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The neuroinvasiveness of California serogroup bunyaviruses is determined by the ability of the virus to replicate in striated muscle after peripheral inoculation of mice. Neuroinvasiveness was mapped to the medium (M) RNA segment of the virus, which encodes the viral glycoproteins, when reassortants were made between La Crosse/original virus, a neuroinvasive isolate, and Tahyna-181/57 virus, a nonneuroinvasive clone. We have tested the murine muscle cell line C2C12 as a surrogate for myotropism and have found that there is a slight, but reproducible difference in the replication of virus clones bearing the M RNA segment of La Crosse/original virus compared to clones bearing the M RNA segment of Tahyna-181/57 virus, as determined by viral titer, antigen expression, and plaque formation.

California bunyaviruses cause encephalitis in mammalian hosts after peripheral infection. The virulence of these viruses is determined by their ability to replicate sequentially in striated muscle, cause viremia, and invade and replicate in the central nervous system. These viruses are also able to infect vector mosquitoes following ingestion of a blood meal containing virus. Bunyaviruses are negative stranded RNA viruses with a trisegmented genome, and the large, medium, and small RNA segments encode the polymerase, the glycoproteins, and the nucleoprotein, respectively. Reassortants between virulent and avirulent virus clones have been used to map virulence determinants in mice as well as determinants of infectivity in mosquitoes. Attenuation in mice and infectivity in mosquitoes of some virus clones maps to the medium RNA segment, implying that the virus glycoproteins, which are involved in virus entry, play a role in virulence. Attenuation in mice and mosquito infectivity of other clones maps to the large RNA segment, suggesting that cell-specific differences in the function of the viral polymerase can also determine virulence and host range.

Bovine anaplasmosis is a vector-borne disease that results in substantial economic losses in other parts of the world but so far not in northern Europe. In August 2002, a fatal disease outbreak was reported in a large dairy herd in the Swiss canton of Grisons. Diseased animals experienced fever, anorexia, agalactia, and depression. Anemia, ectoparasite infestation, and, occasionally, hemoglobinuria were observed. To determine the roles of vector-borne pathogens and to characterize the disease, blood samples were collected from all 286 animals: 50% of the cows were anemic. Upon microscopic examination of red blood cells, Anaplasma marginale inclusion bodies were found in 47% of the cows. The infection was confirmed serologically and by molecular methods. Interestingly, we also found evidence of infections with Anaplasma phagocytophilum , large Babesia and Theileria spp., and Mycoplasma wenyonii . The last two species had not previously been described in Switzerland. Anemia was significantly associated with the presence of the infectious agents detected, with the exception of A. phagocytophilum . Remarkably, concurrent infections with up to five infectious vector-borne agents were detected in 90% of the ill animals tested by PCR. We concluded that A. marginale was the major cause of the hemolytic anemia, while coinfections with other agents exacerbated the disease. This was the first severe disease outbreak associated with concurrent infections with vector-borne pathogens in alpine Switzerland; it was presumably curtailed by culling of the entire herd. It remains to be seen whether similar disease outbreaks will have to be anticipated in northern Europe in the future.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) or prion disease of cattle first recognized in 1986 in the United Kingdom, where it produced a common source epidemic that peaked in January 1993 and has subsided markedly since that time. The epidemic began simultaneously at many geographic locations and was traced to contamination of meat and bone meal (MBM), a dietary supplement prepared from rendering of slaughterhouse offal. It appears that the epidemic was initiated by the presence of the agent of scrapie (a long-standing TSE of sheep) that was first transmitted to cattle, beginning in the early 1980s, when most rendering plants abandoned the use of organic solvents in the preparation of MBM. The epidemic was probably accelerated by the recycling of infected bovine tissues prior to the recognition of BSE. To terminate the epidemic, a prohibition on the feeding of ruminant-derived protein to ruminants was introduced in the United Kingdom in July 1988. The ruminant feed ban accounts for the decline of the epidemic after an interval of about 5 years, approximately equivalent to the average incubation period of BSE. Relatively few cases of BSE have occurred in cattle born after 1993, and it is predicted that the epidemic will terminate about the year 2000 based on an extrapolation of the present declining curve. A comparison of data from the United Kingdom with data from relatively low incidence countries, such as Switzerland, indicates that this epidemic has been mainly confined to the United Kingdom because of a unique concatenation of risk factors, including: 1) a high ratio of sheep to cattle; 2) a relatively high rate of endemic scrapie; 3) the heavy feeding of MBM to dairy cattle; and 4) changes in the rendering process used to prepare MBM. Recently, cases of a variant form of Creutzfeldt-Jakob disease (a TSE of humans) have been reported in the United Kingdom. These cases, at least 10 of which had onset in 1994-1995, are distinguished by their occurrence in subjects under age 40 years, by their clinical presentation, and by their neurohistopathologic picture. The appearance of this novel disease and its concentration in the United Kingdom have raised the question that it might represent the transmission of BSE to humans. However, the cases gave no history indicating an unusual exposure to live cattle, to the preparation of beef products, or of dietary exposure to bovine tissues, and it remains to be determined whether they are associated with BSE.

La Crosse virus, a member of the California serogroup of bunyaviruses, is an important cause of pediatric encephalitis in the midwestern United States. Like all bunyaviruses, La Crosse virus contains two glycoproteins, G1 and G2, the larger of which, G1, is the target of neutralizing antibodies. To develop an understanding of the role of each of the glycoproteins in the generation of a protective immune response, we immunized 1-week-old mice with three different preparations: a vaccinia virus recombinant (VV.ORF) that expresses both G1 and G2, a vaccinia virus recombinant (VV.G1) that expresses G1 only, and a truncated soluble G1 (sG1) protein prepared in a baculovirus system. Whereas VV.ORF generated a protective response that was mostly directed against G1, VV.G1 was only partially effective at inducing a neutralizing response and at protecting mice from a potentially lethal challenge with La Crosse virus. Nevertheless, a single immunization with the sG1 preparation resulted in a robust immune response and protection against La Crosse virus. These results indicate that (i) the G1 protein by itself can induce an immune response sufficient for protection from a lethal challenge with La Crosse virus, (ii) a neutralizing humoral response correlates with protection, and (iii) the context in which G1 is presented affects its immunogenicity. The key step in the defense against central nervous system infection appeared to be interruption of a transient viremia that occurred just after La Crosse virus inoculation.

The California serogroup of viruses includes 14 viruses which take their name from California encephalitis virus, the prototype of the serogroup, which was isolated in California in the 1950s (Hammon and Reeves, 1952). This serogroup is part of the Bunyauirus genus within the family Bunyaviridae (Gonzalez-Scarano and Nathanson, 1990; Kolakofsky, 1991; Whitman and Shope, 1962). La Crosse virus, the most medically significant member of the California serogroup, was isolated in 1964 from a fatal case of encephalitis diagnosed in La Crosse, Wisconsin (Thompson et al., 1965). Although California encephalitis virus was initially reported in association with a few cases of encephalitis, it is not an important human pathogen. On the other hand, La Crosse virus is a common cause of encephalitis in the pediatric age group, with an average of about 75 reported cases of encephalitis each year (Kappus et al., 1983). In years when other arboviruses are not epidemic, La Crosse virus is the most important cause of arbovirus encephalitis in the United States. The virus is estimated to cause more than 100,000 inapparent infections annually which are concentrated in the midwest (Grimstad et al., 1984), which is the principal habitat of Aedes triseriatus, its major mosquito vector. Bunyavirions are roughly spherical particles about 100 nm in diameter consisting of a lipid envelope containing nucleocapsids representing the three different RNA species (L segment, 6.8 kb; M segment, 4.5 kb; and S segment, 0.9 kb) which comprise the negative-sense genome (Elliott, 1990; Gonzalez-Scarano and Nathanson, 1990). The viral envelope contains two glycoproteins, Gl and G2, and

The family Bunyaviridae is comprised of over 250 virus species divided into four genera: Bunyavirus, Phlebovirus, Hantavirus, and Nairovirus, and classified together because of conserved structural features (see below) (1, 2, 3, 4, 5). Most of the bunyaviridae are of no known medical or veterinary importance, and were isolated by routine inoculation of either mice or cell culture of arthropods obtained in the field (6). However, some viruses in several of the genera are important causes of human and animal diseases. Members of the California serogroup (genus Bunyavirus) and Rift Valley fever virus (genus Phlebovirus) can cause encephalitis, and are therefore of principal importance for this book. Recent reviews discuss the medical complications of infections with other Bunyaviridae (2, 7). Studies of the pathogenesis of central nervous disease have been mainly carried out with La Crosse and other members of the California serogroup, and our discussion will be largely confined to these. The members of the California serogroup are listed in Table 1.

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.