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Evolution of Bovine virus diarrhea virus (BVD virus): where is heterogeneity generated?

Ernst Peterhans, Hanspeter Stalder, Phillipp Meier, J. Rüfenacht, Marc Strasser, Patrick Schaller, Christian Hertig
Institute of Veterinary Virology, University of Bern, Länggass-Str. 122, CH-3012 Bern, Switzerland
(ernst.peterhans@ivv.unibe.ch)

BVD virus, a flavivirus related to classical swine fever and hepatitis C viruses, is an important cattle pathogen with a world-wide distribution. Two biotypes of this virus can be differentiated. The cytopathic virus kills its host cells by apoptosis whereas the non-cytopathic virus has no obvious detrimental effects on its host cells. Non-cytopathic BVD virus is capable of interacting with its hosts in two fundamentally different ways: it causes a transient infection in immunologically naive animals; and a persistent infection in animals infected in utero early in gestation. Such animals may be born normal and show no signs of infection although they multiply the virus to titers that may reach 10⁶ in blood. The persistent infection with BVD virus is unique because the virus is immunologically tolerated. Hence, BVD virus faces two fundamentally different situations in its host: a ‘normal’ immune response in acute-transiently infected animals; and immunotolerance in persistently infected animals.

We have investigated viral evolution during acute and transient infection in vivo and tested the effects of viral passage in different types of cultured cells.

Evolution during persistent infection
In persistently infected cattle virus was shown to be composed of a spectrum of quasispecies that changed only very slightly over an observation period of one year, without altering the consensus sequence in the gene encoding the surface glycoprotein E2 .

Evolution during acute infection
Three heifers, seronegative and free of BVD virus, were infected intranasally with 10⁷ TCID₅₀ of BVD virus strain SD-1 (a kind gift of Dr. K. Brock, Wooster, OH, USA). The animals showed lymphopenia for the first 10-15 days post-infection and antibodies to BVD virus were detected from days 13-15 onward. Virus was detected in nasal swabs and blood until days 8-10 post-infection. The sequence analysis failed to reveal any changes in the gene encoding E2, indicating that the virus remained stable in this region of the genome known to be highly variable among BVD viruses.

Transmission to the fetus
This was analyzed in a persistently infected cow and in two calves born to this cow that were also persistently infected. Virus was obtained from the blood of these three animals and additionally from various organs of one of the calves. The analysis of the nucleotide sequence of E2 revealed that (i) virus in the blood of the three animals was closely related yet distinct and (ii) virus present in the blood of the calf differed from that present in the organs.

Evolution during passage in cultured cells
To investigate the effect of submitting the virus to functional pressure, we passaged virus obtained from a persistently infected animal in bovine macrophages, bovine turbinate cells, MDBK cells (transformed bovine kidney cells) and lamb synovial cells. Virus passaged 10 times through bovine macrophages and turbinate cells was unaltered in E2. In contrast, passage through MDBK cells and, to a larger extent, lamb synovial cells led to a number of nucleotide substitutions that also resulted in an altered amino acid sequence. These changes are compatible with the well-known capability of BVD viruses to infect other species of the artiodactyla and that passage to heterologous cells is facilitated by the existence of minor variants that emerge when the conditions favor their growth. In line with this interpretation, backpassage experiments revealed that the observed changes were readily reversible and were due to the presence in the inoculum of variants with differing fitness to grow in the different cell types. This concept was supported by competition experiments using biologically cloned virus.

Overall, our experiments suggest the following model of evolution for BVD virus:
The virus is approaching evolutionary stasis in persistently infected animals, a situation which differs fundamentally from that observed in persistent infections with a high viral load despite an active antiviral immune response, e.g. in HIV-infected humans. No dramatic changes are observed in virus in acutely infected animals. However, passage between animals may result in the selection of variants with improved fitness to growth in new hosts. It is unclear at present whether there exists a bottleneck during viral transmission. The observations made during virus transmission from a persistently infected mother to its offspring however argue against a bottleneck in this situation. The results of the experiments using cultured cells support the view that selection from a pre-existing set of variants may be important for the evolution of BVD virus. Slow evolution through nucleotide replacements, selection during transmission, and preservation during persistent infection may explain the extraordinary heterogeneity of BVD viruses circulating in the cattle population.

Abstract - Presented at the Virus Evolution Workshop
Ardmore, OK
October 21 - 24th, 1999

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e-mail: mroossinck@noble.org

Dr. Marilyn Roossinck
Plant Biology Division
The Noble Foundation
P.O. Box 2180
Ardmore, OK 73402

phone: 580 224-6630