Bovine viral diarrhea virus: Antigenic diversity and practical consequences (Proceedings)

The genetic diversity that occurs among isolates of BVDV is characteristic of RNA viruses that exist in nature as quasispecies (a swarm of viral mutants). The genetic diversity that occurs among BVDV isolates is reflected in the antigenic diversity found among viral isolates worldwide. The persistently infected animal is considered important for maintaining BVDV in nature and as being a primary source of virus for other cattle. Persistently infected cattle may also serves as a source of viral genetic variants that may be "selected" by non-persistently infected cattle when infected with virus. The emergence and establishment of genetic and antigenic variants of BVDV also is affected by selective pressure applied to the virus by the innate and adaptive host immune responses. The array of disease manifestations seen during infection with BVDV, and the corresponding pathogenic processes, may be attributed to viral diversity; however, the definitive viral markers for tissue tropism or virulence have yet to be identified.

Basis for Diversity - BVDV the Quasispecies

Studies have shown that BVDV exists as a quasispecies. The ability to mutate rapidly allows BVDV to quickly produce mutants that are better fit to replicate in the host. Populations of genetic variants of BVDV also have been identified within individual persistently infected cattle. The detection of genetic variants in persistently infected cattle suggests that those animals may enhance the diversity of BVDV by serving as a source of viral variants that can infect other cattle.

While there is a tendency to maintain the master nucleic acid sequence of a virus under neutral conditions, the immune response of the infected host creates a non-neutral condition and may select viral variants. This has been seen on farms that harbor multiple persistently infected cattle, which likely originated from a single outbreak of acute infection in immunocompetent pregnant cattle. Comparison of the BVDV from those animals showed that the viral isolates were similar; however, antigenic differences could be detected among the viral isolates. The selection of the antigenic variants likely occurred during the acute infection of the dams of those persistently infected cattle and resulted in transplacental transmission slightly different BVDV to a group of fetuses.

Genetic and Antigenic Diversity

Viral genotypes and genetic diversity – The high frequency of mutation, propensity for recombination, and selective pressure from immune responses stimulated by natural infection or vaccination has led to the creation of a large assortment of BVDV genetic and antigenic variants. The genetic variants can be grouped based on the homology of aligned nucleic acid sequences from various segments of the viral genome. The array of BVDV form genotypes, subgenotypes within genotypes, and isolates within subgenotypes. Pestiviruses segregate into at least five (possibly six) viral genotypes. Those genotypes are classical swine fever virus, bovine viral diarrhea virus type 1, bovine viral diarrhea virus type 2, border disease virus, and a genotype represented by a single viral isolate termed Giraffe-1.

The viral genotypes are about 60% similar to each other in their base sequence. Subgenotypes within a genotype are designated by a number followed by a lower case letter (BVDV type1a, 1b, etc). Subgenotypes are about 80 to 85% similar to each other. Currently, 11 subgenotypes of BVDV type 1 and two subgenotypes of BVDV type 2 have been identified. Recent phylogenetic surveys suggest that there are regional differences in the distribution of viral genotypes and subgenotypes. The regional distribution of viral genotypes and subgenotypes likely reflects historical routes for movement of cattle, vaccine usage over time, and geographic isolation of cattle populations. There is some linkage of viral genotypes and subgenotypes with clinical manifestations of disease including thrombocytopenia, reproductive failure, or pneumonia.

Antigenic diversity – The genetic diversity seen among BVDV results in extensive antigenic diversity. Most field isolates of BVDV show unique patterns of monoclonal antibody binding when reacted with a large panel of monoclonal antibodies raised against several different viruses. In fact, BVDV isolates that are antigenically alike in monoclonal antibody assays are difficult to find.

Viruses are readily segregated into genotypes by patterns of monoclonal antibody binding. Similarly, segregation of BVDV into genotypes can be done using convalescent serum or post vaccinal serum in viral neutralization assays. Antigenic differences likely exist between subgenotypes, but the diverse antibody response that occurs among cattle after infection or vaccination makes it difficult to consistently separate viruses into subgenotypes using polyclonal antibody. In summary, BVDV exists as an antigenically diverse array of viruses that manage to retain some antigenic similarity with each other and with other pestiviruses.

Consequences of Diversity

Diversity of Clinical Disease – The clinical outcome following infection with BVDV is complex and dependent on multiple factors that are agent, host and environmentally related. Host factors that can influence the clinical outcome of infection include whether the host is immunotolerant or immunocompetent to BVDV, immune status (passive from colostral antibodies or active from exposure or vaccination), pregnancy status in females, gestational age of the fetus at the time of infection, level of environmental stress at the time of infection and concurrent infection with other pathogens. It is well established that variation in virulence exists between different BVDV isolates. However, the basis for clinical variation at the virus level is not understood.

It is important to realize that despite wide genetic and antigenic diversity of BVDV isolates, most viral isolates are capable of inducing some common clinical syndromes. All noncytopathic BVDV isolates appear capable of infecting the fetus resulting in abortion, congenital defects or the development of immunotolerance and subsequent persistent infection. The majority of both type 1 and type 2 BVDV isolates are of low virulence and induce subclinical to very mild disease.

When BVDV infections result in clinical disease, it has historically been referred to as BVD. Most clinical presentations of BVDV infection are mild, consisting of lethargy, anorexia, fever, diarrhea, and decreased milk production in lactating cows. Beginning in 1993, an atypical form of BVDV infection, referred to as severe BVD, was recognized in Canada. The disease had a peracute course, caused high morbidity, and resulted in a substantial number of deaths in all age groups. This new form of BVDV infection killed approximately 25% of veal calves in certain regions of Canada. Viral isolates obtained from these severe acute outbreaks were genotype 2 BVDV. Acute BVDV infections in cattle also can cause a hemorrhagic syndrome. These infections are characterized by severe thrombocytopenia, bloody diarrhea, epistaxis, hemorrhages on mucosal surfaces, hyphema, bleeding from injection sites, pyrexia, leukopenia, and death. Thus far only noncytopathic type 2 BVDV has been associated with the hemorrhagic syndrome.

Although type 2 BVDV has been associated with many of the documented outbreaks of severe BVD and hemorrhagic syndromes, it should be emphasized type 1 BVDV isolates are capable of resulting in severe disease. Outbreaks of severe clinical disease consisting of diarrhea, rapid dehydration and death have been observed in association with isolation of type 1 BVDV (Dr. Kenny Brock, personnel communication). Additionally, type 2 BVDV isolates of low virulence are common and are likely to predominate over virulent type 2 isolates.

It is well established that acute BVDV infection can result in immunosuppression. Comparative experimental studies demonstrate that differences in the effect of BVDV isolates on cells of the immune system can be significant. In calves experimentally inoculated with either a low virulence type 1 virus (TGAN), a low virulence type 2 virus (7937) or a high virulence type 2 virus (890), a corresponding 21%, 49%, and 65% drop in white blood cell count was observed between day of infection and day 12 post infection. These differences are most important when combined with other disease exposures such as those that may occur in a commingle feedlot environment.

In the United States, BVDV has been reported as the most common virus isolated from outbreaks of BRD. Experimentally it has been difficult to reproduce respiratory disease with BVDV alone, but synergistic effects have been documented between BVDV and Mannheimia haemolytica, bovine herpesvirus-1, and bovine respiratory syncytial virus. Experimental studies have suggest that some BVDV isolates have more pulmonary tropism and are more likely to be associated with BRD than others.

BVDV infections have been associated with infertility, early embryonic deaths, a variety of congenital defects and fetal infection with seroconversion. Most importantly, fetal infection between 30 and 125 days of gestation can result in the development of immunotolerance to the virus and the subsequent birth of calves that are persistently infected with BVDV. Cattle persistently infected with BVDV serve as the major virus reservoir and source of virus transmission within and between farms. Differences in reproductive outcomes are most dependent on time of infection. Differences in the ability of individual BVDV isolates to cause reproductive failure have not been well documented although it has been speculated that differences exist. Review of diagnostic laboratory data by Evermann supports this conclusion by finding that Type 1 BVDV isolates were more commonly associated with persistent infections, congenital defects, and weak calves while type 2 BVDV isolates were more commonly found in aborted fetuses. Experimental studies provide evidence that different BVDV isolates have different fetal tissue tropisms and this difference may result in different fetal pathologies and clinical outcomes. In a dose titration study comparing the ability of a type 1 and type 2 isolate to cross the placenta and infect the fetus, fetal infection occurred in 4/4 heifers challenged with 107,105,103,101 CCID50/dose of type 2 virus while occurring in 4/4, 4/4,3/4, and 0/4 heifers challenged with 108,106,104, 102 CCID50/dose of type 1 virus respectively. Results from this study suggested that some BVDV isolates might be more likely to cross the placenta than others, although the reason for this is unknown.

Diagnostic Challenges – Both organism and immune response detection methods are used to diagnose BVDV. Diagnostics target viral antigens (immunoperoxidase microtiter assay, antigen ELISA, immunohistochemistry, fluorescent antibody), genomic material (PCR, in situ hybridization) or BVDV specific antibodies (virus neutralization, antibody ELISA). These assays have varying risks for failure when used to detect an organism with the capability of having a diverse genetic and antigenic makeup, such as BVDV.

Antigen detection assays rely on either monoclonal or polyclonal antibodies to detect BVDV specific antigens. Polyclonal antibodies derived from hyperimmunized swine or calves are generally broadly reactive as they contain antibodies directed against multiple epitopes, many of which are conserved among viruses. Monoclonal antibodies are specific for one epitope and if that epitope varies between viruses, binding of the monoclonal antibody can fail. Most antigen detection assays use polyclonal antibodies or a pool of monoclonal antibodies to provide the broadest reactivity and capability of detecting a diverse population of BVDV isolates.

PCR detects and amplifies genetic sequences that are unique to the organism of interest. The accuracy of PCR is dependent on the ability of PCR primers to specifically bind to target genetic material unique to the organism of interest. The difficulty that can arise with PCR is identifying genetic material that is unique to the organism of interest yet stable enough that it does not change significantly over time. Many PCR methodologies have been reported for detecting BVDV.. Primers have been designed that are capable of detecting a wide variety of field samples. Diagnostic PCR primers primarily have been directed against the 5' untranslated region of BVDV where nucleotide homology can be as high as 95% between isolates thus allowing for high epidemiological sensitivity.

Assays to detect virus neutralizing (VN) antibodies also are affected by BVDV diversity. As a result of non-standard assay procedures, neutralizing BVDV antibody titers reported by different laboratories can vary significantly. A significant variable that can differ from laboratory to laboratory is the reference virus used in the neutralizing assay. In a study by Vaughn, fourteen animal diagnostic laboratories were asked to run BVDV VN's on split serum samples collected from 11 calves. The average of the 11 calves reported by each lab ranged from 1:18 to 1:2028. It was concluded that different BVDV reference strains being used in the VN assays likely account for some of the observed lab-to-lab variation. Virus neutralizing antibody titers may be dramatically different depending on the viral genotype with which the animals are exposed to. In a study using seroconversion in unvaccinated heifers as an indicator for circulating BVDV, it was observed that type 2 VN antibody titers were always highest if the actual virus circulating on the farm was type 2 BVDV (table 1). The same observation was made for type 1 VN antibodies and type 1 virus. Therefore, using a VN assay designed to detect type 1 antibodies (assay using a type 1 reference virus) in a herd where type 2 virus is circulating may yield significantly different results than a VN assay using a type 2 reference virus. When using virus VN assays as a BVDV diagnostic tool, requesting both type 1 and 2 viral neutralizing antibody titers should be considered.

Vaccination Failure – Diversity among BVDV isolates is a suspected cause vaccination failure. However, several studies have shown that a BVDV type 1 immunization induces clinical protection against a type 2 challenge. Although protecting cattle against clinical disease is important, it may not be sufficient in terms of controlling reproductive failure. A key component in controlling BVDV is preventing fetal infections that result in the birth of calves persistently infected with the virus. Preventing fetal infection and the subsequent sequela involves controlling virus exposure and enhancing BVDV specific immunity in susceptible dams. An important issue is the ability of immunity developed against one virus strain to cross protect against heterologous BVDV strains effectively enough to prevent fetal infection. Several field studies suggest that immunological protection against heterologous BVDV challenge may be incomplete with respect to fetal protection. Early vaccines were developed with little knowledge of their ability to provide fetal protection. Currently, efficacy data on fetal protection is not required for approval of vaccines for BVDV in the United States. Experimental studies attempting to address fetal protection are limited and have focused primarily on immunity developed following vaccination. Results of vaccine fetal protection studies have been mixed and are often dependent on the challenge model (see chapter on Reproductive Consequences of BVDV for further details). Most trials have involved killed vaccines and efficacy has ranged from 25-100% . In studies evaluating the fetal protection efficacy of a modified-live vaccine, Cortese and Brock demonstrated 88% and 58% fetal protection in heifers immunized one time with a commercially available type 1 modified-live BVDV vaccine and challenged at 75 days in gestation with type 1 or type 2 BVDV respectively. Except for different challenge viruses, these studies were conducted similarly suggesting that the vaccine was less likely to stimulate a fetal protective immunity against type 2 viruses compared to type 1 viruses. Other studies have not fully evaluated protection against multiple viruses.

Summary

The complexity of clinical presentations associated with BVDV likely arises in part from factors encoded by the virus genome. More importantly, prevention and control of BVDV may be complicated by diagnostic and immunization failure resulting from virus diversity. Evolutionary pressures will continue to drive further diversity, making control of BVDV challenging. Current and the potential for future BVDV strain diversity should be considered when designing BVDV control programs both at the individual farm and national herd level.

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