Attenuation In Mice Research Articles

Salmonella enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis ( S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially Δ phoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and Δ phoP also in pigs. In addition, Δ rpoS and Δ phoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the Δ rpoS and Δ phoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.

Identification and Characterization of Interferon-Induced Proteins That Inhibit Alphavirus Replication

Alpha/beta interferon (IFN-alpha/beta) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-alpha/beta can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized had potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.

Mutations in Influenza Virus M1 CCHH, the Putative Zinc Finger Motif, Cause Attenuation in Mice and Protect Mice against Lethal Influenza Virus Infection

Mutations in CCHH, the putative zinc finger motif, apparently do not play an important role in virus replication in MDCK cells in culture (E. K.-W. Hui, K. Ralston, A. K. Judd, and D. P. Nayak, J. Gen. Virol. 84:3105-3113, 2003). In this report, however, we demonstrate that the CCHH motif plays a critical role in virulence in mice and that some CCHH mutants are highly attenuated in BALB/c mice. Some of the mutant viruses replicated the least in mice lungs, induced little or no lung lesions, and caused highly reduced morbidity and mortality. Furthermore, growth patterns of mutant viruses in different cell lines (MDCK, MLE12, 3LL, A549, and 293T) varied. Mutant viruses that were attenuated in mice also grew poorly in mouse and human cells in culture. However, wild-type (WT) and all mutant viruses replicated to the same titer in MDCK (canine) cells or embryonated chicken eggs. Attenuation in mice correlated with reduced growth in mouse cells in culture, suggesting that potential attenuation in a given host can be predicted from the growth characteristics of the virus in cultured cells (preferably lung cells) from the same species. In challenge experiments, mice immunized by infection with attenuated mutant viruses were fully protected from lethal challenge with WT virus. In summary, the replication and attenuating properties of these mutants suggest that the CCHH motif provides a critical determinant for virulence in mouse and that mutations in the CCHH motif yield potential vaccine candidates for the development of live species-specific attenuated influenza virus vaccines.

Direct repeat‐mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis

Francisella tularensis, the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella. In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat-mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage-like cell lines. Importantly, the pilin-negative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.

A non‐RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis

The Snm secretion system is a crucial virulence determinant of Mycobacterium tuberculosis. Genes encoding all known components of this alternative secretion pathway are clustered at the same genetic locus, known as RD1. Here, we show that a mutant M. tuberculosis strain containing a transposon insertion in the Rv3615c gene, which is situated outside the RD1 locus, results in loss of Snm secretion. Complementation analysis revealed that both Rv3615c and the downstream gene Rv3614c are required for Snm secretion. Thus, we have renamed the two genes snm9 and snm10 respectively. The snm9::Tn mutant phenocopies bona fide snm mutants, exhibiting attenuation in mice, macrophage growth defects and failure to suppress cytokine induction. Furthermore, yeast two-hybrid analysis revealed a physical interaction between Snm10 and Snm7 (Rv3882c), suggesting that Snm10 may function in complex with other Snm proteins during secretion. Thus, snm9 and snm10 are the first genes located outside the RD1 locus identified as critical components of Snm secretion. These data indicate that Snm secretion consists of an elaborate network of interactions that likely arose from multiple duplication events during the evolution of M. tuberculosis.

Role for Nucleotide Excision Repair in Virulence of Mycobacterium tuberculosis

Mutations in Mycobacterium tuberculosis uvrB result in severe sensitivity to acidified nitrite, a source of nitric oxide (6). In this study, we show that a uvrB mutant is exquisitely sensitive to UV light but not to several sources of reactive oxygen species in vitro. Furthermore, a uvrB mutant was attenuated in mice as judged by an extension of life span. Attenuation in mice was partially reversed by genetic inactivation of nitric oxide synthase 2 (iNOS) and almost completely reversed in mice lacking both iNOS and phagocyte oxidase. Thus, a gene predicted to encode a key element of DNA repair is required for resistance of M. tuberculosis to both reactive nitrogen and reactive oxygen species in mice.

Growth, virulence, and immunogenicity of Listeria monocytogenes aro mutants.

Mutants of Listeria monocytogenes with deletions in genes of the common branch of the biosynthesis pathway leading to aromatic compounds were constructed as possible virulence-attenuated carrier strains for protein antigens or vaccine DNA. aroA, aroB, and in particular aroE mutants showed strongly reduced growth rates in epithelial cells and even in rich culture media. The metabolism of the aro mutants under these conditions was predominantly anaerobic. Aerobic metabolism and a wild-type growth rate were, however, regained upon the addition of vitamin K2, suggesting that the aro mutants are deficient in oxidative respiration due to the lack of menaquinone. Replication of the aro mutants in the host cell's cytosol and cell-to-cell spread were drastically slowed down, and all aro mutants showed high virulence attenuation in mice, i.e., the 50% lethal dose in BALB/c mice was increased at least 10(4)-fold for the aroA, aroB, and aroA/B mutants and >10(5)-fold for the aroE mutant compared to the parent strain. Nevertheless, mice preimmunized with aro mutant bacteria elicited good T-cell response and full protection against a subsequent challenge with the virulent wild-type strain. A total of 5 x 10(6) aroA, aroB, and aroA/B mutant bacteria were sufficient to obtain a protective T-cell response, while 5 x 10(8) aroE or aroA/E mutants were necessary to achieve comparable numbers of antigen-specific T cells. These numbers were well tolerated without causing any signs of disease, indicating that Listeria strains with deletions in genes of the basic branch of the aromatic amino acid pathway could be useful vaccine carriers for inducing T-cell immunity.

Alteration of DNA adenine methylase (Dam) activity in Pasteurella multocida causes increased spontaneous mutation frequency and attenuation in mice.

Pasteurella multocida is one of the primary bacterial pathogens associated with bovine respiratory disease (BRD) complex. Relatively few virulence factors of P. multocida have been characterized, and there is a need for improved vaccines for prevention of BRD. In other Gram-negative species, DNA adenine methylase (Dam) regulates the expression of virulence genes, and appropriate expression of Dam is required for virulence. In this study, the authors cloned and sequenced the P. multocida A1 dam gene and demonstrated that it is able to restore Dam function in an Escherichia coli dam mutant. When P. multocida dam was placed under the control of a constitutively expressed promoter on a plasmid, it caused an increased spontaneous mutation rate in P. multocida. In addition, the plasmid-mediated alteration of Dam production in P. multocida caused it to be highly attenuated in mice. These findings indicate that appropriate expression of Dam is required for virulence of P. multocida, which is believed to be the first report that Dam is required for virulence of a species in the Pasteurellaceae. Therefore, Dam may function as a virulence gene regulator in the Pasteurellaceae, similar to previously reported findings from other Gram-negative species.

West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy.

A candidate live attenuated vaccine strain was constructed for West Nile virus (WN), a neurotropic flavivirus that has recently emerged in the U.S. Considerable attenuation for mice was achieved by chimerization with dengue virus type 4 (DEN4). The genes for the structural premembrane and envelope proteins of DEN4 present in an infectious cDNA clone were replaced by the corresponding genes of WN strain NY99. Two of 18 cDNA clones of a WN/DEN4 chimera yielded full-length RNA transcripts that were infectious when transfected into susceptible cells. The two infectious clones shared a motif in the transmembrane signal domain located immediately downstream of the NS2B-NS3 protease cleavage site that separates the DEN4 capsid protein and the WN premembrane protein of the chimera. This motif, Asp and Thr at a position 3 and 6 amino acids downstream of the cleavage site, respectively, was not present in the 16 noninfectious cDNA clones. The WN/DEN4 chimera was highly attenuated in mice compared with its WN parent; the chimera was at least 28,500 times less neurovirulent in suckling mice inoculated intracerebrally and at least 10,000 times less virulent in adult mice inoculated intraperitoneally. Nonetheless, the WN/DEN4 chimera and a deletion mutant derived from it were immunogenic and provided complete protection against lethal WN challenge. These observations provide the basis for pursuing the development of a live attenuated WN vaccine.

Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes

The influenza A virus pandemic of 1918-1919 resulted in an estimated 20-40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site.

The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3'-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine.

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3.

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) cells. The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype. Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.

The two amino acid substitutions in the L protein of cpts530/1009, a live-attenuated respiratory syncytial virus candidate vaccine, are independent temperature-sensitive and attenuation mutations

cpts530/1009 is a live-attenuated, temperature-sensitive ( ts) RSV vaccine candidate that was shown previously to be attenuated for seronegative humans. It was generated by two rounds of chemical mutagenesis: first, a partially attenuated, cold-passaged ( cp), non- ts RSV mutant ( cpRSV) was mutagenized to yield the ts derivative cpts530, and then cpts530 was mutagenized to yield cpts530/1009, which is more ts. Previous nucleotide (nt) sequence analysis of cpts530 showed that it has a single nt change compared to cpRSV that results in an amino acid substitution at residue 521 in the L protein. Reverse genetics confirmed that this mutation is responsible for the ts phenotype of cpts530. Here, determination of the complete 15,222-nt sequence of cpts530/1009 identified a single change compared to cpts530, namely a point mutation at nt 12002, which results in a methionine-to-valine substitution at amino acid 1169 in the L protein. The contribution of the 1009 mutation to the level of temperature sensitivity and attenuation exhibited by cpts530/1009 was evaluated by its introduction alone or with the 530 and cp mutations into the full-length cDNA clone of wild-type (wt) RSV. Subsequent analysis of infectious viruses recovered from the mutant cDNAs indicated that (i) the 1009 mutation indeed was a ts mutation and the level of temperature sensitivity specified by the 1009 mutation was less than that specified by the 530 mutation, (ii) the 530 and 1009 mutations each contributed to attenuation in the upper respiratory tract of mice and their effects were additive, (iii) viruses bearing the 1009 mutation were more attenuated in the lower respiratory tract of mice than viruses bearing the 530 mutation and (iv) the combination of the 530 and 1009 mutations in the cpRSV background resulted in the same level of temperature sensitivity and attenuation in mice as that observed for the biologically-derived cpts530/1009 mutant. These data show that the genetic basis of the attenuation and temperature sensitivity of the cpts530/1009 candidate vaccine virus is the sum of the contributions of seven identified amino acid substitutions, i.e. the 5 cpRSV mutations, the 530 mutation and the 1009 mutation.

A Single Nucleotide Substitution in the Transcription Start Signal of the M2 Gene of Respiratory Syncytial Virus Vaccine Candidatecpts248/404 Is the Major Determinant of the Temperature-Sensitive and Attenuation Phenotypes

Respiratory syncytial virus (RSV)cpts248/404 is a live-attenuated, temperature-sensitive (ts) vaccine candidate derived from cold-passagedcpRSV by two rounds of chemical mutagenesis and biological selection. Previous sequence analysis showed that these two steps introduced three single nucleotide substitutions into thecpRSV parent. Two of these occurred within the coding sequence for the L protein, and each resulted in a single amino acid substitution: Gln-831-Leu (248 mutation) and Asp-1183-Glu (404-L mutation). The third mutation resulted in a nucleotide substitution at position 9 of thecis-acting gene start signal of the M2 gene (404-M2 mutation). In the present study, the genetic basis of attenuation ofcpts248/404 was defined by the introduction of each of these mutations (singly or in combination) into a full-length cDNA clone ofcpRSV. Recombinant RSV derived from each mutant cDNA was analyzed to determine the contribution of each mutation to thetsand attenuation phenotypes of the virus. This analysis showed that the 248 mutation specifies a significant reduction of plaque formation at 38°C and is responsible for an intermediate level of attenuation in mice. In contrast, the 404-L mutation did not contribute to thetsor attenuation phenotype alone or in combination with other mutations and is thus an incidental change. Unexpectedly, the 404-M2 mutation alone specified complete restriction of plaque formation at 37°C and a high level of attenuation in mice. This indicates that the level of temperature sensitivity and attenuation ofcpts248/404 can be attributed primarily to the 404-M2 mutation. Thus thecpts248/404 virus contains a set oftsand non-tsattenuating mutations, which likely accounts for its genetic stability. The recombinant version of this virus, rA2cp248/404, was phenotypically indistinguishable fromcpts248/404 and represents a background into which additional mutations can be introduced as needed to obtain the desired level of attenuation for successful immunization of the very young human infant.

Spontaneous and Engineered Deletions in the 3′ Noncoding Region of Tick-Borne Encephalitis Virus: Construction of Highly Attenuated Mutants of a Flavivirus

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.

Vaccination of mice with attenuated mutants ofListeria monocytogenes: requirement for induction of macrophage la expression

Isogenic mutant strains ofListeria monocytogenesdemonstrating graded attenuation in mice were used to analyse the correlation between bacterial virulence, ability to induce class II MHC (la) molecules in antigen presenting cells and ability to vaccinate against secondary infection. The mutants used differed only in the amino acid sequence of the thiol–activated hemolysin, Listeriolysin O (LLO). The results indicate thatL. monocytogenesmutants of reduced virulence have the potential to act as vaccines only if they are sufficiently persistant to induce la expression in antigen presenting cells. The findings also suggest that specific mutagenesis of virulence factors, including LLO, could provide an approach for creatingListeria monocytogenesstrains with potential for use as attenuated live vaccines.

Lethality of PE2 incorporation into Sindbis virus can be suppressed by second-site mutations in E3 and E2

Sindbis virions contain two glycoproteins, E1 and E2. E2 is produced initially as a precursor, PE2, from which the amino-terminal 64 amino acids are cleaved by a cellular protease at a late stage in virion maturation. A mutation at E2 position 1 (Arg to Asn) was placed into Sindbis virus AR339 by site-directed mutagenesis of a full-length AR339 cDNA clone, pTRSB, to produce pTRSB-N. The mutation created a signal for N-linked glycosylation immediately adjacent to the PE2 cleavage signal. Virions derived from pTRSB-N were glycosylated at E2 position 1, and they quantitatively incorporated PE2 in place of E2. When pTRSB-N transcripts were electroporated into BHK-21 cells, TRSB-N particles were released with nearly normal efficiency; however, the specific infectivity of TRSB-N particles was very low. Analysis of seven infectious revertants of TRSB-N revealed that reversion was linked to (i) mutations that eliminated the signal for N-linked glycosylation and thus restored the PE2 cleavage phenotype or (ii) conservation of the PE2 cleavage defect combined with incorporation of suppressor mutations in E3 or E2. The genotype of each revertant was reconstructed in the genetic background of TRSB-N, and each reverting mutation also was replaced individually into the genetic background of wild-type virus (TRSB). Each PE2-containing revertant was attenuated in newborn CD-1 mice and replicated poorly in cultured mosquito cells (C6/36). Reverting mutations in the genetic background of TRSB did not reduce virulence in mice or growth in mosquito cells, suggesting that the phenotypes of attenuation in mice and reduced growth in mosquito cells were linked to failure of PE2 cleavage and not to the reverting mutations themselves.

Molecular determinants of the virulence and infectivity of California serogroup bunyaviruses.

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.

A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice

The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.