Lower Respiratory Tract Of Hamsters Research Articles

New conjugates based on N4-hydroxycytidine with more potent antiviral efficacy in vitro than EIDD-2801 against SARS-CoV-2 and other human coronaviruses

The spread of COVID-19 continues due to genetic variation in SARS-CoV-2. Highly mutated variants of SARS-CoV-2 have an increased transmissibility and immune evasion. Due to the emergence of various new variants of the virus, there is an urgent need to develop broadly effective specific drugs for therapeutic strategies for the prevention and treatment of COVID-19. Molnupiravir (EIDD-2801, MK-4482), is an orally bioavailable ribonucleoside analogue of β-D-N4-hydroxycytidine (NHC), has demonstrated efficacy against SARS-CoV-2 and was recently approved for COVID-19 treatment. To improve antiviral potency of NHC, we developed a panel of NHC conjugates with lipophilic vectors and ester derivatives with amino- and carboxylic-acids. Most of the synthesized compounds had comparable or higher (2–20 times) antiviral activity than EIDD-2801, against different lineages of SARS-CoV-2, MERS-CoV, seasonal coronaviruses OC43 and 229E, as well as bovine coronavirus. For further studies, we assessed the most promising compound in terms of activity, simplicity and cost of synthesis - NHC conjugate with phenylpropionic acid (SN_9). SN_9 has shown high efficacy in prophylactic, therapeutic and transmission models of COVID-19 infection in hamsters. Importantly, SN_9 profoundly inhibited virus replication in the lower respiratory tract of hamsters and transgenic mice infected with the Omicron sublineages XBB.1.9.1, XBB.1.16 and EG.5.1.1. These data indicate that SN_9 represents a promising antiviral drug candidate for COVID-19 treatment, and NHC modification strategies deserve further investigation as an approach to develop prodrugs against various coronaviruses.

Molnupiravir inhibits SARS-CoV-2 variants including Omicron in the hamster model.

The recent emergence of the SARS-CoV-2 Omicron variant of concern (VOC), which contains a heavily mutated spike protein capable of escaping preexisting immunity, identifies a continued need for interventional measures. Molnupiravir (MK-4482), an orally administered nucleoside analog, has demonstrated efficacy against earlier SARS-CoV-2 lineages and was recently approved for SARS-CoV-2 infections in high-risk adults. Here, we assessed the efficacy of MK-4482 against the earlier Alpha, Beta, and Delta VOCs and Omicron in the hamster COVID-19 model. Omicron replication and associated lung disease in vehicle-treated hamsters was reduced compared with replication and lung disease associated with earlier VOCs. MK-4482 treatment inhibited virus replication in the lungs of hamsters infected with Alpha, Beta, or Delta VOCs. Importantly, MK-4482 profoundly inhibited virus replication in the upper and lower respiratory tract of hamsters infected with the Omicron VOC. Consistent with its mutagenic mechanism, MK-4482 treatment had a more pronounced inhibitory effect on infectious titers compared with viral RNA genome load. Histopathologic analysis showed that MK-4482 treatment caused a concomitant reduction in the level of lung disease and viral antigen load in infected hamsters across all VOCs examined. Together, our data indicate the potential of MK-4482 as an effective antiviral against known SARS-CoV-2 VOCs, especially Omicron, and likely future SARS-CoV-2 variants.

SARS-CoV-2 Omicron variant causes mild pathology in the upper and lower respiratory tract of hamsters

Since its discovery in 2019, multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been identified. This study investigates virus spread and associated pathology in the upper and lower respiratory tracts of Syrian golden hamsters at 4 days post intranasal SARS-CoV-2 Omicron infection, in comparison to infection with variants of concern (VOCs) Gamma and Delta as well as ancestral strain 614 G. Pathological changes in the upper and lower respiratory tract of VOC Omicron infected hamsters are milder than those caused by other investigated strains. VOC Omicron infection causes a mild rhinitis with little involvement of the olfactory epithelium and minimal lesions in the lung, with frequent sparing of the alveolar compartment. Similarly, viral antigen, RNA and infectious virus titers are lower in respiratory tissues of VOC Omicron infected hamsters. These findings demonstrate that the variant has a decreased pathogenicity for the upper and lower respiratory tract of hamsters.

Intranasal administration of a single dose of a candidate live attenuated vaccine derived from an NSP16-deficient SARS-CoV-2 strain confers sterilizing immunity in animals.

Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA, adenoviral vector and inactivated vaccines fail to induce. Here, we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene, which encodes 2′-O-methyltransferase, is catalytically disrupted by a point mutation. This virus, designated d16, was severely attenuated in hamsters and transgenic mice, causing only asymptomatic and nonpathogenic infection. A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters, thus preventing viral spread in a contact-based transmission model. It also robustly stimulated humoral and cell-mediated immune responses, thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model. The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants. Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice. Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain, to which new features might be introduced to improve safety, transmissibility, immunogenicity and efficacy.

Characterization of a new SARS-CoV-2 variant that emerged in Brazil

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a key role in viral infectivity. It is also the major antigen stimulating the host's protective immune response, specifically, the production of neutralizing antibodies. Recently, a new variant of SARS-CoV-2 possessing multiple mutations in the S protein, designated P.1, emerged in Brazil. Here, we characterized a P.1 variant isolated in Japan by using Syrian hamsters, a well-established small animal model for the study of SARS-CoV-2 disease (COVID-19). In hamsters, the variant showed replicative abilities and pathogenicity similar to those of early and contemporary strains (i.e., SARS-CoV-2 bearing aspartic acid [D] or glycine [G] at position 614 of the S protein). Sera and/or plasma from convalescent patients and BNT162b2 messenger RNA vaccinees showed comparable neutralization titers across the P.1 variant, S-614D, and S-614G strains. In contrast, the S-614D and S-614G strains were less well recognized than the P.1 variant by serum from a P.1-infected patient. Prior infection with S-614D or S-614G strains efficiently prevented the replication of the P.1 variant in the lower respiratory tract of hamsters upon reinfection. In addition, passive transfer of neutralizing antibodies to hamsters infected with the P.1 variant or the S-614G strain led to reduced virus replication in the lower respiratory tract. However, the effect was less pronounced against the P.1 variant than the S-614G strain. These findings suggest that the P.1 variant may be somewhat antigenically different from the early and contemporary strains of SARS-CoV-2.

Chimeric Bovine/Human Parainfluenza Virus Type 3 Expressing Respiratory Syncytial Virus (RSV) F Glycoprotein: Effect of Insert Position on Expression, Replication, Immunogenicity, Stability, and Protection against RSV Infection

A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109-114, 2012; C.-F. Yang et al., Vaccine 31:2822-2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity. The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this knowledge has general application to the development and clinical evaluation of other mononegavirus vectors and vaccines.

Generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters

Human metapneumovirus (HMPV) causes acute respiratory tract illness primarily in young children, immunocompromised individuals and the elderly. Vaccines would be desirable to prevent severe illnesses in these risk groups. Here, we describe the generation and evaluation of cold-passage (cp) temperature-sensitive (ts) HMPV strains as vaccine candidates. Repeated passage of HMPV at low temperatures in Vero cells resulted in the accumulation of mutations in the viral genome. Introduction of these mutations in a recombinant HMPV by reverse genetics resulted in a ts-phenotype, judged on the decreased shut-off temperature for virus replication in vitro. As an alternative approach, three previously described cp-respiratory syncytial virus (cp-HRSV) mutations were introduced in a recombinant HMPV, which also resulted in a low shut-off temperature in vitro. Replication of these ts-viruses containing either the cp-HMPV or cp-HRSV mutations was reduced in the upper respiratory tract (URT) and undetectable in the lower respiratory tract (LRT) of hamsters. Nevertheless, high titres of HMPV-specific antibodies were induced by both ts-viruses. Upon immunization with the ts-viruses, the LRT of hamsters were completely protected against challenge infection with a heterologous HMPV strain, and URT viral titres were reduced by 10 000-fold. In conclusion, we provide proof-of-principle for two candidate live-attenuated HMPV vaccines that induce cross-protective immunity to prevent infection of the LRT in Syrian golden hamsters. Further mapping of the molecular determinants of attenuation of HMPV should be the subject of future studies.

A Severe Acute Respiratory Syndrome Coronavirus That Lacks the E Gene Is Attenuated In Vitro and In Vivo

A deletion mutant of severe acute respiratory syndrome coronavirus (SARS-CoV) has been engineered by deleting the structural E gene in an infectious cDNA clone that was constructed as a bacterial artificial chromosome (BAC). The recombinant virus lacking the E gene (rSARS-CoV-DeltaE) was rescued in Vero E6 cells. The recovered deletion mutant grew in Vero E6, Huh-7, and CaCo-2 cells to titers 20-, 200-, and 200-fold lower than the recombinant wild-type virus, respectively, indicating that although the E protein has an effect on growth, it is not essential for virus replication. No differences in virion stability under a wide range of pH and temperature were detected between the deletion mutant and recombinant wild-type viruses. Although both viruses showed the same morphology by electron microscopy, the process of morphogenesis seemed to be less efficient with the defective virus than with the recombinant wild-type one. The rSARS-CoV-DeltaE virus replicated to titers 100- to 1,000-fold lower than the recombinant wild-type virus in the upper and lower respiratory tract of hamsters, and the lower viral load was accompanied by less inflammation in the lungs of hamsters infected with rSARS-CoV-DeltaE virus than with the recombinant wild-type virus. Therefore, the SARS-CoV that lacks the E gene is attenuated in hamsters, might be a safer research tool, and may be a good candidate for the development of a live attenuated SARS-CoV vaccine.

Generation of recombinant human parainfluenza virus type 1 vaccine candidates by importation of temperature-sensitive and attenuating mutations from heterologous paramyxoviruses.

Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.

Construction of a live-attenuated bivalent vaccine virus against human parainfluenza virus (PIV) types 1 and 2 using a recombinant PIV3 backbone

PIV1 and PIV2 are important agents of pediatric respiratory tract disease. We are developing live-attenuated vaccines against these viruses. We earlier constructed a PIV3/PIV1 antigenic chimeric virus, designated rPIV3-1, in which the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of wild type rPIV3 were replaced by their PIV1 counterparts. In the present study, rPIV3-1 was used as a vector to express the HN protein of PIV2 to generate a single virus capable of inducing immunity to both PIV1 and PIV2. The PIV2 HN open reading frame was expressed from an extra gene cassette, under the control of PIV3 cis-acting transcription signals, inserted between the F and HN genes of rPIV3-1. The recombinant derivative, designated rPIV3-1.2HN, was readily recovered and exhibited a level of temperature sensitivity and in vitro growth similar to that of its parental virus. The rPIV3-1.2HN virus was restricted in replication in both the upper and lower respiratory tracts of hamsters compared with rPIV3-1, identifying an attenuating effect of the PIV2 HN insert in hamsters. rPIV3-1.2HN elicited serum antibodies to both PIV1 and PIV2 and induced resistance against challenge with wild type PIV1 or PIV2. Thus, rPIV3-1.2HN, a virus attenuated solely by the insertion of the PIV2 HN gene, functioned as a live attenuated bivalent vaccine candidate against both PIV1 and PIV2.

Mutations in the C, D, and V Open Reading Frames of Human Parainfluenza Virus Type 3 Attenuate Replication in Rodents and Primates

Human parainfluenza virus type 3 (HPIV3) is a single-stranded negative-sense RNA virus belonging to the Respirovirus genus of the Paramyxoviridae family in the order Mononegavirales. The P gene encodes at least four proteins, including the C protein, which is expressed from an open reading frame (ORF) that overlaps the P ORF, and the D protein, which is encoded when the P ORF is fused to the D ORF by transcriptional editing. The P mRNA also contains a third ORF for the V protein, although it is unclear how or whether this ORF is accessed. We have used recombinant DNA technology to recover five mutant viruses that either interrupt or alter the C, D, and V ORFs. In one mutant virus, rC-KO, expression of the C protein was abrogated by changing the start codon from methionine to threonine and introducing two stop codons at amino acid positions 7 and 26 of the C ORF. In a second mutant virus, rF164S, a point mutation was introduced into the C ORF changing amino acid position 164 from phenylalanine (F) to serine (S), which corresponds to the F170S mutation described in the C protein of Sendai virus (Itoh et al., J. Gen. Virol. 78, 3207–3215). rC-KO was significantly attenuated in vitro and in vivo (rodents and primates), whereas rF164S was attenuated only in vivo. Interestingly, the rF164S mutant was more attenuated in the upper than in the lower respiratory tract of hamsters and monkeys. This pattern is the converse of that seen with temperature-sensitive attenuating mutations, and thus inclusion of this novel mutation in a recombinant live-attenuated vaccine candidate might prove useful in reducing residual virulence in the upper respiratory tract. Both rC-KO and rF164S conferred protection against challenge with wild-type HPIV3. In three other viruses, the D and V ORFs were interrupted singly or in combination. Although interruption of the D and V ORFs individually did not affect virus replication in vitro or in vivo, interruption of both together attenuated replication in vivo. These results indicate that the C, D, and V proteins of HPIV3 each has a role in virus replication in vitro, in vivo, or both, and define mutations that might be useful for the development of a vaccine against HPIV3.

Identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated cold-passage 45 (cp45) human parainfluenza virus 3 candidate vaccine.

The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29). cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for the ts, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, and att phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3' N region (i.e., including the leader, N GS, and N coding changes) contributes to the ts phenotype. A recombinant virus containing all the cp45 mutations except those in L was more ts than cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3' N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non-ts mutations. Attenuating ts mutations are located in the L protein, and non-ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non-ts attenuating mutations in cp45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.

Three amino acid substitutions in the L protein of the human parainfluenza virus type 3 cp45 live attenuated vaccine candidate contribute to its temperature-sensitive and attenuation phenotypes.

Studies were initiated to define the genetic basis of the temperature-sensitive (ts), cold adaptation (ca), and attenuation (att) phenotypes of the human parainfluenza virus type 3 (PIV3) cp45 live attenuated vaccine candidate. Genetic data had previously suggested that the L polymerase protein of cp45, which contains three amino acid substitutions at positions 942, 992, and 1558, contributed to its temperature sensitivity (R. Ray, M. S. Galinski, B. R. Heminway, K. Meyer, F. K. Newman, and R. B. Belshe, J. Virol. 70:580-584, 1996; A. Stokes, E. L. Tierney, C. M. Sarris, B. R. Murphy, and S. L. Hall, Virus Res. 30:43-52, 1993). To study the individual and aggregate contributions that these amino acid substitutions make to the ts, att, and ca phenotypes of cp45, seven PIV3 recombinant viruses (three single, three double, and one triple mutant) representing all possible combinations of the three amino acid substitutions were recovered from full-length antigenomic cDNA and analyzed for their ts, att, and ca phenotypes. None of the seven mutant recombinant PIVs was cold adapted. The substitutions at L protein amino acid positions 992 and 1558 each specified a 105-fold reduction in plaque formation in cell culture at 40 degrees C, whereas the substitution at position 942 specified a 300-fold reduction. Thus, each of the three mutations contributes individually to the ts phenotype. The triple recombinant which possesses an L protein with all three mutations was almost as temperature sensitive as cp45, indicating that these mutations are the major contributors to the ts phenotype of cp45. The three individual mutations in the L protein each contributed to restricted replication in the upper or lower respiratory tract of hamsters, and this likely contributes to the observed stability of the ts and att phenotypes of cp45 during replication in vivo. Importantly, the recombinant virus possessing L protein with all three mutations was as restricted in replication as was the cp45 mutant in both the upper and lower respiratory tracts of hamsters, indicating that the L gene of the cp45 virus is a major attenuating component of this candidate vaccine.

Recovery of Infectious Human Parainfluenza Virus Type 3 from cDNA

Infectious HPIV3 was produced by the intracellular coexpression of four plasmid-borne cDNAs. These separately encoded a complete HPIV3 genome (negative-sense), the HPIV3 nucleocapsid protein N, the phosphoprotein P, and the polymerase protein L. The cDNA-encoded HPIV3 genome differed from the JS wildtype (wt) strain of HPIV3 used in its construction by seven point mutations: four of these are silent mutations in the HN or L gene coding regions that serve as markers of a cDNA-derived virus, two were introduced to create an amino acid substitution that ablates an epitope recognized by the HN-specific monoclonal neutralizing antibody 423/6, and the remaining point mutation results in an incidental amino acid substitution in the HN protein at amino acid position 263. The four plasmids were transfected into HEp-2 cell monolayers and their expression was driven by T7 RNA polymerase supplied by a vaccinia virus recombinant. The titer of virus present in the harvested transfection supernatant was low (<5 PFU/ml), and the recovered recombinant virus (rJS) retained each of the seven mutations present in the cDNA from which it was derived. Despite the introduced and incidental mutations, rJS retained the wt phenotypes as regards replication at elevated temperaturein vitroand efficient replication in the upper and lower respiratory tract of hamsters. rJS was also recovered from a cDNA encoding a complete antigenome (positive-sense) with slightly greater efficiency than from the negative-sense construct. The ability to generate infectious HPIV3 from cDNA should greatly enhance our ability to develop new live-attenuated parainfluenza virus vaccines, including chimeric PIV1 and PIV2 vaccines, and to understand the genetic basis of attenuation of PIV3 candidate vaccines.

Sequential addition of temperature-sensitive missense mutations into the PB2 gene of influenza A transfectant viruses can effect an increase in temperature sensitivity and attenuation and permits the rational design of a genetically engineered live influenza A virus vaccine

We have previously described a strategy for the recovery of a synthetic influenza A virus wild-type (wt) PB2 gene (derived from influenza A/Ann Arbor/6/60 [AA] virus) into an infectious virus. It was possible to introduce an attenuating temperature-sensitive (ts) mutation at amino acid residue 265 of the AA wt PB2 gene and to rescue this mutant gene into infectious virus. Application of this new technology to influenza A virus vaccine development requires that multiple attenuating mutations be introduced to achieve a satisfactorily attenuated virus that retains the attenuation (att) phenotype following replication in vivo. In this report, we demonstrate that putative ts mutations at amino acids 112, 556, and 658 each indeed specify the ts and att phenotypes. Each of these mutations was introduced into a cDNA copy of the AA mutant mt265 PB2 gene to produce three double-mutant PB2 genes, each of which was rescued into an infectious virus. In general, the double-mutant PB2 transfectant viruses were more ts and attenuated in the lower respiratory tracts of hamsters than the single-mutant transfectant viruses, and the ts phenotype of two of three double-mutant PB2 transfectant viruses was stable even after prolonged replication in the upper respiratory tracts of immunocompromised mice. Two triple-mutant PB2 transfectant viruses with three predicted amino acid substitutions resulting from five nucleotide substitutions in the cDNA were then generated. The triple-mutant PB2 transfectant viruses were more ts and more attenuated than the double-mutant PB2 transfectant viruses. These results indicate that sequential introduction of additional ts mutations into the PB2 gene can yield mutants that exhibit a stepwise increase in temperature sensitivity and attenuation compared with the preceding mutant(s) in the series. Furthermore, the level of temperature sensitivity of the transfectant viruses correlated significantly with the level of attenuation of these viruses in hamsters. Although the triple-mutant PB2 transfectant viruses were attenuated in hamsters, intranasal administration of these viruses elicited a vigorous serum hemagglutination-inhibiting antibody response, and this was associated with resistance of the lower respiratory tract to subsequent wt virus challenge. These observations suggest the feasibility of using PB2 reverse genetics to generate a live influenza A virus vaccine donor strain that contains three attenuating mutations in one gene. It is predicted that reassortant viruses derived from such a donor virus would have the properties of attenuation, genetic stability, immunogenicity, and protective efficacy against challenge with wt virus.

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Attenuation and Phenotypic Stability of Influenza B/Texas/l/84 Cold-Adapted Reassortant Virus: Studies in Hamsters and Chimpanzees

The temperature sensitivity, level of attenuation, and phenotypic stability of an influenza B/Texas/1/84 X B/Ann Arbor/1/66 cold-adapted (ca) reassortant virus that received the RNA segments that encode the hemagglutinin and neuraminidase surface glycoproteins from the B/Texas/84 wild-type virus and the remaining six RNA segments from the B/Ann Arbor/66 ca donor virus were evaluated. In comparison to wild-type virus, the ca reassortant was restricted in replication in the upper and lower respiratory tracts of hamsters and chimpanzees. This clearly demonstrated that the six transferable RNA segments of the B/Ann Arbor/66 ca donor virus specify the attenuation phenotype for animals with a 37 degrees C core body temperature. In addition, the ca virus retained the temperature-sensitive (ts) phenotype after replication in hamsters and chimpanzees. To further evaluate its phenotypic stability, the ca reassortant virus was isolated after 6-15 d of replication in hamsters that were immunosuppressed with cyclophosphamide, and the isolates were tested for their temperature sensitivity in vitro and for their level of replication in immunocompetent hamsters. The isolated viruses retained their ts and attenuation phenotypes even after prolonged replication in vivo. Thus, the B/Ann Arbor/66 ca donor virus can transfer the desired properties of attenuation and phenotype stability to its reassortants. The findings support the continued evaluation of ca reassortant influenza B virus vaccines in humans, including fully susceptible children.