CHAPTER 14

1. CHAPTER 14 Bunyavirus

2. Definitions of the virus: • The family Bunyaviridae is the largest virus family, with more than 350 member viruses included in five genera:Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus. • The features of the bunyaviruses pertain both to the nature of the virions and to their biological properties.

3. Viruses in three genera (Orthobunyavirus, Nairovirus, and Phlebovirus) are maintained in arthropod–vertebrate–arthropod cycles (so-called arboviruses). Viruses in the genus Hantavirus are an exception, in that they are maintained in vertebrate–vertebrate cycles without arthropod vectors; the hantaviruses also exhibit great specificity in vertebrate reservoir hosts, and therefore also have distinct geographic and ecologic niches.

6. Arthropod-borne bunyaviruses are transmitted by specific mosquitoes, ticks, midges, or biting flies, whereas the hantaviruses are disseminated by specific rodents. Bunyaviruses cause transient infection in their vertebrate hosts, whether mammal or bird, and life-long persistent infection in their arthropod vectors, whereas hantaviruses cause persistent infection in their rodent reservoir hosts.

7. Most bunyaviruses never infect domestic animals or humans, but those that do can cause important diseases from congenital fetal malformation to systemic “hemorrhagic fever” disease syndromes. Genomic features are used to define genera, particularly the organization of each RNA genome segment and the sequences of conserved nucleotides at the termini of each segment. Classical serological methods are used to classify these viruses further.

8. Antigenic determinants on the nucleocapsid protein are relatively conserved, and so serve to define broad groupings among the viruses, whereas shared epitopes on the envelope glycoproteins, which are the targets in neutralization and hemagglutination-inhibition assays, define serogroups. Genetic reassortment occurs when mosquitoes are coinfected with related bunyaviruses. Within its particular ecologic niche, each bunyavirus evolves by genetic drift and selection.

9. The genus Orthobunyavirus contains viruses that share common genetic features and are serologically unrelated to viruses in other genera of the Bunyaviridae. These viruses are mosquito-borne, but some are transmitted by Culicoides spp. The genus includes pathogens of domestic animals and humans, including Akabane viruse. The genus Phlebovirus contains viruses which are transmitted by mosquitoes. The genus contains Rift Valley fever virus and the sandfly fever viruses.

10. The genus Nairovirus are tick-borne, including the pathogens Nairobi sheep disease and Crimean-Congo hemorrhagic fever viruses. The genus Hantavirus are transmitted by persistently infected reservoir rodents via urine, feces, and saliva; the same transmission pattern has occurred among rats. In humans, these viruses from Asia cause hemorrhagic fever with renal syndrome, those from Europe are associated with the “neuropathica epidemica.” The hantaviruses from the Americas cause the “hantavirus pulmonary syndrome.”

12. Bunyavirus virions are spherical, 80–120 nm in diameter, and are composed of a lipid envelope with glycoprotein spikes, inside which are three circular ribonucleoprotein (RNP) complexes comprised of individual genome RNA segments. These RNP complexes are stabilized by a panhandle structure generated by non-covalent bonds between inverted palindromic sequences on the 3’ and 5’-ends of each RNA genome segment. The terminal sequences are identical for three RNA segments within each virus species, and are recognized by the viral polymerase for virus genome replication and initiation of virus mRNA transcription.

13. The genome of bunyaviruses is 11–19 kb and consists of three segments of negative-sense (or ambisense), single-stranded RNA, designated large (L), medium (M), and small (S). The L RNA encodes a single large protein (L), the RNA-dependent RNA polymerase (transcriptase). The M RNA encodes a polyprotein that is processed to form two glycoproteins (Gn and Gc) and, in some cases, a non-structural protein (NSm). The S RNA encodes the nucleocapsid (N) protein and, for members of the Orthobunyavirus and Phlebovirus genera, a non-structural (NSs) protein.

14. The N and NSs proteins of viruses in the genus Phlebovirus are each translated from a separate subgenomic mRNA. The N protein is encoded in the 3’ half of the S RNA, and its messenger RNA (mRNA) is transcribed using genomic RNA as template. However, the NSs protein, occupying the 5’ half of the same S RNA molecule, is encoded in the reverse complementary sense, with the NSs mRNA being transcribed only after the synthesis of full-length viral genome RNA intermediates; thus the S segment RNA exhibits an ambisense coding strategy.

17. Bunyaviruses have four virion proteins, including two external glycoproteins (Gn,Gc), the L protein (transcriptase), and the N protein (nucleoprotein). Virions also contain lipids and carbohydrates as side chains on the glycoproteins. The Gn glycoprotein is responsible for receptor binding of California serogroups bunyaviruses. The non-structural NSs protein of Rift Valley fever virus interferes with the innate host-cell antiviral response via inhibition of the cell signaling molecules (protein kinase and the transcription factor, TFIIH), leading to global suppression of the type I interferon response.

18. The viruses are quite sensitive to heat and acid conditions, and are inactivated readily by detergents, lipid solvents, and common disinfectants. Most bunyaviruses replicate well in many kinds of cells, including Vero cells, BHK-21 cells, and, except for hantaviruses, C6/36 mosquito (Aedes albopictus) cells. Except for hantaviruses and some nairoviruses, these viruses are cytolytic for mammalian cells, but are non-cytolytic for invertebrate cells.

19. Viral entry into its host cell is by receptor-mediated endocytosis; all subsequent steps take place in the cytoplasm. Cell receptors include integrins and other cell receptor proteins such as gC1qR/p32, which is expressed on endothelial cells, dendritic cells, lymphocytes, and platelets. Because the genome of the single-stranded, negative-sense RNA viruses cannot be translated directly, the first step after penetration of the host cell and uncoating is the activation of the virion RNA polymerase (transcriptase) and its transcription of viral mRNAs from each of the three virion RNAs.

20. The exception is that in the genus Phlebovirus the 5’-half of the S RNA is not transcribed directly; instead, the mRNA for the NSs protein is transcribed after synthesis of full-length complementary RNA. The RNA polymerase also has endonuclease activity, cleaving 5’- methylated caps from host mRNAs and adding these to viral mRNAs to prime transcription (cap snatching). After primary viral mRNA transcription and translation, replication of the virion RNA occurs and a second round of transcription begins, amplifying the genes that encode structural

21. necessary for virion synthesis. Virions mature by budding through intracytoplasmic vesicles associated with the Golgi complex and are released by the transport of vesicles through the cytoplasm and release by exocytosis from the apical and/or basolateral plasma membranes. Akabane virus is best known for its teratogenic effects in ruminants, with seasonal epizootics of reproductive loss (embryonic/fetal mortality, abortion) and congenital arthrogryposis and hydranencephaly being well described in cattle in Australia, Japan, and Israel.

22. Akabane virus infection of non-pregnant ruminants typically is inapparent, but infection of pregnant cattle or sheep can lead to one of two outcomes: death of the fetus and abortion, or birth, sometimes premature, of progeny with congenital defects. Affected fetuses characteristically have extensive cavitary defects of the central nervous system (hydranencephaly) and severe musculoskeletal abnormalities (arthrogryposis), thus abortion or birth is often accompanied by dystocia. Fetuses born with hydranencephaly (“bubble brain”) usually are unable to stand after birth; those less severely affected may manifest marked

23. incoordination and a variety of other neurologic deficits. Although the vectors of Akabane virus is believed that is transmitted in Japan by Aedes spp. and Culex spp. mosquitoes, and in Australia by the midge, Culicoides brevitarsis. As Akabane virus is an arthropod-borne virus infection, its transmission is seasonal. After the bite of an infected mosquito, the virus infects the pregnant ruminant without producing clinical signs, and reaches the fetus from the maternal circulation. The most severe fetal lesions in cattle result from infection at

24. 3–4 months of gestation, and earlier in sheep and goats, when the central nervous system is developing. Fetal infection results in both encephalomyelitis and polymyositis, and virus replication within the developing central nervous system leads to destruction of the developing brain and subsequent hydranencephaly. In general, the earlier the virus reaches the developing cerebrum, the worse the teratogenic defect. Arthrogryposis, is characterized by muscular atrophy and the abnormal fixation of several limbs, usually in flexion.

25. In enzootic areas, diagnosis of Akabane virus infection was suggested by clinical, pathologic, and epidemiologic observations (seasonal occurrence), but most often by gross pathologic examination. Diagnosis is confirmed by the detection of a specific neutralizing antibody in serum collected from aborted fetuses or from newborn calves, kids, or lambs before ingestion of colostrum. Alternatively, diagnosis may be made by detecting an increase in antibody titer between paired maternal sera. Virus is difficult or impossible to isolate after calves, kids, or lambs are born, but can be recovered from

26. the placenta, fetal brain, or muscle of animals taken before normal parturition by cesarean section or after slaughter of the dam. Virus isolation is carried out in cell cultures or by intracerebral inoculation of suckling mice. Epizootics of Rift Valley fever in sheep, goats, and cattle have occurred at regular intervals in southern and eastern African countries from the time when intensive livestock husbandry was introduced at the beginning of the 20th century.

27. In an epizootic, virus is amplified in wild and domesticanimals by many species of Culex and other Aedesmosquitoes. These mosquitoes become very numerousafter heavy rains or when improper irrigation techniquesare used; they feed indiscriminately on viremic sheep andcattle (and humans). A very high level of viremia is maintainedfor 3–5 days in infected sheep and cattle, allowingmany more mosquitoes to become infected. In its epizootic cycles, Rift Valley fever virus is also spread mechanically by fomites and by blood and tissues of infected animals.

28. Infected sheep have a very high level of viremia, and transmission at the time of abortion, via contaminated placentae and fetal and maternal blood, is a particular problem. Abattoir workers and veterinarians are often infected directly. The capacity of Rift Valley fever virus to be transmitted without the involvement of an arthropod vector raises concerns over the possibility for its importation into nonenzootic areas via contaminated materials, animal products, viremic humans, or non-livestock animal species.

29. Once it is established in previously free regions, it would be difficult or impossible to eradicate the virus, because of the many mosquito species capable of efficient virus transmission and the phenomenon of transovarial transmission. The incubation period is short in animals infected with Rift Valley fever virus—typically less than 3 days. Infected sheep develop fever, inappetence, mucopurulent nasal discharge, and bloody diarrhea.

30. Rift Valley fever virus is zoonotic and causes an important human disease that occurs coincidentally with outbreaks. The human disease begins after a very short incubation period (2–6 days) with fever, severe headache, chills, “back-breaking” myalgia, diarrhea, vomiting, and hemorrhages. The clinical disease lasts 5 days, followed by a prolonged convalescence and complete recovery. A small percentage develop more severe diseases. The case-fatality rate is about 1–2%. Vaccination can be used to prevent human disease, especially those most at risk, such as veterinarians and abattoir workers.

33. Research and diagnostic procedures with Rift Valley fever virus are restricted to certain national laboratories. The virus is a BioSafety Level 3 pathogen, and must be handled in the laboratory under strict biocontainment conditions. Rift Valley fever virus replicates rapidly and to very high titer in target tissues. After entry by mosquito bite, percutaneous injury, or through the oropharynx via aerosols, there is an incubation period of 30–72 hours, during which virus invades the parenchyma of the liver and lymphoreticular organs.

34. Extensive hepatocellular necrosis is common in terminally affected sheep. The spleen is enlarged and there are gastrointestinal and subserosal hemorrhages. Encephalitis, evidenced by neuronal necrosis and perivascular inflammatory infiltration, is a late event that occurs in a small proportion of animals surviving the hepatic infection. Control is based primarily on livestock vaccination, but vector control is also used during outbreaks. In addition, environmental management can be a useful control strategy, including assessment of the risk of creating new larval habitats in enzootic areas.

35. Attenuated-virus Rift Valley fever vaccines produced in mouse brain and in embryonated eggs are effective and inexpensive for use in sheep, but they cause abortions in pregnant ewes. Inactivated-virus vaccines produced in cell cultures avoid the problem of abortion, but are expensive. Both types of vaccines have been produced in Africa in large quantities, but to be fully effective vaccines must be delivered in a systematic way to entire animal populations,

36. preferably on a regular schedule before the start of the mosquito season, or at least at the first indications of virus activity.