Genetic characterisation of bovine herpesvirus 1 in New Zealand

AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV- 1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in Argentina.

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

Antigenic characterisation of virus isolates from vaccinated dogs dying of rabies

Four rabies virus isolates from dogs that succumbed to rabies infection in Nigeria within one year of anti-rabies vaccination were characterised by monoclonal antibodies (MAbs). The samples were screened for rabies and rabies-related viral antigens by the indirect fluorescent antibody test, performed with MAb 502-2, which recognises the nucleocapsid (NC) protein of all known Lyssaviruses and with MAb 422-5 which identifies African rabies-related viruses. All four canine virus isolates displayed positive fluorescence with MAb 502-2 and were negative with MAb 422-5. In the anti-NC MAb characterisation with a panel of 34 additional MAbs, all isolates displayed positive staining with 32 of the MAbs, were negative with MAb 102-27 and all displayed poor immunofluorescence with MAb 377-7. On the basis of reactivity with a panel of 40 anti-glycoprotein (G) MAbs the isolates were separated into four distinct viral subtypes. None of these canine isolates was identified as the common attenuated Flury LEP rabies strain used for domestic animal vaccination and none resembled other previously characterised rabies viruses from Nigeria.     

DNA restriction enzyme analysis of a bovine herpesvirus 1 strain isolated from encephalitis in Hungary

The DNA of a bovine herpesvirus 1 (BHV-1) strain isolated from calf encephalitis in Hungary was analysed with restriction enzymes. The cleavage pattern of the encephalitis strain Na/67 differed from those of all the other Hungarian BHV-1 isolates investigated so far. The EcoRI and HindIII cleavage patterns of virus strain Na/67 were found to be similar to the patterns of two other encephalitis strains (N569 and A663 from Australia and Argentina, respectively) characterized earlier. Strain Na/67 is the first isolate in Europe which showed the restriction enzyme pattern of BHV-1.3 previously supposed to be characteristic of encephalitis strains.     

Restriction endonuclease analysis of a porcine isolate of bovine herpesvirus type 1.

Deoxyribonucleic acid (DNA) was extracted from bovine herpesvirus type 1 (BHV-1) isolated from a stillborn porcine fetus, from the Cooper reference strain of BHV-1, and from an Ontario bovine respiratory isolate. Each DNA was digested with the restriction endonucleases HindIII, EcoRI, HpaI and BamHI. Except for very minor differences in the patterns produced after digestion with EcoRI and HindIII, the DNA of the porcine isolate reacted in a similar manner to the bovine viruses, and it was concluded that the porcine virus is genetically similar to bovine isolates of BHV-1.     

Bovine herpesvirus 4 isolates: a comparison of three major glycoproteins.

Twenty-four Belgian field isolates of bovine herpesvirus 4 (BHV-4), together with four reference strains were compared by radio-immunoprecipitation and western blotting using a polyvalent antiserum and monoclonal antibodies raised against major glycoproteins. Most of these strains showed the same protein profile as the European reference strain Movar 33/63. For two strains the molecular weight of gp 6, p (gp 10/gp 17) and gp 10 were the same as those of the American reference strain DN 599. No relationship could be established between the protein profiles and origin of the isolates or with the restriction patterns. This study provides a view of the molecular weight variations of the major BHV-4 glycoproteins among field isolates.     

A study of the predominant genotypes of bovid herpesvirus 1 found in the U.K

The genotypic classification of 84 U.K. isolates of bovid herpesvirus 1 was determined by the restriction endonuclease technique. Preliminary studies with six enzymes (EcoRI, HindIII, HpaI, BamHI, PstI and BstEII) showed that the principal genotypic variants, including the live vaccine strains used in the U.K., could be identified from the digestion patterns with just two endonucleases: HindIII and HpaI. Isolates from the 1960s were all categorised as genotype 2b. From 1977 onwards, genotype 1 has predominated in mainland Britain, with occasional type 2b isolates. Type 2b viruses were isolated from both respiratory and genital disease cases. There was no evidence of genotypes 2a or 3 in the U.K.     

Clinical, pathological and antigenic aspects of bovine viral diarrhea virus (BVDV) type 2 isolates identified in Brazil

Nucleotide sequencing and phylogenetic analysis of Brazilian bovine viral diarrhea virus (BVDV) field isolates identified four viruses belonging to the genotype 2. Comparison of 5' UTR sequences from these isolates to those of North American BVDV type 2 revealed genomic variations that correlated with the geographic origins of the isolates. Two of the Brazilian type 2 viruses were isolated from clinical cases of gastroenteric/respiratory disease and two were isolated from healthy bovine fetuses. The clinical cases affected young animals (8- and 18-months-old) and were characterized by diarrhea, respiratory signs, extensive oral and digestive tract erosions, conjunctival and vulvar congestion, occasional digestive bleeding and vulvar and heart petechial hemorrhage. Antigenic analysis of these isolates with a panel of 10 monoclonal antibodies revealed marked antigenic differences in the major envelope glycoprotein, gp53/E2, compared to standard laboratory and vaccine BVDV strains. In addition, virus-specific antisera raised to Brazilian BVDV type 2 viruses displayed very low serological cross-reactivity with standard BVDV type 1 strains. Differences up to 64-fold in cross-neutralization titers were observed between BVDV type 1 and Brazilian BVDV type 2 isolates. The identification of BVDV type 2 among Brazilian cattle may have important implications for epidemiological studies, diagnostic and immunization strategies. Furthermore, the low neutralizing activity of BVDV type 1 antisera against the recently identified Brazilian BVDV type 2 isolates raises the question about the degree of protection conferred by BVDV vaccines, most of them based on a single type 1 strain.     

Antigenic Characterization of Rabies Virus Isolates from Vaccinated Dogs in Plateau State,Nigeria

Rabies isolates (genotype 1 lyssaviruses) from vaccinated dogs that died of rabies infection in the Plateau area of Nigeria were characterized using monoclonal antibodies (MAbs). The isolates were examined for rabies (genotype 1) and rabies-related (genotypes 2, 3 and 4) viruses by the indirect fluorescent antibody test carried out with MAb 502-2, which recognizes the nucleocapsid protein of all known lyssaviruses, and with MAb 422-5, which identifies only rabies-related viruses. All three isolates showed positive immunofluorescence with MAb 502-2 and were negative with MAb 422-5, indicating that they were all rabies (genotype 1) viruses.Characterization with a panel of 36 anti-nucleocapsid monoclonal antibodies showed that all three isolates reacted positively with 35 of the anti-nucleocapsid MAbs, including MAb 102-27 and MAb 377-7. Characterization using a panel of 44 anti-glycoprotein MAbs differentiated the isolates sharply from LEP Flury and PM vaccine viruses. The pattern of anti-glycoprotein reactivity of the isolates showed them to belong to one distinct viral subtype, except for a minor variation in one isolate that was not neutralized by MAb 1101-3. None of the three isolates was identified as the Flury low egg passage (LEP) vaccine strain used for vaccinating dogs in Nigeria. In fact, all the three isolates had the typical pattern of reactivity of isolates from unvaccinated dogs, including MASS 83, a rabies virus isolated in Nigeria and characterized at the Wister Institute before this study.     

Biological and biochemical comparison of bovid herpesvirus-4 strains

Bovid herpesvirus-4 (BHV-4) isolates V.Test and LVR140, isolated from genital disease, respectively, in bull and in cow, and the reference strains Movar 33/63 and DN599 were compared by several methods: cross-serological relationship studied by indirect immunofluorescence; kinetics of intracellular and extracellular viral production; comparison of the mean plaque size; restriction analysis of viral DNA with restriction enzymes EcoRI, BamHI and HindIII. BHV-4 strains were serologically identical and the kinetics of viral production were very similar. Comparison of the mean plaque size allowed classification into 3 classes (Class I, Movar33/63; Class II, LVR140; Class III, V.Test and DN599) and restriction analysis of viral DNA revealed clear differences between the electrophoretic patterns of the four BHV-4 strains. The differentiation between BHV-4 strains can therefore be achieved by a biological method (mean plaque size) and by restriction analysis. The two genital isolates are easily differentiated by the two methods.     

Antigenic variation of Mycoplasma gallisepticum, as detected by use of monoclonal antibodies.

A panel of monoclonal antibodies (MAb) developed against Mycoplasma gallisepticum strain PG31 was used to probe the antigenic profiles of 5 recognized strains (PG31, R, S6, F, A5969) and 6 field isolates of M gallisepticum. Monoclonal antibody G9 predominantly recognized antigens at apparent molecular mass positions of 90 to 98 kDA. The MAb reacted with all strains and isolates, but the molecular mass position of the antigens varied among some mycoplasmas. Monoclonal antibody G12 reacted with all strains and isolates of M gallisepticum and had an identical banding pattern. However, MAb G10 and G11 reacted selectively only with a limited number of strains and/or isolates. Surface distribution of the MAb-recognized antigens was revealed by immunoelectron microscopy. Partial physicochemical characterization of MAb G9-recognized antigens identified glycopeptide characteristics. Monoclonal antibody G9 reacted with surface antigens and, hence, participated in agglutination of M gallisepticum. However, the degree of agglutination varied among the various strains and isolates, indicating a quantitative or conformational limitation or an alteration in the anomeric expression of the epitopes. Antigenic variation in M gallisepticum may be mediated by immunologic selective pressures, or a proclivity for habit niche in the host.     

Comparative study of two strains of Bovid herpesvirus-4

Two strains of Bovid herepesvirus-4 (BHV-4), i.e. the prototype strain DN-599, obtained from a steer suffering of a respiratory disease, and the strain 85/BH 16TV, originated from a cow with vulvovaginitis, were compared in studies which included restriction endonuclease analysis, experimental infection and reciprocal cross protection tests. The restriction endonuclease analysis revealed that the resultant DNA patterns of the isolates were generally similar with only a difference in one fragment. The two strains were capable of causing respiratory tract infection in calves, even if they displayed a different level of virulence: the strain 85/BH 16TV being the most virulent while the strain DN-599 the least. The two viral strains were mutually protective in that the calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. Finally, both viral strains failed to evoke the production of neutralizing antibody in the experimental calves.     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in argentina

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

Comparison of Haemophilus paragallinarum isolates by restriction endonuclease analysis of chromosomal DNA

Chromosomal DNA from Haemophilus paragallinarum was examined by restriction endonuclease analysis (REA) using the enzymes BamHI, EcoRI, HindIII, or SmaI. The enzyme SmaI had no apparent effect upon the DNA from eight representative H. paragallinarum isolates. The remaining enzymes cut the H. paragallinarum DNA to varying degrees, with the most useful patterns for distinguishing isolates being given by HindIII. The REA patterns given by HindIII were stable under both in vitro and in vivo conditions. The use of the enzyme HindIII showed that eight Australian isolates of H. paragallinarum were genetically similar. In contrast, 14 isolates of H. paragallinarum from outside Australia were markedly different from each other and the Australian isolates. A plasmid of approximately 6 kilobase pairs in size was found in one isolate of H. paragallinarum.     

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.     

Evaluation of two monoclonal antibodies for serotyping Haemophilus paragallinarum

Two monoclonal antibodies (MAbs) were evaluated for their ability to serotype 108 isolates of Haemophilus paragallinarum. One MAb (E5C12D10) was raised against a Page serovar A strain and the other (F2E6) against a Page serovar C strain. In both dot blot and hemagglutination-inhibition tests, MAb E5C12D10 recognized the type strains of Page serovar A and Kume serovars A-1, A-2, A-3, and A-4. MAb F2E6 recognized the type strains of Page serovar C and Kume serovars C-1, C-2, and C-3. Neither antibody recognized the type strains of Page serovar B or Kume serovars B-1 and C-4. When evaluated with 97 field isolates in a dot blot test, the MAbs serotyped 81 isolates, which was better than agglutinin typing by the Page scheme (69 isolates serotyped). The field isolates that did not react with the MAbs were either Page serovar B/Kume serovar B-1 (three isolates), Page serovar C/Kume serovar C-4 (12 isolates), or nontypable by either the Page or Kume scheme (one isolate).     

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.     

A serological comparison of some animal herpesviruses

Bovine herpesvirus 1 (BHV-1) isolates (Cooper-type strain 4975 and Oxford) were compared in neutralization tests with the bovine herpesvirus 4 (BHV-4) isolate (85/16 TV) and the herpesviruses of red deer (D2839/1) and goats (E/CH). Hyperimmune antiserum was prepared in rabbits against the plaque-selected viruses and endpoint and kinetic neutralization test were made. BHV-4 was clearly different from the other four viruses. The closely-related BHV-1 strains were also related in these tests to the red deer herpesvirus. The Oxford strain seemed rather closer antigenically than the Cooper-type strain to the red deer herpesvirus. Antiserum to the caprine herpesvirus failed to neutralize either BHV-1 strain or red deer virus, but antiserum to the Cooper-type and red deer herpesviruses did neutralize caprine virus to a limited extent.