Molecular analysis of dengue virus attenuation after serial passage in primary dog kidney cells.

A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2A⌬30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2A⌬30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (⌬30) in the 3 untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 was utilized to generate attenuating mutations which may be useful in further attenuation of the 2A⌬30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and a panel of 1,248 clones were isolated. Twenty ts mutant viruses were identified that were ts in both simian Vero and human liver HuH-7 cells (n ‫؍‬ 13) or only in HuH-7 cells (n ‫؍‬ 7). Each of the 20 ts mutant viruses possessed an attenuation phenotype, as indicated by restricted replication in the brains of 7-day-old mice. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and nonstructural genes as well as in the 5 and 3 UTRs, with more than one change occurring, in general, per mutant virus. A ts mutation in the NS3 protein (nucleotide position 4995) was introduced into a recombinant DEN4 virus possessing the ⌬30 deletion, thereby creating rDEN4⌬30-4995, a recombinant virus which is ts and more attenuated than rDEN4⌬30 virus in the brains of mice. We are assembling a menu of attenuating mutations that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus.The mosquito-borne dengue (DEN) viruses (serotypes 1 to 4 [DEN1 to -4]) are members of the Flavivirus genus and contain a single-stranded positive-sense RNA genome of approximately 10,600 nucleotides (nt) (43). The genome organization of DEN viruses is 5Ј-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3Ј (where UTR is untranslated region, C is capsid, prM is premembrane, E is envelope, and NS is nonstructural) (10, 48). A single viral polypeptide is cotranslationally processed by viral and cellular proteases, generating three structural proteins (C, M, and E) and seven NS proteins. The disease burden associated with DEN virus infection has increased over the past several decades in tropical and semitropical countries. Annually, there are an estimated 50 to 100 million cases of DEN fever (DF) and 500,000 cases of the more severe and potentially lethal DEN hemorrhagic fever/DEN shock syndrome (DHF/DSS) (19).The site of viral replication in DEN virus-infected humans and the pathogeneses of DF and DHF/DSS are still incompletely understood (26). In humans, DEN virus infects lymphocytes (32, 55), macrophages (21, 52), dendritic cells (34, 63), and hepatocytes (36, 39). The liver is clearly involved in DEN virus infection of humans, as indicated by the occurrence of transient elevations in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the ...

Section: Discussionsupporting

confidence: 67%

“…This DEN2 virus vaccine strain has been used to generate a chimeric virus with C, prM, and E genes from DEN1 (24). In separate studies, the sequence of the DEN1 virus vaccine strain 45AZ5 PDK-27 was determined and compared to those of parental viruses, but the mutations responsible for attenuation have not yet been identified (47).…”

Section: Discussionmentioning

confidence: 99%

Chemical Mutagenesis of Dengue Virus Type 4 Yields Mutant Viruses Which Are Temperature Sensitive in Vero Cells or Human Liver Cells and Attenuated in Mice

Blaney

Johnson

Firestone

et al. 2001

“…In humans, DEN4⌬30 present in serum of vaccinees retained its ⌬30 mutation, confirming its genetic stability in vivo (5). The attenuating mutations in other existing dengue live, attenuated vaccine candidates are based on less-stable point mutations (4,15). Third, since the ⌬30 mutation is common to all of the four viruses of the tetravalent vaccine, recombination between the any of the four vaccine serotypes would not lead to loss of the attenuating mutation or reversion to a wt phenotype.…”

mentioning

confidence: 83%

A Live, Attenuated Dengue Virus Type 1 Vaccine Candidate with a 30-Nucleotide Deletion in the 3′ Untranslated Region Is Highly Attenuated and Immunogenic in Monkeys

Whitehead

Falgout

Hanley

et al. 2003

139

The ⌬30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3 untranslated region (3 UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1⌬30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4⌬30 vaccine candidate. rDEN1⌬30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3 UTR, and both vaccines were highly protective against challenge with wt DEN1. Both rDEN1⌬30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1⌬30 nor rDEN1mutF showed restricted replication following intrathoracic inoculation in the mosquito Toxorhynchites splendens. The ability of the ⌬30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the ⌬30 mutation into wt DEN viruses belonging to each of the four serotypes.There are four serotypes of dengue virus (dengue virus type 1 [DEN1], DEN2, DEN3, and DEN4) that annually cause an estimated 50 to 100 million cases of dengue fever and 500,000 cases of the more severe form of dengue virus infection known as dengue hemorrhagic fever/dengue shock syndrome (6). Dengue virus is widely distributed throughout the tropical and semitropical regions of the world, and the number of dengue virus infections continues to increase due to the expanding range of its Aedes aegypti mosquito vector. A vaccine is not available for the control of dengue disease despite its importance as a reemerging disease. The goal of immunization is to protect against dengue virus disease by the induction of a long-lived neutralizing antibody response against each of the four serotypes. Simultaneous protection against all four serotypes is required, since an increase in disease severity can occur in persons with preexisting antibodies to a heterotypic dengue virus. Such immunization can be achieved economically with a live, attenuated virus vaccine.Dengue viruses are positive-sense RNA viruses belonging to the Flavivirus genus. The approximately 11,000-base genome contains a single open reading frame encoding a polyprotein which is processed by proteases of both viral and cellular origin into three structural proteins (C, prM, and E) and at least seven nonstructural (NS) proteins. Both ends of the dengue virus genome contain an untranslated region (UTR), and the overall genome organization is 5Ј-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3Ј. The 3Ј UTR is nearly 400 bases in length and is predicted to contain several stemloop structures conserved among dengue virus serotypes (3,9,14,17). One such stem-loop structure, identified as TL2 in the proposed secondary structure of the 3Ј UTR (14), was previously removed by deletion of 30 nucleotides from the DEN4 genome (3Ј nucleotides 172 to 143) (12) and has subsequently been designated as the ⌬30 mutation (5). The resulting virus, rDEN...

“…Investigation of this chimeric-virus approach for novel flavivirus vaccines has been promising, with the development of experimental vaccines for tick-borne encephalitis virus and dengue and Japanese enceph-alitis viruses (5,12,18,19,(42)(43)(44). With increasing knowledge of genetic determinants of virulence for dengue virus and other flaviviruses (6,7,29,34,36,46), this approach in theory allows for modification of the vaccine to reduce or eliminate unacceptable neurovirulence or other undesirable tropisms. In the present study, we describe the properties of two YFV/dengue virus chimeras which we have characterized in cell culture and tested as experimental vaccines in laboratory mice.…”

mentioning

confidence: 99%

Yellow Fever Virus/Dengue-2 Virus and Yellow Fever Virus/Dengue-4 Virus Chimeras: Biological Characterization, Immunogenicity, and Protection against Dengue Encephalitis in the Mouse Model

Chambers

Liang

Droll

et al. 2003

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10 5 PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.