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The aim of this study was to analyze the population pharmacokinetics of total and unbound concentrations of prophylactic cefazolin (CFZ) in patients with prostatectomy or nephrectomy. We also aimed to calculate a pharmacodynamics target unbound concentration that exceeded the minimum inhibitory concentration (MIC), to design an effective dosing regimen. Briefly, 614 total concentration and 610 unbound concentration samples from 152 individuals were evaluated, using a nonlinear mixed-effects model. The obtained pharmacodynamics index target value reflected the probability of maintaining CFZ unbound trough concentrations exceeding MIC 90 , 0.5 mg/L, and MIC 50 , and 1.0 mg/L, to account for methicillin-susceptible Staphylococcus aureus (MSSA) or Escherichia coli . Population pharmacokinetics were estimated using a two-compartment model with nonlinear protein binding. Unbound systemic clearance (CL) was significantly associated with creatinine clearance, while the maximum protein-binding constant was significantly associated with albumin levels. The probability of achieving an unbound concentration exceeding the MIC 50 for E. coli or MIC 90 for MSSA in a patient with normal renal function following a 1 g CFZ infusion over 15 min was above 90% at 3 h after the initial dose. Our findings indicated that population pharmacokinetic parameters are useful for determining unbound CFZ pharmacokinetics and evaluating intraoperative CFZ redosing intervals., Competing Interests: The authors declare no conflict of interest.

The purpose of this study was to investigate the population pharmacokinetics of prophylactic flomoxef based on serum and liver tissue concentrations and to demonstrate a pharmacodynamic target concentration in the serum and liver tissue exceeding the MIC in order to design an effective dosing regimen. Serum samples ( n  = 210) and liver tissue samples ( n  = 29) from 43 individuals were analyzed using a nonlinear mixed-effects model. The pharmacodynamics index target value was regarded as the probability of maintaining flomoxef serum trough and liver tissue concentrations exceeding the MIC 90 values, 0.5 mg/L and 1.0 mg/L, for Escherichia coli and methicillin-susceptible Staphylococcus aureus, respectively. The final population pharmacokinetic model was a two-compartment model with linear elimination. Creatinine clearance (CL CR ) was identified as a significant covariate influencing total clearance when CL CR was less than 60 mL/min. The probability of achieving concentrations in the serum and liver tissue exceeding the MIC 90 for E. coli or methicillin-susceptible S. aureus for a 1 g bolus dose was above 90% at 2 h after the initial dose. Our findings suggest that population pharmacokinetic parameters are helpful for evaluating flomoxef pharmacokinetics and determining intraoperative flomoxef redosing intervals.

The objectives of this study were to evaluate the population pharmacokinetics of prophylactic cefazolin (CFZ) from its serum and hip joint capsule concentrations in patients undergoing total hip arthroplasty and to establish the pharmacodynamic target concentration exceeding the MIC for designing an effective dosing regimen for serum and the hip joint capsule. We analyzed 249 serum samples and 125 hip joint capsule samples from 125 individuals using a nonlinear mixed-effects model. The pharmacodynamic index target value obtained from our results indicates the probability of maintaining CFZ trough and hip joint capsule concentrations exceeding the MIC of 1 mg/liter to account for methicillin-susceptible S. aureus (MSSA). We estimated the population pharmacokinetics using a two-compartment model. The estimated population pharmacokinetic parameters were as follows: clearance (CL) (liters/h) = 1.46 × (creatinine clearance [CL cr ] [ml/min]/77) 0.891 , volume of distribution of the central compartment (V c ) (liters) = 7.5, central-hip joint capsule compartment clearance (Q) (liters/h) = 3.38, and volume of distribution in the hip joint capsule compartment (V JC ) (liters) = 36.1. The probability of achieving concentrations exceeding the MIC 90 for MSSA was approximately 100% for serum and 100% for the hip joint capsule at 3 h after the initial dose. Our findings suggest that population-based parameters are useful for evaluating CFZ pharmacokinetics and that individual dosages should be determined based on the dosage regimen that achieves and maintains adequate tissue CFZ concentration., (Copyright © 2021 American Society for Microbiology.)

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1β (IL-1β) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1β maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1β than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1β secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1β activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1β secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1β activation. IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type 2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also lead to identification of novel therapeutic targets for paramyxovirus infection and related diseases., (Copyright © 2018 American Society for Microbiology.)

Adenoviruses are DNA viruses with a lytic infection cycle. Following the fate of incoming as well as recently replicated genomes during infections is a challenge. In this study, we used the ANCHOR3 technology based on a bacterial partitioning system to establish a versatile in vivo imaging system for adenoviral genomes. The system allows the visualization of both individual incoming and newly replicated genomes in real time in living cells. We demonstrate that incoming adenoviral genomes are attached to condensed cellular chromatin during mitosis, facilitating the equal distribution of viral genomes in daughter cells after cell division. We show that the formation of replication centers occurs in conjunction with in vivo genome replication and determine replication rates. Visualization of adenoviral DNA revealed that adenoviruses exhibit two kinetically distinct phases of genome replication. Low-level replication occurred during early replication, while high-level replication was associated with late replication phases. The transition between these phases occurred concomitantly with morphological changes of viral replication compartments and with the appearance of virus-induced postreplication (ViPR) bodies, identified by the nucleolar protein Mybbp1A. Taken together, our real-time genome imaging system revealed hitherto uncharacterized features of adenoviral genomes in vivo The system is able to identify novel spatiotemporal aspects of the adenovirus life cycle and is potentially transferable to other viral systems with a double-stranded DNA phase. IMPORTANCE Viruses must deliver their genomes to host cells to ensure replication and propagation. Characterizing the fate of viral genomes is crucial to understand the viral life cycle and the fate of virus-derived vector tools. Here, we integrated the ANCHOR3 system, an in vivo DNA-tagging technology, into the adenoviral genome for real-time genome detection. ANCHOR3 tagging permitted the in vivo visualization of incoming genomes at the onset of infection and of replicated genomes at late phases of infection. Using this system, we show viral genome attachment to condensed host chromosomes during mitosis, identifying this mechanism as a mode of cell-to-cell transfer. We characterize the spatiotemporal organization of adenovirus replication and identify two kinetically distinct phases of viral genome replication. The ANCHOR3 system is the first technique that allows the continuous visualization of adenoviral genomes during the entire virus life cycle, opening the way for further in-depth study., (Copyright © 2018 Komatsu et al.)

Unlabelled: Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes., Importance: The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Porphyromonas gingivalis, a major periodontal pathogen, may contribute to atherogenesis and other inflammatory cardiovascular diseases. However, little is known about interactions between P. gingivalis and endothelial cells. E-selectin is a membrane protein on endothelial cells that initiates recruitment of leukocytes to inflamed tissue, and it may also play a role in pathogen attachment. In the present study, we examined the role of E-selectin in P. gingivalis adherence to endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) to induce E-selectin expression. Adherence of P. gingivalis to HUVECs was measured by fluorescence microscopy. TNF-α increased adherence of wild-type P. gingivalis to HUVECs. Antibodies to E-selectin and sialyl Lewis X suppressed P. gingivalis adherence to stimulated HUVECs. P. gingivalis mutants lacking OmpA-like proteins Pgm6 and -7 had reduced adherence to stimulated HUVECs, but fimbria-deficient mutants were not affected. E-selectin-mediated P. gingivalis adherence activated endothelial exocytosis. These results suggest that the interaction between host E-selectin and pathogen Pgm6/7 mediates P. gingivalis adherence to endothelial cells and may trigger vascular inflammation.

In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of histones, does not affect the level of histone H3 bound on viral chromatin, although CAF-1 is accumulated at viral DNA replication foci together with PCNA. Chromatin immunoprecipitation assays using epitope-tagged histone H3 demonstrated that histone variant H3.3, which is deposited onto the cellular genome in a replication-independent manner, is selectively associated with both incoming and newly synthesized viral DNAs. Microscopic analyses indicated that histones but not USF1, a transcription factor that regulates viral late gene expression, are excluded from viral DNA replication foci and that this is achieved by the oligomerization of the DNA binding protein (DBP). Taken together, these results suggest that histone deposition onto newly synthesized viral DNA is most likely uncoupled with viral DNA replication, and a possible role of DBP oligomerization in this replication-uncoupled histone deposition is discussed.

Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X(3)-Trp-X(9)-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

Sendai virus (SeV) C protein is a multifunctional protein that plays important roles in regulating viral genome replication and transcription, antagonizing the host interferon system, suppressing virus-induced apoptosis, and facilitating virus assembly and budding. We here report a novel role of SeV C protein, the limitation of double-stranded RNA (dsRNA) generation for maintaining the rate of protein synthesis in infected cells. It was found that the intracellular protein synthesis rate was maintained even after wild-type (wt) SeV infection, but markedly suppressed following C-knockout SeV infection. This indicates the requirement of C protein for maintaining protein synthesis after infection. In contrast to wt SeV infection, C-knockout SeV infection caused phosphorylation of both the translation initiation factor eIF2alpha and dsRNA-dependent protein kinase (PKR). Phosphorylation of eIF2alpha occurred mainly due to the action of PKR, since knockdown of PKR by small interfering RNA limited eIF2alpha phosphorylation. C protein, however, could inhibit neither poly(I):poly(C)-activated nor Newcastle disease virus-induced phosphorylation of PKR and eIF2alpha, suggesting that C protein does not target common pathways leading to PKR activation. Immunofluorescent staining experiments with a monoclonal antibody specifically recognizing dsRNA revealed generation of a large amount of dsRNA in cells infected with C-knockout SeV but not wt SeV. The dsRNA generation as well as phosphorylation of PKR and eIF2alpha induced by C-knockout SeV was markedly suppressed in cells constitutively expressing C protein. Taken together, these results demonstrate that the SeV C protein limits generation of dsRNA, thereby keeping PKR inactive to maintain intracellular protein synthesis.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen (LANA) tethers viral terminal repeat (TR) DNA to mitotic chromosomes to mediate episome persistence. The 1,162-amino-acid LANA protein contains both N- and C-terminal chromosome attachment regions. The LANA C-terminal domain self-associates to specifically bind TR DNA and mitotic chromosomes. Here, we used alanine scanning substitutions spanning residues 1023 to 1145 to investigate LANA self-association, DNA binding, and C-terminal chromosome association. No residues were essential for LANA oligomerization, as assayed by coimmunoprecipitation experiments, consistent with redundant roles for amino acids in self-association. Different subsets of amino acids were important for DNA binding, as assayed by electrophoretic mobility shift assay, and mitotic chromosome association, indicating that distinct C-terminal LANA subdomains effect DNA and chromosome binding. The DNA binding domains of LANA and EBNA1 are predicted to be structurally homologous; certain LANA residues important for DNA binding correspond to those with roles in EBNA1 DNA binding, providing genetic support for at least partial structural homology. In contrast to the essential role of N-terminal LANA chromosome targeting residues in DNA replication, deficient C-terminal chromosome association did not reduce LANA-mediated DNA replication.

In latent infection, Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA1)-specific binding to KSHV terminal repeat DNA mediates multicopy episome persistence. We now use electrophoretic mobility shift assays to investigate LANA1 binding to its 20-bp cognate sequence. Mutations at positions 6, 7, and 8 ((6)CCC(8)) severely reduced LANA1 binding, whereas mutations at other positions only modestly reduced binding. Since (6)CCC(8) is in the 5' half of an inverted repeat sequence, these results are consistent with an asymmetric role for the inverted repeat in LANA1 binding.

Sendai virus (SeV) C protein functions as an interferon (IFN) antagonist and renders cells unresponsive to both alpha/beta IFN (IFN-alpha/beta) and IFN-gamma. We have recently found the physical association of the C protein with signal transducer and activator of transcription 1 (STAT1) in infected cells. However, involvement of the C-STAT1 interaction in the blockade of IFN signaling has remained unclear. We generated here a series of C mutant proteins that retained or lost the STAT1-binding capacity and examined their effects on IFN-alpha signaling. All of the C mutant proteins with no STAT1-binding capacity lost the ability to inhibit the IFN-alpha response. In contrast, the C mutant proteins retaining the STAT1-binding capacity suppressed IFN-alpha-stimulated tyrosine phosphorylation of both STAT2 and STAT1 to various degrees. Remarkably, their anti-IFN-alpha capacities correlated well with the inhibitory effect on phosphorylation of STAT2 rather than STAT1. In infected cells, the levels of tyrosine-phosphorylated (pY) STAT2 were below the detection level irrespective of duration of IFN-alpha stimulation, whereas the levels of pY-STAT1 strikingly increased after long-term IFN-alpha stimulation. These results suggest that the STAT2 activation process is a crucial target for the blockade of IFN-alpha signaling. An in vitro binding assay with extracts from (STAT1-deficient) U3A and (STAT1-expressing) U3A-ST1 cells suggested the requirement of STAT1 for the C-STAT2 interaction. Furthermore, expression of STAT1 enhanced the inhibitory effect of the C protein on STAT2 activation in U3A cells. The C protein thus appears to participate in the inhibitory process for STAT2 activation through the STAT1 interaction.

Kaposi's sarcoma-associated herpesvirus (KSHV) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. K12 (T0.7) is the most abundant transcript expressed in latent KSHV infection. We characterize here the K12 transcript from a PEL tumor prior to passage in cell culture. The PEL tumor KSHV K12 transcript contained additional complex nucleotide repeat elements compared to the previously described K12 message of the BCBL-1 PEL cell line. The PEL tumor lacked kaposin B, the predominant BCBL-1 K12 protein product, but encoded kaposins A and C. The K12 transcript was spliced and the splicing event occurred in all KSHV isolates tested. The 5' end of the K12 transcript was mapped by 5' RACE (rapid amplification of cDNA ends) and S1 nuclease protection assays and was at the site of an active promoter. This work demonstrates that the K12 transcript contains variable, complex repeat elements, is spliced and is expressed from a novel KSHV promoter.

Archaeal community structures in microhabitats in a deep-sea hydrothermal vent chimney structure were evaluated through the combined use of culture-independent molecular analyses and enrichment culture methods. A black smoker chimney was obtained from the PACMANUS site in the Manus Basin near Papua New Guinea, and subsamples were obtained from vertical and horizontal sections. The elemental composition of the chimney was analyzed in different subsamples by scanning electron microscopy and energy-dispersive X-ray spectroscopy, indicating that zinc and sulfur were major components while an increased amount of elemental oxygen in exterior materials represented the presence of oxidized materials on the outer surface of the chimney. Terminal restriction fragment length polymorphism analysis revealed that a shift in archaeal ribotype structure occurred in the chimney structure. Through sequencing of ribosomal DNA (rDNA) clones from archaeal rDNA clone libraries, it was demonstrated that the archaeal communities in the chimney structure consisted for the most part of hyperthermophilic members and extreme halophiles and that the distribution of such extremophiles in different microhabitats of the chimney varied. The results of the culture-dependent analysis supported in part the view that changes in archaeal community structures in these microhabitats are associated with the geochemical and physical dynamics in the black smoker chimney.

We demonstrate here that Sendai virus (SeV) blocks alpha interferon (IFN-alpha) signaling to signal transducers and activators of transcription (STATs) in HeLa cells. IFN-alpha-stimulated tyrosine phosphorylation of STATs and subsequent formation of the IFN-stimulated gene factor 3 transcription complex were inhibited in SeV-infected cells, resulting in inefficient induction of IFN-stimulated gene products. None of the components of the signaling pathway-type I IFN receptor subunits Jak1, Tyk2, Stat1, Stat2, and p48-was degraded. Moreover, tyrosine phosphorylation of Jak1 in response to IFN-alpha was unaffected at the early phase of infection, suggesting that oligomerization of the receptor subunits proceeded normally. In contrast to Jak1, IFN-alpha-stimulated tyrosine phosphorylation of Tyk2 was partially inhibited. Therefore, this partial inhibition of activation of Tyk2 probably contributes to the subsequent failure in the activation of STATs.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.

Vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS) when intranasally applied. We have examined cellular inflammatory changes in the CNS following VSV infection. As early as 1 day postinfection (p.i.), astrocytes were activated in the olfactory bulb (OB). This was followed by activation of microglia, first observed in the OB at day 3 p.i. Expression of inducible nitric oxide synthase was observed in activated microglia in the OB at day 3 p.i., and increased inducible nitric oxide synthase expression coincided with decreased virus titers in tissue homogenates. Expression of major histocompatibility complex (MHC) class I molecules on astrocytes and microglial, endothelial, and ependymal cells was also rapidly induced and followed by induced expression of MHC class II molecules on astrocytes and microglial and endothelial cells. Consistent with the pattern of viral dissemination, MHC molecules were expressed temporally from the rostral-to-caudal direction. Infiltration of CD8+ cells was observed as early as 1 day p.i. in the OB. CD4+ cells were detected in the OB at day 4 p.i. Increasing T-cell infiltration coincided with decreased virus titers. In contrast, B-cell infiltration of the CNS was not detected until day 14 p.i., after the virus was cleared and mice were showing behavioral signs of recovery. Breakdown of the blood-brain barrier was detected beginning at day 6 p.i., was most severe at day 8 p.i., and was followed by full recovery. Collectively, these data show that both innate immunity (production of nitric oxide) and acquired immunity (expression of MHC molecules and T-cell infiltration) are activated following VSV infection in the CNS.

Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure -->2-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1-->3)-alpha- Man-(1-->. The E. coli O9 rfb cluster has been sequenced, and six genes, in addition to the previously described rfbK and rfbM, were identified. They correspond to six open reading frames (ORFs) encoding polypeptides of 261, 431, 708, 815, 381, and 274 amino acids. They are all transcribed in the counter direction to those of the his operon. No gene was found between rfb and his. A higher G+C content indicated that E. coli O9 rfb evolved independently of the rfb clusters from other E. coli strains and from Shigella and Salmonella spp. Deletion mutagenesis, in combination with analysis of the in vitro synthesis of the O9 mannan in membranes isolated from the mutants, showed that three genes (termed mtfA, -B, and -C, encoding polypeptides of 815, 381, and 274 amino acids, respectively) directed alpha-mannosyl transferases. MtfC (from ORF274), the first mannosyl transferase, transfers a mannose to the endogenous acceptor. It critically depended on a functional rfe gene (which directs the synthesis of the endogenous acceptor) and initiates the growth of the polysaccharide chain. MtfB (from ORF381) then transfers two mannoses into the 3 position of the previous mannose, and MtfA (from ORF815) transfers three mannoses into the 2 position. Further chain growth needs only the two transferases MtfA and MtfB. Thus, there are fewer transferases needed than the number of sugars in the repeating unit. Analysis of the predicted amino acid sequence of the ORF261 and ORF431 proteins indicated that they function as components of an ATP-binding cassette transport system. A possible correlation between the mechanism of polymerization and mode of membrane translocation of the products is discussed.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.

A protein factor which induces high levels of gamma interferon (IFN-gamma) in resting splenic nonadherent cells was isolated from the sera of mice with generalized inflammation caused by endotoxic shock. The factor was highly purified by ammonium sulfate precipitation followed by ion-exchange column chromatography on DEAE-Sepharose, molecular sieving on Ultrogel AcA 44, and hydrophobic column chromatography with phenyl-Sepharose. It was further purified to apparent homogeneity by polyacrylamide gel electrophoresis. It induced IFN-gamma production in a dose-dependent manner in the presence of interleukin-2, monoclonal anti-CD3 antibody (anti-CD3 MAb), or concanavalin A (ConA) in spleen cells deprived of plastic plate- and nylon wool-adherent cells. Anti-CD3 MAb induced the highest level of production of the three. The factor, interleukin-2, anti-CD3 MAb, or ConA alone induced a trace of or no detectable IFN-gamma in these cells. The factor also exhibited an accessory function during proliferation in these cells in the presence of a suboptimal dose of ConA. However, the factor failed to stimulate IFN-gamma production when staphylococcal enterotoxin A, a superantigenic T-cell mitogen, was employed. Treatment with pronase or heat abolished these activities. These studies confirm the existence of a soluble protein factor which is able to exhibit a novel accessory function in IFN-gamma production in resting T or natural killer cells. It will be of interest to compare this factor with the recently cloned human natural killer stimulatory factor (NKSF/IL-12).

Introduction of the romA gene cloned from Enterobacter cloacae into Escherichia coli K-12 resulted in almost complete inhibition of OmpF expression and a concomitant increase in resistance to quinolones, beta-lactams, chloramphenicol, and tetracyclines. In addition, the romA gene reduced the susceptibility to these multiple drugs even in the OmpF porin-deficient mutants of E. coli K-12. Results indicate the presence of romA-sensitive penetration pathway(s) for these multiple drugs other than the OmpF porin in E. coli.

The genes determining the biosynthesis of type 2 (K2) capsular polysaccharide [3----beta Glc1,4----beta Man(1,3----beta GlcUA) 1,4----alpha Glc1----] of Klebsiella pneumoniae Chedid (O1:K2), which is highly virulent for mice, were cloned and introduced into Escherichia coli HB101 and into four noncapsulated mutants derived from K. pneumoniae reference strains of K1, K7, K9, and K28. The recombinant plasmid pCPS7B06 carried 23 kb of a chromosomal DNA fragment of strain Chedid and encoded a part of the Klebsiella cps gene cluster. However, pCPS7B06 encoded enough genetic information for the production of Klebsiella K2 capsular polysaccharide on the cell surfaces of four noncapsulated mutants of K. pneumoniae. On the other hand, both pCPS7B06 and pROJ3 carrying the rmpA gene locus derived from a resident large plasmid of Chedid were required for the biosynthesis of Klebsiella K2 capsular polysaccharide on the cell surface of E. coli HB101. The insertion inactivation analysis using Tn5 revealed that the cps gene cluster occupied more than 15 kb of the chromosome of Chedid. We conclude that rmpA, which has been known to enhance the biosynthesis of colanic acid in E. coli, is also involved in the biosynthesis of Klebsiella capsular polysaccharide in E. coli HB101.

To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium.

The introduction of a newly cloned Enterobacter cloacae chromosomal gene romA, into Escherichia coli and E. cloacae resulted in enhancement of resistance to quinolones, beta-lactams, chloramphenicol, and tetracycline. The primary effect of romA on a multicopy vector in E. coli was almost complete inhibition of OmpF expression in the outer membrane. From the experiments with ompR and envZ mutants or with ompF-lacZ and ompC-lacZ fusion plasmids, it was concluded that this inhibition is posttranscriptional. The introduction of romA on a multicopy vector into strains with micF deletion elicited only a moderate decrease in OmpF protein expression. This indicates that reduction of OmpF expression by romA is partly mediated posttranscriptionally by the activation of micF. Moreover, the overexpression of RomA protein from an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter resulted in nearly complete inhibition of expression of OmpC and OmpA, as well as OmpF. Taken together with an observation in a recent study that overexpressed OmpC inhibited the synthesis of OmpA and LamB, a possible inhibitory mechanism at the translational stage of the synthesis of outer membrane proteins should also be considered. By Southern hybridization, romA was generally detected in the chromosomes of all E. cloacae strains tested but not in the E. coli K-12 chromosome. Sequence data show that there is an open reading frame specifying 368 amino acids residues including a putative signal peptide. RomA appears to belong to the outer membrane protein family since it was extractable from an outer membrane preparation, but no sequence homology to other outer membrane proteins was detected.

The chemotherapeutic properties of miloxacin (5,8-dihydro-5-methoxy-8-oxo-2H-1,3-dioxolo-[4,5-g]quinoline-7-carboxylic acid) have been compared with those of oxolinic acid and nalidixic acid. The in vitro activities of miloxacin (minimum inhibitory concentrations) against a variety of gram-negative bacteria, especially Enterobacteriaceae and Haemophilus, were comparable to those of oxolinic acid and 8 to 16 times greater than those of nalidixic acid. Miloxacin was more active than oxolinic acid against some anaerobes and less active against staphylococci. Miloxacin exhibited significant activities when administered orally to mice infected with Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, or Serratia marcescens. Its efficacy was comparable to that of oxolinic acid and two to four times greater than that of nalidixic acid. Miloxacin was less active against a Pseudomonas aeruginosa infection and inactive at the maximum test doses against a Streptococcus pyogenes infection.

Serial dilutions of hemorrhagic fever with renal syndrome viruses were inoculated into Vero-E6 cells in microplates. After 2 weeks of incubation, infected cells were disrupted by freezing and thawing, and virus antigens were detected by immune adherence hemagglutination. The infectivity titers of the virus as determined by this method were in close agreement with those obtained by the immunofluorescent antigen endpoint method. Then, a neutralization method was established. Japanese hemorrhagic fever with renal syndrome isolates, strains SR-11 and TR-352, were found to be distinct from Hantaan virus, strain 76-118, by the neutralization test.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.

Miloxacin, a synthetic antibacterial agent structurally related to oxolinic acid, has a broad spectrum of activity in vitro against gram-negative bacteria and considerable activity in vivo against infections with these bacteria. These observations led to studies on the absorption and excretion of miloxacin in mice, rats, and dogs after administration of a single oral dose. Studies on oxolinic acid have been included for comparison. Peak serum levels of miloxacin, attained 1 h after administration of 20, 50, and 100 mg/kg to rats and dogs, were approximately 20, 40, and 60 micrograms/ml, respectively. Peak levels in mice receiving the same dose were 15, 60, and 80 micrograms/ml at 0.5 h. Peak serum levels of oxolinic acid were attained 0.5 to 1 h later than the above times at comparable doses and were one-half to one-fourth those of miloxacin. Urinary recovery of miloxacin at the above doses ranged from 3.2 to 6.5% during the 24-h posttreatment period. Recoveries of oxolinic acid were one-half to one-fifth those of miloxacin. At a 50-mg/kg dose, rats excreted 4.6% of the miloxacin in bile in the 20-h posttreatment period.

Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation.

The therapeutic effects of imipenem-cilastatin (MK-0787-MK-791) on experimental intrauterine infections in progesterone-treated virgin rats and postpartum rats were studied. The relative efficacy of imipenem-cilastatin for the treatment of such intrauterine infections was compared with that of cefazolin and ampicillin for the treatment of infections caused by Escherichia coli and Streptococcus faecalis, respectively. Treatment with imipenem-cilastatin significantly inhibited the proliferation of E. coli and S. faecalis in uteri, as compared with the proliferation in untreated controls. Cefazolin failed to affect the E. coli infection. With the S. faecalis infection, ampicillin effectively reduced bacterial growth, as compared with that in untreated controls. However, ampicillin was inferior to and comparable to imipenem-cilastatin in progesterone-treated virgin rats and postpartum rats, respectively. A further experiment with S. faecalis infections in rats made neutropenic by intraperitoneal injection of cyclophosphamide showed that the therapeutic effectiveness of imipenem-cilastatin was superior to that of ampicillin and was not influenced by neutropenia. Our results suggest that imipenem-cilastatin may be a useful agent for the treatment of obstetric and gynecologic infections.

The chromosomally encoded beta-lactamase gene of Klebsiella oxytoca E23004, a strain resistant to cefoperazone and aztreonam, was cloned and expressed in Escherichia coli HB101. The molecular mass and pI of this enzyme were 28 kilodaltons and 7.4, respectively. Although the beta-lactamase of K. oxytoca hydrolyzed many cephalosporins, including broad-spectrum drugs, the nucleotide sequence and deduced amino acid sequence lacked homology with chromosomal class C beta-lactamase genes (ampC) of E. coli or Citrobacter freundii. Rather, about 45% nucleotide sequence homology and 40% deduced amino acid sequence homology were observed between the K. oxytoca beta-lactamase and TEM-1, a class A beta-lactamase which does not efficiently hydrolyze cephalosporins. Values of Km, relative Vmax, and relative Vmax/Km for the K. oxytoca beta-lactamase indicated that the enzyme is a penicillinase but that it can hydrolyze cefoperazone effectively and other broad-spectrum cephems weakly. Hence, the chromosomal beta-lactamase of K. oxytoca E23004 belongs to class A but differences in its amino acid sequence provide a broader spectrum of activity.

PC-904, sodium 6-{d(-)-alpha-(4-hydroxy-1,5-naphthyridine-3-carboxamido) phenylacetamido}-penicillanate, is a novel semisynthetic penicillin derivative that possesses a broad spectrum of in vitro and in vivo antibacterial activities. In low concentrations, PC-904 inhibits growth against large proportions of the gram-positive and gram-negative organisms susceptible to carbenicillin and gentamicin. In addition, PC-904 is several times more potent than carbenicillin against organisms such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Shigella, Salmonella, Neisseria gonorrhoeae, and Bacteroides fragilis. Most striking are the inhibitory effects of PC-904 against P. aeruginosa and K. pneumoniae. Against these two clinical isolates, PC-904 is, respectively, 35 and 100 times more active than carbenicillin. The minimum inhibitory concentrations of PC-904 against P. aeruginosa are comparable to those of gentamicin. PC-904 acts bactericidally. The effect of inoculum size on the antibacterial activity is often small and generally comparable to carbenicillin. The rate of binding to serum protein is high (88 to 98%), but the effect of the addition of serum on the drug's activity is not marked, because such binding is reversible. It is confirmed that PC-904 has a very potent in vivo antibacterial activity against gram-negative and gram-positive organisms. Against systemic infections with P. aeruginosa, K. pneumoniae, and E. coli in mice, PC-904 is 7 to 10 times, over 8 times, and 2 to 15 times more active than carbenicillin, respectively.

The rfb gene, involved in the synthesis of the O-specific polysaccharide (a mannose homopolymer) of Escherichia coli O9 lipopolysaccharide (LPS), was cloned in E. coli K-12 strains. The O9-specific polysaccharide covalently linked to the R core of K-12 was extracted from the K-12 strains harboring the O9 rfb gene. All the other genes required for the synthesis of rfe-dependent LPS are therefore considered to be present in the K-12 strains. It was found that bacteria harboring some clones with deletions of the ca. 20-kilobase-pair (kbp) BglII-StuI fragment no longer synthesized the O9-specific polysaccharide. However, bacteria harboring clones del 21, del 22, and del 25, which carry deletions of the 10-kbp PstI-StuI fragment, synthesized an O-specific polysaccharide antigenically distinct from E. coli O9 LPS. Although this new O-specific polysaccharide consisted solely of mannose and the mannose residues were combined only through alpha-1,2 linkage, it was still composed of a repeating oligosaccharide unit, possibly a trisaccharide unit,----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----. It is therefore likely that this new O-specific polysaccharide was derived from a part of the O9-specific polysaccharide----3)alpha Man-(1----3)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----and that the deleted part of the clones was responsible for the synthesis of alpha-1,3 linkages of the O9-specific polysaccharide.

A new chemotherapeutic agent, AB206, shows potent antibacterial activity against gram-negative bacteria, including most of the nalidixic acid-resistant strains tested. It strongly inhibits deoxyribonucleic acid (DNA) synthesis in Escherichia coli, but only slightly inhibits ribonucleic acid and protein synthesis. Its activity on DNA synthesis in vivo and in vitro is greater than that of nalidixic acid. AB206 also strongly inhibits in vivo DNA synthesis in nalidixic acid-susceptible and -resistant clinical isolates of Proteus and Serratia. AB206 shows high penetrability into E. coli cells, as demonstrated by antibacterial activity with or without ethylenediaminetetraacetic acid, inhibition of in vivo and in vitro DNA synthesis, and uptake of the drug into cells, as compared to nalidixic acid. It appears that the high antibacterial activity of AB206 may be explained both by its potent inhibitory action against DNA synthesis and also by its high penetrability into bacterial cells.

Penicillin-binding protein 5 in Pseudomonas aeruginosa had moderately penicillin-sensitive D-alanine carboxypeptidase activity. As in Escherichia coli, a defect in this enzyme activity was not lethal.

SM-1652 (sodium 7-[D(-)-alpha-(4-hydroxy-6-methylpyridine-3-carboxamido)-alpha-(4-hydroxyphenyl)acetamido]-3-[(1-methyl-1H-tetrazol-5-yl) thiomethyl]-3-cephem-4-carboxylate) is a new semisynthetic cephalosporin derivative with a broad spectrum of antibacterial activity. Its in vitro activity against gram-positive bacteria was comparable to that of cefazolin. SM-1652 exceeded cefazolin in potency and broadness of antibacterial activity against such Enterobacteriaceae as indole-positive Proteus spp., Enterobacter cloacae, and Serratia marcescens. A remarkable feature of the spectrum of SM-1652 is its high activity against Pseudomonadaceae. Against 200 clinical isolates of Pseudomonas aeruginosa, SM-1652 was significantly more active than cefoperazone, cefotaxime, and sulbenicillin and as active as cefsulodin. The activities of SM-1652 against Pseudomonas maltophilia and Pseudomonas cepacia were superior to those of cefoperazone, cefotaxime, cefsulodin, sulbenicillin, and gentamicin. SM-1652 was relatively stable to hydrolysis with plasmid-mediated penicillinases and cephalosporinases produced by gram-negative bacteria.

A sensitive and reliable high-pressure liquid chromatography (HPLC) assay for miloxacin and its two principal metabolites, 5,8-dihydro-8-oxo-2H-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid (M-1) and 1,4-dihydro-1,6-dimethoxy-7-hydroxy-4-oxoquinoline-3-carboxylic acid (M-2), in human serum and urine was developed. A strong anion-exchange Zipax SAX column using a mobile phase of 0.01 M citric acid solution containing 0.03 M sodium nitrate with pH 5.0 was used to achieve separation of the three compounds. The retention times of miloxacin, M-1, and M-2 were 3.8, 9.3, and 5.9 min, respectively. Serum and urine concentrations of these compounds as low as 10 ng/ml were measured. When results from the HPLC assay were compared with those from the microbiological assay of serum and urine samples from human subjects receiving miloxacin orally, the correlation coefficients were 0.94 for the serum and 0.99 for the urine. The HPLC assay method presents an alternative to the microbiological assay and permits future pharmacokinetic investigations of miloxacin.

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.