Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus.

Tarbatt, Catherine J.; Glasgow, Gwendoline M.; Mooney, Dorothy A.; Sheahan, B. J.; Atkins, Gregory J.

doi:10.1099/0022-1317-78-7-1551

Cited by 23 publications

(28 citation statements)

References 32 publications

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“…The present analysis of the nonstructural genome, however, clearly established that these are two essentially different SFV strains, although they both derive from the original Bradish isolate (4). The nsp regions of these two strains showed major differences in the sequence per se; also, whereas in rA774 the avirulence was primarily mediated by nsp3, with less pronounced contributions by nsp1 or nsp2 and with the structural genome being fully functional, in CA7 the attenuating domains were found distributed in both structural and nonstructural parts of the genome (46). However, in contrast to rA774, the nsp3-nsp4 region of CA7 was not found to contain major attenuating determinants.…”

Section: Discussionmentioning

confidence: 72%

“…The nsp4 gene of rA774 exhibited 99.1 and 97.4% nucleotide identity with CA7 and SFV4, respectively, with the deduced amino acid sequences differing by only two amino acids in both (Table 1). Interestingly, in both rA774 and CA7 (46), there was an adenine residue in nsp4 (nt 7350 and 7371, respectively) immediately preceding the transcription start site of 26S RNA, while SFV4 had a guanidine at this site (44). As this could have been critical for transcription efficiency, we replaced in rA774 the whole 26S RNA-encoding region, including the preceding 28 nt, with the equivalent SFV4 sequence.…”

Section: Resultsmentioning

confidence: 99%

“…We obtained this strain from H. E. Webb (London, United Kingdom), and it has since then been passaged several times in MBA-13 cells and subsequently plaque-purified three times on the same cells for later molecular analyses and construction of the full-length genome. The CA7 strain analyzed previously by Tarbatt et al (46) was separated from the Bradish isolate after the initial seven passages in mouse brain (4) and passaged once more in mouse brain before three plaque-purifications and five or fewer passages in BHK cells (G.…”

Section: Methodsmentioning

confidence: 99%

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Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

Tuittila

Santagati²,

Röyttä

et al. 2000

J Virol

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Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.Semliki Forest virus (SFV) is an enveloped positive-stranded RNA virus of the family Togaviridae. The SFV prototype was isolated in 1942 from a pool of mosquitoes in Uganda (40). The A7(74) strain (4) is a derivative of an SFV strain isolated from mosquitoes in Mozambique in 1959 (26). Several strains of SFV, such as L10 (4) and SFV4 (24), cause lethal encephalitis in mice of all ages, leading to death of the animals in a few days (3, 11), whereas A7(74) infection of adult mice is asymptomatic per se but leads to axonal demyelination mediated by CD8 ϩ T cells (2). However, the severity of A7(74) infection is strictly age dependent, being lethal for neonatal mice less than 2 weeks old (8, 27), probably because the strain is capable of virion formation in propagating neurons, unlike in mature neurons (27).SFV nonstructural proteins (nsP) are translated as a polyprotein (nsP1234) from the genomic 42S RNA, and they form essential components of viral RNA replication and transcription complexes (14). The nsP1 protein is a methyl-and guanylyltransferase (1, 18), whereas the nsP2 is a proteinase (45) and nucleoside triphosphatase (33). The nsp3 gene product is a phosphoprotein, and it has been proposed to function together with nsP1 in anchoring the replication complex proteins to cytoplasmic membrane structures (30, 31). In Sindbis virus (SIN), p123 a...

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Section: Discussionmentioning

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

Tuittila

Santagati²,

Röyttä

et al. 2000

J Virol

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show abstract

“…In suckling mouse brain and in culture, many infected brain cells rapidly undergo apoptosis (3, 17), whereas in immunocompromised adult mice, virus can persist in CNS cells without any apparent cell death for many months (4, 12). Age-related neurovirulence may be related to changes in the propensity of CNS cells to undergo apoptosis on infection (2, 3).The SFV4, A7, and A7(74) strains have been cloned and sequenced (14,16,31,32,39,40,41). Between the strains there are multiple changes scattered throughout the genome (41).…”

mentioning

confidence: 99%

“…The SFV4, A7, and A7(74) strains have been cloned and sequenced (14,16,31,32,39,40,41). Between the strains there are multiple changes scattered throughout the genome (41).…”

mentioning

confidence: 99%

A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus

Fazakerley

Boyd

Mikkola

et al. 2002

J Virol

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The replicase protein nsP2 of Semliki Forest virus (SFV) has a 648 RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.The molecular and cell biology of the alphavirus Semliki Forest virus (SFV) have been extensively studied, as has the pathogenesis of SFV infection in laboratory mice (6, 10). Generally, the virus is neuroinvasive, and extraneural inoculation results in a high-titer plasma viremia with infection of central nervous system (CNS) cells by virus passage across the bloodbrain barrier (24,25,11,36). Strains of SFV vary in their neurovirulence in adult mice. Irrespective of the route of inoculation, adult mice infected with the L10, V13, or prototype strains or virus derived from the prototype molecular clone SFV4 rapidly succumb to a fulminant encephalitis, whereas adult mice infected with the A7 or A7(74) strain develop a subclinical encephalitis (8,27). The nominally avirulent strains spread more slowly in the CNS, and host responses prevent widespread fatal infection and eventually eliminate infectious virus (7, 11), though they generate lesions of immune-mediated demyelination (12,37). In SJL mice these lesions are active for many months (9, 35). In contrast to their differences in adult mice, all strains of SFV are virulent in embryonic, neonatal, and suckling mice (5,11,13,15). For the adultavirulent strains A7 and A7 (74) there is a sharp age-related transition (22,23). In suckling mouse brain and in culture, many infected brain cells rapidly undergo apoptosis (3, 17), whereas in immunocompromised adult mice, virus can persist in CNS cells without any apparent cell death for many months (4, 12). Age-related neurovirulence may be related to changes in the propensity of CNS cells to undergo apoptosis on infection (2, 3).The SFV4, A7, and A7(74) strains have been cloned and sequenced (14,16,31,32,39,40,41). Between the strains there are multiple changes scattered throughout the genome (41). The difference in virulence between SFV4 and A7 or A7(74) appears to be polygenic, and to date, changes in the E2 gene, the 5Ј untranslated region, and the nsP3 gene have been shown to be determinants of virulence between these particular strains (32,33,40,41). As with all viruses, there will al...

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The Significance of the 3′-Nontranslated Region and E2 Amino Acid Mutations in the Virulence of Semliki Forest Virus in Mice

et al. 1998

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We have recently shown that the 3'-nontranslated region (3'-NTR) of the avirulent Semliki Forest virus A7(74) [SFVA7(74)] contains a unique sequence of 101 nucleotides and five repetitive nucleotide units whereas the 3'-NTR of the neurovirulent SFV4 has only two repeats. A chimeric virus was constructed by replacing the entire 3'-NTR of the SFV4 clone with the A7(74) 3'-NTR. The hybrid replicated efficiently in the central nervous system (CNS) of adult Balb/c mice and, similarly to SFV4, led to high mortality after intraperitoneal inoculation. In contrast, another chimeric virus, CME2, containing the E2 gene of the avirulent SFVA7(74) virus in the SFV4 clone was recently shown to be avirulent for mice. Several derivatives with single-site or a constellation of amino acid mutations were constructed. Two single-site E2 mutants, Val37lle and Asn212Ser, displayed an attenuated phenotype in mice with mortality reduced from 90 to 48 and 43%, respectively. None of the multiple site mutants were significantly attenuated. Adult female mice showed a greater resistance to SFV infection than male mice. The SFV hybrid viruses, CM3NTR and CME2, reached the CNS similarly to the parental viruses, but the single-site E2 mutants were only sporadically found in the CNS. We conclude that in mice the 3'-NTR does not play a significant role in the pathogenesis of Semliki Forest virus and that specific E2 amino acid mutations reduce the virulence, especially in female mice. The results additionally suggest that individual amino acid mutations in the E2 glycoprotein affect the efficiency of migration into the CNS.

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Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus.

Cited by 23 publications

References 32 publications

Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus

The Significance of the 3′-Nontranslated Region and E2 Amino Acid Mutations in the Virulence of Semliki Forest Virus in Mice

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