Flavivirus genomic RNAs contain a conserved stem-loop structure within the 3'-noncoding region (3'-SL). One cDNA-derived DEN2 virus that contained a 7-bp substitution of WN genomic 3'-SL nt sequences for the analogous nt sequences of wt DEN2 3'-SL was severely restricted for growth in mosquito cells but replicated like wt D2 virus in monkey kidney cells (L Zeng, B Falgout, L Markoff [1998] J Virol 72:7510-22). The mosquito cell growth-restricted mutant (DEN2mutF) was additionally characterized. In summary, these data suggested that the DEN2mutF virus had a unique host-range restriction for replication in mosquito cells and in adult mosquitoes. Last year, the deletion and substitution mutations that differentiated DEN2mutF with respect to the wt D2NGC genome were introduced into an infectious full-length DNA derived from the genome of DEN1 strain WestPac virus. This """"""""DEN1mutF"""""""" virus grew to wt titers in LLCMK2 cells but did not replicate in cultured mosquito cells, recapitulating the phenotype of the original DEN2mutF virus. Last year, DEN1mutF virus and its wt human virulent parent virus were tested in Rhesus macaques for their immunogenicity and ability to cause viremia. Monkeys are the best available animal model for prediction of attenuation of dengue viruses in humans; they do not become detectably ill from dengue infection, but levels of viremia in monkeys have been shown to correlate directly with virulence in humans. DEN1mutF virus had all properties of a suitable vaccine candidate; it was highly immunogenic, inducing similar titers of neutralizing antibodies in sera compared to wt, and severely impaired in its ability to cause viremia. The DEN1mutF virus is of particular interest to vaccine developers, since DEN1 virus is the most common cause of serious dengue illness in SE Asia. This year, monkeys that were immunized with a single dose of DEN1mutF virus were challenged at one year and again at 17 months after vaccination (different monkeys were challenged in each instance) with the human virulent wt DEN1 parent virus. All DEN1mutF-immunized monkeys had an anamnestic immune response to the challenge virus, and none of them became viremic. In contrast, 5 of 6 control monkeys exhibited high levels of viremia in response to the challenge dose of virus. A revertant of DEN1mutF virus was also created by serial passage in mosquito cells. After three blind passages of 8 days each, or a total of 24 days, a fully reverted DEN1mutF virus was detected, DEN1mutFRev. The complete genomes of DEN1mutF and DEN1mutFR viruses were sequenced and compared to that of the wt parent D1 virus. DEN1mutF RNA contained one mutation in the envelope (E) protein gene sequence, in addition to the expected mutations in the 3'-SL, compared to wt. D1mutFR RNA contained three additional mutations vs D1mutF, in NS1, NS5, and in the 3'-SL. Any or all of these mutations could affect RNA synthesis, the presumptive defect in mutF viruses when grown in mosquito cells, based on earlier data. The mutation in E was corrected in the context of D1mutF infectious DNA and then shown to have no effect on the phenotype of D1mutF virus. This year, a set of DEN1mutant virus genomes was constructed, containing all possible combinations of the 3 mutations detected in the DEN1mutFR genome compared to that of DEN1mutF. We currently await results which will determine which of the mutations were required for the observed phenotypic reversion of DEN1mutFR virus. Additional """"""""MutantF"""""""" viruses. LVVD collaborates with virologists at WRAIR with respect to their dengue vaccine development program, via an IAG. Previously, the WRAIR vaccine candidates were created by serial passage of dengue viruses in Primary Dog Kidneys cells (PDK). WRAIR has been testing PDK-derived candidate dengue vaccines under IND for several years already. They have been unsuccessful in identifying a suitably attenuated D1 vaccine candidate; wt D1 virus that had been passaged 20 times in PDK cells was insufficiently attenuated, whereas PDK-27 dengue-1 virus was over-attenuated (did not induce an immune response). LVVD had created an infectious DNA clone of the dengue-1 PDK-20 (under-attenuated) virus. The mutF mutational changes were inserted into the cloned DNA by standard techniques using homologous recombination in yeast. Subsequently, D1/PDK-20/mutF virus was generated by transfection of in vitro synthesized full-length RNA into LLCMK2 cells. This mutant virus exhibited the same host-range restricted phenotype as that observed for D1mutF and D2mutF viruses. Subsequently, the human virulent wt D1 virus, D1mutF virus, and D1/PDK-20/mutF virus were tested in a large monkey study. The wt parent virus induced viremia in all monkeys, whereas viremia was significantly reduced in monkeys infected with either of the two mutant viruses (suggesting that both viruses would be attenuated in humans and confirming the results described above for D1mutF). All monkeys had a D1-specific immune response, but the response in D1mutF-infected monkeys was more vigorous than that seen in D1/PDK-20/mutF-infected monkeys. This suggested that the D1/PDK-20/mutF virus might be more attenuated in humans than the D1mutF virus. To resolve this question, Phase 1 clinical trials are being planned for each of the mutant viruses, to compare their levels of attenuation and immunogenicity in humans. This year, scientists in LVVD created DEN3 and DEN4 mutant viruses containing the mutant F set of mutations, and these viruses were found to be host range-restricted mutants, like DEN2mutF virus and DEN1mutF virus. The level of attenuation of the former viruses in animals has yet to be determined, but this finding shows that it may be possible to construct a tetravalent mutant F dengue vaccine. LVVD also collaborates with the Laboratory of Infectious Diseases, NIAID, in relation to the science of dengue vaccine development. LID has a D4 vaccine virus which has a 30-nt deletion in the 3'-noncoding region of the genome, in a region that is entirely upstream from the 3'-SL. This virus, D4d30, is currently in Phase I clinical trials and under IND. LVVD and LID have collaborated to """"""""swap"""""""" mutations. Thus the two labs have created two new mutant viruses: D1d30 virus, which has the analogous deletion mutation found in the D4d30 virus, and D4mutF virus, which has the mutF mutations introduced into the genome of the D4 strain 814669 virus utilized by LID scientists to create their D4d30 candidate vaccine virus. This year (2002), the levels of attenuation of D1d30 and D1mutF viruses were compared in monkeys. The D1d30 virus was found to be slightly more attenuated than D1mutF virus, compared to wt DEN1 strain WP virus. West Nile virus is a known cause of fever/arthralgia/rash syndrome and encephalitis in N Africa and the middle East. However, no known case of WNV infection of humans had occurred in N America until the past few years, when isolated cases of WN encephalitis have been documented in NYC, NJ and MD. This year, clinically evident infection with WNV spread throughout the eastern two-thirds of the US, with a dramatic increase in deaths. We obtained a full-length DNA copy of the WNV strain Wengler genome from Dr. Vladimir Yamshchikov at the University of Virginia. Dr. Li Yu created a large number of mutants in the WNV genomic DNA and evaluated the growth phenotypes of the WN mutant viruses that were thus derived in LLCMK2, BHK, vero, and C6/36 cells. He initially created WN mutants that were the """"""""mirror image"""""""" of the D2 3'-SL mutants created by Dr. Lingling Zeng. In this way, we hoped to test the hypotheses related to 3'-SL function in viral replication that arose from the original study of the 3'-SL in the context of the D2 genome, where chimeric D2/WN 3?-SLs were substituted for the wt D2 3'-SL. Findings in brief are as follows: For WN, substitution of the """"""""bottom"""""""" half of the WN 3'-SL by DEN2 nt sequences resulted in a viable mutant virus that replicated efficiently in both monkey and mosquito cells. This was in contrast to results previously obtained in the context of the D2 genome. A second WN mutant virus, containing a substitution of D2 nt sequences for the wt WN nt sequences in the """"""""top"""""""" half of the WN 3'-SL was non-viable. A revertant of this virus displayed a host range restricted phenotype in mosquito cells, a la DEN2mutF virus, and its genome contained spontaneous mutations in the region of the 3'-SL involved in mutF. Comparison of the results for WNV with those earlier obtained with DEN2 V suggests that conserved bulges in the long stem of the 3'-SL , at the boundary of the top and bottom portions previously defined, are required for virus replication. Mouse neurovirulence studies are planned in the immediate future to determine whether any of the 3'-SL mutant viruses have potential merit as vaccine candidates.
