Cold-Adapted Variants of Influenza A Virus: Evaluation in Adult Seronegative Volunteers of A/Scotland/840/74 and A/Victoria/3/75 Cold-Adapted Recombinants Derived from the Cold-Adapted A/Ann Arbor/6/60 Strain

Influenza A/Scotland/74 (H3N2) and A/Victoria/75 (H3N2) cold-adapted (ca) recombinant viruses, prepared by mating the A/Ann Arbor/6/60 (H2N2) ca donor virus and influenza A wild-type virus, were evaluated in adult seronegative volunteers (serum hemagglutination-inhibiting antibody titer, 相似文献   

Mutational analysis of the West Nile virus NS4B protein

West Nile virus NS4B is a small hydrophobic nonstructural protein approximately 27 kDa in size whose function is poorly understood. Amino acid substitutions were introduced into the NS4B protein primarily targeting two distinct regions; the N-terminal domain (residues 35 through 60) and the central hydrophobic domain (residues 95 through 120). Only the NS4B P38G substitution was associated with both temperature-sensitive and small-plaque phenotypes. Importantly, this mutation was found to attenuate neuroinvasiveness greater than 10,000,000-fold and lower viremia titers compared to the wild-type NY99 virus in a mouse model. Full genome sequencing of the NS4B P38G mutant virus revealed two unexpected mutations at NS4B T116I and NS3 N480H (P38G/T116I/N480H), however, neither mutation alone was temperature sensitive or attenuated in mice. Following incubation of P38G/T116I/N480H at 41 °C, five mutants encoding compensatory substitutions in the NS4B protein exhibited a reduction in the temperature-sensitive phenotype and reversion to a virulent phenotype in the mouse model.     

Attenuation of the recombinant human parainfluenza virus type 3 cp45 candidate vaccine virus is augmented by importation of the respiratory syncytial virus cpts530 L polymerase mutation.

A phenylalanine to leucine mutation at position 521 in the L polymerase of cpts530, a live-attenuated respiratory syncytial virus (RSV) cold-passaged (cp), temperature-sensitive (ts) candidate vaccine, specifies the ts and attenuation (att) phenotypes. Sequence alignment of this region in the L proteins of several distantly related paramyxoviruses revealed that this phenylalanine is conserved. Using reverse genetics, the analogous phenylalanine at position 456 in the L protein of wild-type PIV3 was mutagenized to leucine (F456L). The resulting virus, designated r456(L), was ts (40 degrees C shut-off temperature of plaque formation), and its replication in the upper, but not the lower, respiratory tract of hamsters was 10-fold reduced compared with that of the recombinant wild-type PIV3 (rwt). Thus the phenylalanine to leucine mutation specified a similar level of temperature sensitivity and attenuation in two distantly related paramyxoviruses. We next sought to determine whether the addition of this mutation to the L protein of two rPIV3 candidate vaccine viruses, one bearing the three cp45 ts missense mutations in the L protein (rcp45(L)) and the other bearing all 15 cp45 mutations (rcp45), would further attenuate the viruses in vivo. Each rcp45 derivative to which the F456L mutation was added exhibited an increased level of temperature sensitivity. Furthermore rcp45(L)-456 and rcp45-456 were 100- to 1000-fold more restricted in replication in hamsters than their rcp45(L) and rcp45 parents. Despite the high level of restriction of replication in hamsters, immunization with rcp45-456 induced a moderate level of resistance to replication of PIV3 challenge virus. In contrast to the highly restricted replication observed in hamsters, rcp45-456 was only fivefold more restricted in the respiratory tract of chimpanzees than rcp45 and induced a comparable, moderate to high level of PIV3-specific serum antibodies. rcp45 and rcp45-456 viruses isolated from chimpanzees throughout the 2-week course of replication maintained the level of temperature sensitivity of their respective input viruses, illustrating their phenotypic stability. Thus the acquisition of the F456L mutation by the cp45 virus resulted in a small, incremental increase in its level of attenuation, indicating its possible usefulness in the fine tuning of the level of attenuation of the cp45 vaccine candidate. The ability to transfer mutations identified in heterologous paramyxoviruses, which in this case represent different subfamilies, greatly enhances our ability to rapidly develop novel parainfluenza virus candidate vaccines.     

Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation

The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the vesicular stomatitis virus large polymerase protein essential for mRNA cap methyltransferase activity. J. Virol. 79, 13373-13384], a new region between L amino acids 1450-1481 was identified which is critical for mRNA cap methylation.     

Relationship of genotype of recombinants of influenza A/Hong Kong/68-ts-1[E]virus used as live virus vaccines to virulence in humans.

Influenza A/Hong Kong/68-ts-1[E] virus is a temperature-sensitive mutant developed for use as a live virus vaccine (B. R. Murphy, E. G. Chalhub, S. R. Nusinoff, J. Kasel, and R. M. Chanock, J. Infect. Dis. 128:479--487, 1973). This virus and temperature-sensitive recombinants derived by mating it with A/Udorn/72, A/Georgia/74, or A/Victoria/75 wild-type virus have been administered to volunteers in clinical trials on the assumption that the ts-1[E] temperature-sensitive genetic lesions on a polymerase gene (P3) and on the nucleoprotein gene (NP) would determine a satisfactory and reproducible level of attentuation regardless of the genetic constitution of ts-1[E] recombinants at other loci (B. R. Murphy, D. D. Richman, S. B. Spring, and R. M. Chanock, Postgrad. Med. 52:381--388, 1976). In this paper, the parental origin of genes in the ts-1[E] recombinants was determined by using the technique of polyacrylamide gel electrophoresis of virion ribonucleic acid segments in the presence of a denaturing agent (urea). When tested in individuals who lacked immunity to hemagglutinin antigen, attenuation of the ts-1[E] recombinants appeared to correlate with inheritance of the ts-1[E] temperature-sensitive genes at the P3 and NP loci and with the level of preinfection neuraminidase immunity. There was no evidence that other genes from the ts-1[E] donor virus played a role in attenuation.     

Evaluation of bovine, cold-adapted human, and wild-type human parainfluenza type 3 viruses in adult volunteers and in chimpanzees.

In an attempt to evaluate the level of attenuation of live parainfluenza type 3 virus (PIV3) vaccine candidates, we compared the responses of partially immune adult volunteers inoculated intranasally with 10(6) to 10(7) 50% tissue culture infective dose (TCID50) of bovine PIV3 (n = 18) or cold-adapted (ca) PIV3 (n = 37) with those of 28 adults administered 10(6) to 10(7) TCID50 of wild-type PIV3. The candidate vaccine viruses and the wild-type virus were avirulent and poorly infectious for these adults even though all of them had a low level of nasal antibodies to PIV3. To determine whether the ca PIV3 was attenuated, we then administered 10(4) TCID50 of ca PIV3 (cold-passage 12) or wild-type PIV3 intranasally and intratracheally to two fully susceptible chimpanzees, respectively, and challenged the four primates with wild-type virus 1 month later. Compared with wild-type virus, which caused upper respiratory tract illness, the ca PIV3 was highly attenuated and manifested a 500-fold reduction in virus replication in both the upper and lower respiratory tracts of the two immunized animals. Despite restriction of virus replication, infection with ca PIV3 conferred a high level of protective immunity against challenge with wild-type virus. The ca PIV3 which had been passaged 12 times at 20 degrees C did not retain its ts phenotype. These findings indicate that ca PIV3 may be a promising vaccine candidate for human beings if a passage level can be identified that is genetically stable, satisfactorily attenuated, and immunogenic.     

Avian retrovirus integration protein: structure-functional analysis of viable mutants

A replication-competent avian retrovirus mutant, containing a single amino acid substitution at amino acid residue 115 in the 3' endonuclease (IN) region of the polymerase (pol) gene, was characterized. DNA transfection experiments demonstrated that the mutant virus exhibited a delayed growth phenotype at 41 degrees while replicating efficiently at 35 degrees. Examination of virus-infected cells at the molecular level demonstrated that the mutant virus at either temperature was capable of synthesizing viral DNA as efficiently as wild-type Rous sarcoma virus, strain Prague A. This result suggested that the same mutation, which was also present in the IN moeity of the polymerase beta polypeptide, did not affect DNA synthesis. Further analyses demonstrated that at either temperature the mutant virus integrated its DNA at about 10-20% of wild-type level, although possibly less efficiently at 41 degrees than at 35 degrees. The mutation at residue 115 (Pro to Ser) appeared to lower the ability of IN to function in the integration of viral DNA relative to wild-type virus. No definitive conclusion could be made as to whether IN in this mutant possessed a temperature-sensitive lesion which caused the observed replication defect at 41 degrees.     

Cold-passaged human parainfluenza type 3 viruses contain ts and non-ts mutations leading to attenuation in rhesus monkeys.

Cold-passaged (CP) mutants derived from the JS strain of wild type wt parainfluenza type 3 virus (PIV3) are being evaluated as candidate live virus vaccines. The wt virus was serially passaged 45 times at low temperature and mutant clones with the cold-adapted (CA), temperature-sensitive (ts), and attenuation (ATT) phenotypes were selected following passage levels 12, 18 and 45 (cp12, cp18, and cp45). The cp45 virus was more ts than the cp12 or cp18 mutants, although all 3 mutant viruses were clearly attenuated in rhesus monkeys compared to wild type virus. The mean peak titers of the cp12 and cp18 viruses administered by the intratracheal route were at least 6000-fold lower than JSwt in both the upper and lower respiratory tracts. The cp45 virus was not recovered from monkeys administered virus by the i.t. route alone; however, when the cp45 virus was administered by the intranasal route, it replicated in the upper respiratory tract to a level comparable to that of the cp12 and cp18 viruses, but continued to be markedly restricted in the lower respiratory tract. These data indicate that the cp12 and cp18 viruses contain predominantly non-ts attenuating mutations whereas the cp45 mutant has both non-ts and ts attenuating mutations. Each of the CP mutants induced a high level of resistance to wild type virus challenge. Also, the ATT phenotype of the cp12 and cp18 viruses as measured in rhesus monkeys was stable after replication in chimpanzees or humans, respectively, although the ts phenotype was not. Based on its greater level of temperature sensitivity in vitro and its greater degree of attenuation in rhesus monkeys, the cp45 virus appears to be the most promising vaccine candidate for humans.     

Phenotypic properties resulting from directed gene segment reassortment between wild-type A/Sydney/5/97 influenza virus and the live attenuated vaccine strain

Widespread use of a live-attenuated influenza vaccine (LAIV) in the United States (licensed as FluMist) raises the possibility that vaccine viruses will contribute gene segments to the type A influenza virus gene pool. Progeny viruses possessing new genotypes might arise from genetic reassortment between circulating wild-type (wt) and vaccine strains, but it will be difficult to predict whether they will be viable or exhibit novel properties. To begin addressing these uncertainties, reverse-genetics was used to generate 34 reassortant viruses derived from wt influenza virus A/Sydney/5/97 and the corresponding live vaccine strain. The reassortants contained different combinations of vaccine and wt PB2, PB1, PA, NP, M, and NS gene segments whereas all strains encoded wt HA and NA glycoproteins. The phenotypes of the reassortant strains were compared to wt and vaccine viruses by evaluating temperature-sensitive (ts) plaque formation and replication attenuation (att) in ferrets following intranasal inoculation. The results demonstrated that the vaccine virus PB1, PB2, and NP gene segments were dominant when introduced into the wt A/Sydney/5/97 genetic background, producing recombinant viruses that expressed the ts and att phenotypes. A dominant attenuated phenotype also was evident when reassortant strains contained the vaccine M or PA gene segments, even though these polypeptides are not temperature-sensitive. Although the vaccine M and NS gene segments typically are not associated with temperature sensitivity, a number of reassortants containing these vaccine gene segments did exhibit a more restricted ts phenotype. Overall, no reassortant strains were more virulent than wt, and in fact, 33 of the 34 recombinant viruses replicated less efficiently in infected ferrets. These results suggest that genetic reassortment between wt and vaccine strains is unlikely to produce viruses having novel properties that differ substantially from either progenitor, and that the likely outcome of reassortment will be attenuated viruses.     

Identification and functional analysis of an interaction between domains of the 126/183-kDa replicase-associated proteins of tobacco mosaic virus

The Tobacco mosaic virus (TMV) 126-kDa and read-through 183-kDa replicase-associated proteins have been shown to interact [Watanabe, T., Honda, A., Iwata, A., Ueda, S., Hibi, T., Ishihama, A. (1999). J. Virol. 73, 2633-2640]. To identify and investigate the sequence required for this interaction, five segments covering different portions of the 126/183-kDa open reading frame, including the methyl-transferase, intervening region (IR), helicase-like (HEL), and polymerase domains, were screened via the yeast two-hybrid system against a library of TMV protein segments. Only one specific interaction between the HEL domain clone and a TMV library clone, IRnHEL, encoding the C-terminal half of the IR and the N-terminal portion of the HEL domain was identified. Sequence and deletion analysis revealed that the interacting clones share a region containing the helicase NTP-binding motif and that this region was essential for the interaction. To determine the functional significance of this interaction, mutants of the HEL domain segment that conferred a temperature-sensitive (ts) defect in the yeast interaction were identified and cloned into a recombinant TMV strain. Of the five selected mutants, three (V823I/S824N/V1042M, A877V, V1087I) produced a ts replication phenotype in protoplasts while the other two (A1073V, T884I) abolished TMV replication at both the permissive and the nonpermissive temperatures. An additional mutation, K839S, designed to disrupt the shared NTP-binding motif, nearly abolished the two-hybrid interaction and prevented virus replication, suggesting that NTP-binding and/or the structure of this motif is a contributing factor in the interaction. Taken together, these results provide support for an interaction between TMV replicase-associated proteins that involves specific structural features of the HEL and IR domains.     

Recombinant parainfluenza virus 5 (PIV5) expressing the influenza A virus hemagglutinin provides immunity in mice to influenza A virus challenge

Parainfluenza virus type 5 (PIV5), formerly known as simian virus 5 (SV5), is a non-segmented negative strand RNA virus that offers several advantages as a vaccine vector. PIV5 infects many cell types causing little cytopathic effect, it replicates in the cytoplasm of infected cells, and does not have a DNA phase in its life cycle thus avoiding the possibility of introducing foreign genes into the host DNA genome. Importantly, PIV5 can infect humans but it is not associated with any known human illness. PIV5 grows well in tissue culture cells, including Vero cells, which have been approved for vaccine production, and the virus can be obtained easily from the media. To test the feasibility of using PIV5 as a live vaccine vector, the hemagglutinin (HA) gene from influenza A virus strain A/Udorn/72 (H3N2) was inserted into the PIV5 genome as an extra gene between the hemagglutinin-neuraminidase (HN) gene and the large (L) polymerase gene. Recombinant PIV5 containing the HA gene of Udorn (rPIV5-H3) was recovered and it replicated similarly to wild type PIV5, both in vitro and in vivo. The HA protein expressed by rPIV5-H3-infected cells was incorporated into the virions and addition of the HA gene did not increase virus virulence in mice. The efficacy of rPIV5-H3 as a live vaccine was examined in 6-week-old BALB/c mice. The results show that a single dose inoculation provides broad and considerable immunity against influenza A virus infection.     

A study of molecular mechanisms of rubella virus attenuation evidenced from the Russian C-77 strain

Live attenuated vaccine is used for vaccination. The temperature-sensitive (ts) phenotype is characteristic for almost all vaccinal rubella strains. The acquisition of the temperature-sensitive phenotype during adaptation to cold is strongly correlated with attenuation of the wild-type virus. Nevertheless, the molecular mechanisms of rubella virus attenuation have been insufficiently studied. Hence, it is still urgent to study the mechanisms of wild-type virus attenuation and search for key mutations leading to attenuation, as well as detect phenotypic signs that may serve as attenuation markers and be used, along with sequencing, for vaccinal strain genetic stability supervision. This study presents complete genome sequencing of the wild-type (wt) and cold-adapted (ca) C-77 rubella strain variants isolated in Russia; probable genetic determinants have been revealed. After comparison of the wt and ca genomes of the C-77 strain, 13 nucleotide differences were detected, of which six resulted in amino acid substitutions; four differences out of the six that result in these substitutions virtually do not occur in wild-type strains of the rubella virus. Tyr1042Cys substitution is of special interest in the protease domain of the C-77 strain, which likely plays a crucial role in cold-adapted phenotype acquisition by the C-77 strain.     

Characterization of DNA complementary to nucleotide sequences adjacent to poly(A) at the 3'-terminus of the avian sarcoma virus genome.

The temperature-sensitive influenza virus A/Hong Kong/68 (H3N2) ts-1[E] has been used as a prototype live attenuated influenza virus vaccine. Using recently developed techniques to map the genome of influenza viruses and to “genotype” influenza virus recombinants, the temperature-sensitive lesions in the virus were identified. These defects, responsible for the attenuation of the virus, are located in the genes for the P3 protein and the nucleoprotein and are associated with virus-specific RNA synthesis. Hong Kong/68 (H3N2) ts-1[E] virus can also serve as a donor of “attenuation characteristics” for the selection of recombinant strains which have different surface antigens and may be used as vaccine strains in the future. The temperature-sensitive mutations of Hong Kong/68 (H3N2) ts-1[E] virus were previously transferred to recombinant viruses carrying the HO hemagglutinin. The RNAs of 8 of these temperature-sensitive recombinants were analyzed. One of these viruses, R1, classified in group 1 of the Hong Kong mutant virus set was found to possess a ts defect only in the P3 protein. R8, a member of group 2 of the Hong Kong mutant virus set had a ts mutation in the nucleoprotein.     

Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees

Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of vaccines at a 3-week interval, both strains induced antibody in 75 to 95% of the children. On coinfection of chicken embryos with both recombinants, only weak interference was observed. Administration to children of the bivalent vaccine containing H1N1 and H3N2 recombinants induced efficient production of antibody to H1 and H3 hemagglutinins and N1 and N2 neuraminidases without adverse reactions. The recombinants studied were genetically stable as judged by retention of the temperature-sensitive phenotypes and a lack of reversion of the genes carrying temperature-sensitive mutations in all of the reisolates from vaccinated children.     

Cold-adapted reassortants of influenza A virus: Pathogenicity of A/Ann Arbor/6/60×A/Alaska/6/77 reassortant virusesIn vivo andIn vitro

Summary Cold-adapted reassortants of A/Ann Arbor/6/60×A/Alaska/6/77 viruses made in MDCK cells have recently been assessed genotypically and for temperature-sensitive and cold-adapted phenotypes. These reassortants were used to infect ferrets and hamsters and to inoculate organ cultures of hamster tracheal rings, in order to assess their degree of virulence. Virulence in the three model systems corresponded quite well, and a correlation between loss of virulence and particular A/AA/6/60 genes present in the reassortants was noted. Two different reassortants containing either RNA 2 or RNA 5 (NA gene) alone from A/AA/6/60 showed little attenuation from the wild-type parent. A reassortant containing both RNA2 and the NA gene from A/AA/6/60 and all remaining wild-type genes showed some small decrease in virulence compared to the wild-type virus. However a reassortant containing these two A/AA/6/60 genes and RNA 3 as an additional gene from this parent, had a level of attenuation comparable to that of the cold-adapted virus.With 1 Figure     

Analysis of varicella zoster virus attenuation by evaluation of chimeric parent Oka/vaccine Oka recombinant viruses in skin xenografts in the SCIDhu mouse model

Varicella-zoster virus (VZV) is the only human herpes virus for which a vaccine has been licensed. A clinical VZV isolate, designated the parent Oka (pOka) strain was passed in human and non-human fibroblasts to produce vaccine Oka (vOka). The pOka and vOka viruses exhibit similar infectivity in cultured cells but healthy susceptible individuals given vaccines derived from vOka rarely develop the cutaneous vesicular lesions characteristic of varicella. Inoculation of skin xenografts in the SCIDhu mouse model of VZV pathogenesis demonstrated that vOka had a reduced capacity to replicate in differentiated human epidermal cells in vivo (Moffat, J.F., Zerboni, L., Kinchington, P.R., Grose, C., Kaneshima, H., Arvin A.M., 1998a. Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol. 72:965-74). In order to investigate the attenuation of vOka in skin, we made chimeric pOka and vOka recombinant viruses from VZV cosmids. Six chimeric pOka/vOka viruses were generated using cosmid sets that incorporate linear overlapping fragments of VZV DNA from cells infected with pOka or vOka. The cosmid sets consist of pOka and vOka DNA segments that have identical restriction sites. As expected, the growth kinetics and plaque morphologies of the six chimeric pOka/vOka viruses were indistinguishable in vitro. However, the chimeric viruses exhibited varying capacities to replicate when evaluated in skin xenografts in vivo. The presence of ORFs 30-55 from the pOka genome was sufficient to maintain wild-type infectivity in skin. Chimeric viruses containing different vOka components retained the attenuation phenotype, suggesting that vOka attenuation is multi-factorial and can be produced by genes from different regions of the vOka genome.     

Respiratory syncytial virus strain A2 is resistant to the antiviral effects of type I interferons and human MxA.

Respiratory syncytial virus (RSV) belongs to Paramyxoviridae family of enveloped negative-strand RNA viruses and causes severe bronchiolitis and pneumonia in children younger than 2 years of age. As members of Paramyxoviridae family, RSV and parainfluenza type 3 (PIV3) have similar modes of infection and replication. A variety of negative-strand RNA virus infections, including that of PIV3, are inhibited by human MxA protein, a type I interferon (IFN)-inducible GTPase. We tested whether the MxA protein, induced either by type I human IFNs or by stable transfection of human MxA gene in human (U-87) or simian (Vero) cells, confers resistance to these cells against infection by RSV strain A2. RSV infection was resistant to antiviral effects induced by 0-10,000 U/ml type I IFNs (IFN-alpha or -beta) in both human lung epithelial, A549, and fibroblast, MRC-5 cells. RSV virus yield was reduced only by 10- to 20-fold, and viral protein synthesis was not significantly affected under conditions of IFN treatment where PIV3 yield was reduced by 1000- to 10,000-fold. Human or simian cell lines constitutively expressing MxA were protected against infection by PIV3 but not by RSV. Our results indicate that RSV A2 is resistant to the antiviral effects of MxA, even though RSV and PIV3 have similar replication strategies. In IFN-treated coinfected cultures, IFN-resistant RSV A2 did not prevent the IFN-mediated inhibition of PIV3 multiplication. Hence the resistance of RSV A2 to type I IFNs does not appear to be due to soluble factors released into the medium or a disruption in the cellular antiviral machinery brought about by RSV A2 infection.     

Extensive antigenic diversity among human parainfluenza type 2 virus isolates and immunological relationships among paramyxoviruses revealed by monoclonal antibodies

A panel of 128 monoclonal antibodies (MAbs) directed against hemagglutinin-neuraminidase (HN), fusion (F), matrix (M), and polymerase (P) proteins, and nucleoprotein (NP) of the Toshiba strain of human parainfluenza type 2 virus (PIV2) was prepared to examine the antigenic relationships among clinical isolates of PIV2 and among paramyxoviruses by indirect enzyme-linked immunosorbent assays. The HN proteins of 18 clinical isolates of PIV2 showed extensive antigenic diversity: 23 of 33 anti-HN MAbs showed no or limited reactivity to many isolates, while other structural proteins were antigenically well conserved. Some anti-HN MAbs recognizing conserved epitopes of the isolates exhibited two types of neutralizing activity, that is, these antibodies inhibited viral infectivity through attachment inhibition or fusion inhibition. This result also showed the presence of a potential third function of the HN protein which might affect the fusing activity of the F protein besides the hemagglutinating and neuraminidase activities. Many of the anti-NP and anti-P MAbs reacted with simian virus 41 (SV41) and simian virus 5 (SV5), whereas a few reacted with mumps virus or PIV4. Two of 6 anti-F MAbs reacted with SV41. None of the 128 MAbs showed reactivity with PIV1, PIV3, Newcastle disease virus (NDV), and measles virus. This result confirmed antigenic proximity of SV5 and SV41 to PIV2 and revealed comparatively restricted immunological relatedness among PIV2, PIV4, and mumps virus.     

Genetics of cold-adapted B/Ann Arbor/1/66 influenza virus reassortants: the acidic polymerase (PA) protein gene confers temperature sensitivity and attenuated virulence

The cold-adapted B/Ann Arbor/1/66 influenza virus (ca B/AA/1/66) expresses temperature-sensitive (ts), cold-adapted (ca) and attenuation phenotypes. Reassortants which inherit one or more genes from ca B/AA/1/66 and all other genes from a virulent, wild-type influenza virus, B/Houston/1732/76, were produced and evaluated in order to identify the gene(s) responsible for the ts, ca and attenuation phenotypes. Only reassortants which inherited the PA gene from ca B/AA/1/66 expressed the ts phenotype in MDCK cells at 39 degrees C. None of the reassortants tested expressed the ca phenotype in embryonated eggs at 25 degrees C. The virulence of several reassortants was evaluated in ferrets. Inheritance of the PA gene from ca B/AA/1/66 was correlated with significant febrile attenuation and the apparent restriction of viral replication in the lower respiratory tract. Isolation of a virulent, non-ts revertant virus inheriting only the PA gene from ca B/AA/1/66 established a direct relationship between expression of the ts phenotype and attenuated virulence. Evidence for the contribution of at least one other gene from ca B/AA/1/66 to attenuation was observed. Thus, based on the methods used to determine reassortant gene compositions, these results indicate that the PA gene is primarily responsible for attenuation of ca B/AA/1/66 and its reassortants.