣ发生反应,也能与抗Ｍ４１、Ｇｒａｙ、Ｈｏｌｔｅ、Ｔ、Ｈ５２、Ｎ１１５和分离株Ｃ９００１、ＤＬＺ９１１１的多抗血清反应,同时抗Ｈ９５株的４株单抗、多抗血清也能与上述毒株反应。卵磷脂酶Ｃ处理的Ｈ９５株的血凝活性能被相应的Ｈ９５株的单克隆抗体和高免血清所抑制,也能被Ｍ４１单抗和高免血清所抑制。通过ＲＴ－ＰＣＲ获得了Ｈ９５毒株的免疫原基因Ｓ１,经Ｓｏｕｔｈｅｒｎｂｌｏｔｉｎｇ和ＩＢＶ的Ｓ探针检测呈阳性。     

Serologic survey of selected viral agents in recently captured wild North American river otters (Lontra canadensis).

Blood samples were collected from 64 wild North American river otters (Lontra [Lutra] canadensis) from northern and eastern New York State and analyzed for serologic evidence of exposure to selected viral agents during a 1995 1996 translocation program. No clinical signs of disease nor lesions suggestive of prior viral exposure were seen. Titers were detected for antibodies against canine distemper virus, canine herpesvirus-1, and canine parvovirus-2 but not for antibodies against canine adenovirus-1, canine coronavirus, canine parainfluenza virus, rabies virus, feline herpesvirus-1, feline calicivirus, or feline coronavirus. This is the first report of titers for antibodies against canine herpesvirus-1 in North American river otters, and it suggests a low prevalence of antibody titers against most canine viruses in otter populations in northern and eastern New York. Confounding variables in this study could include exposure to domestic dogs associated with the project, prolonged time spent in captivity, and concurrent bacterial or parasitic infection. Stress-associated humoral immune suppression could have altered serologic profiles, especially in otters exposed to dogs after trapping but before venipuncture.     

Characterisation of monoclonal antibodies to epizootic hemorrhagic disease virus of deer (EHDV) and bluetongue virus by immunisation of mice with EHDV recombinant VP7 antigen.

Immunisation of mice with recombinant VP7 antigen of epizootic hemorrhagic disease virus of deer (EHDV) induced serum antibody responses to EHDV. However, from the 19 monoclonal antibodies (Mab) produced from these mice, 15 were specific for EHDV and four for bluetongue virus (BTV). No Mabs were identified with the specificity for an epitope of VP7 shared by both EHDV and BTV in spite of the fact that they share a large portion of homology in VP7 amino acids composition. These Mabs were divided into five groups based on their specificity and interaction with each other. Group II Mabs, consisting of 13 Mabs, recognises a potential serogroup specific, linear epitope of EHDV VP7 antigen. One of the Mabs to BTV (Group V) was identified as BTV VP7 specific with the possibility of being the serogroup specific and recognizes a potential conformational epitope. Two Mabs from these VP7 specific groups were further analysed and found to be useful in a competitive enzyme-linked immunosorbent assay (C - ELISA) for detection of specific antibodies against EHDV and BTV in bovine sera.     

Antigenic characterization of foot-and-mouth disease virus serotype Asia1 field isolates using polyclonal and monoclonal antibodies

Foot-and-mouth disease virus (FMDV) serotype Asia1 field isolates (n = 100) were compared using a panel of 11 monoclonal antibodies (Mab) in sandwich ELISA. The majority (over 89%) of the isolates showed either homologous (76% and above reactivity) or reduced affinity (20-75% reactivity) for the Mabs 2A, 13, 40, 34 and 81, suggesting that these Mab binding epitopes are conserved, whereas a more variable reactivity was observed for the Mabs B3, 1A, 24, 72, 82 and 89. Polyclonal relationship ('r' value) of the field isolates in liquid phase blocking (LPB) ELISA was examined, and the mean 'r' value was 0.62 relative to vaccine virus IND 63/72. Some of the field isolates (n = 34) were tested in virus neutralization test (VNT) and showed an 'r' value of >0.40. Although a minor antigenic difference was observed in the Mab profiling study, there has not been large antigenic divergence between reference virus and field viruses, thereby providing evidence of wide antigenic coverage of the vaccine strain.