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Escherichia coli ST131 is a multidrug-resistant lineage associated with the global spread of extended-spectrum β-lactamase-producing organisms. Particularly, ST131 clade C1 is the most predominant clade in Japan, harboring bla CTX-M-14 at a high frequency. However, the process of resistance gene acquisition and spread remains unclear. Here, we performed whole-genome sequencing of 19 E. coli strains belonging to 12 STs and 12 fimH types collected between 1997 and 2016. Additionally, we analyzed the full-length genome sequences of 96 ST131- H 30 clade C0 and C1 strains, including those obtained from this study and those registered in public databases, to understand how ST131 clade C1 acquired and spread bla CTX-M-14 . We detected conjugative IncFII plasmids and IncB/O/K/Z plasmids carrying bla CTX-M-14 in diverse genetic lineages of E. coli strains from the 1990s to the 2010s, suggesting that these plasmids played an important role in the spread of bla CTX-M-14 . Molecular phylogenetic and molecular clock analyses of the 96 ST131- H 30 clade C0 and C1 strains identified 8 subclades. Strains harboring bla CTX-M-14 were clustered in subclades 4 and 5, and it was inferred that clade C1 acquired bla CTX-M-14 around 1993. All 34 strains belonging to subclade 5 possessed bla CTX-M-14 with IS Ecp1 upstream at the same chromosomal position, indicating their common ancestor acquired bla CTX-M-14 in a single IS Ecp1 -mediated transposition event during the early formation of the subclade around 1999. Therefore, both the horizontal transfer of plasmids carrying bla CTX-M-14 to diverse genetic lineages and chromosomal integration in the predominant genetic lineage have contributed to the spread of bla CTX-M-14 ., Competing Interests: The authors declare no conflict of interest.

Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius , the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus Simplexvius , the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection., Competing Interests: The authors declare no conflict of interest.

Although Shewanella spp. are most frequently isolated from marine environments; more rarely, they have been implicated in human infections. Shewanella spp. are also recognized as the origin of genes for carbapenem-hydrolyzing class D β-lactamases. Due to the spread globally among Enterobacterales in recent years, risk assessments of both clinical and environmental Shewanella strains are urgently needed. In this study, we analyzed the whole-genome sequences of 10 clinical isolates and 13 environmental isolates of Shewanella spp. and compared them with those of Shewanella species strains registered in public databases. In addition, the levels of bla transcription and β-lactamase activity of a carbapenem-resistant Shewanella algae isolate were compared with those of carbapenem-susceptible S. algae clade isolates. All clinical isolates were genetically identified as S. algae clade (S. algae, Shewanella chilikensis, and Shewanella carassii), whereas all but one of the environmental isolates were identified as various OXA-55-like spp. outside the S. algae clade. Although all isolates of the S. algae clade commonly possessed an approximately 12,500-bp genetic region harboring Shewanella , genetic structures outside this region were different among species. Among S. algae clade isolates, only one showed carbapenem resistance, and this isolate showed a high level of bla transcription and β-lactamase activity. Although this study documented the importance of the S. algae clade in human infections and the relationship between enhanced production of OXA-55-like and resistance to carbapenems in S. algae, further studies are needed to elucidate the generalizability of these findings. OXA-55-like , genetic structures outside this region were different among species. Among S. algae clade isolates, only one showed carbapenem resistance, and this isolate showed a high level of bla OXA-55-like spp. with those originating from environmental sources. All 10 clinical isolates were genetically identified as members of the Shewanella algae clade (S. algae, IMPORTANCE Shewanella spp., which are known to carry chromosomally located bla species members outside the S. algae clade. Although all the S. algae clade isolates possessed an approximately 12,500-bp genetic region harboring OXA genes, have mainly been isolated from marine environments; however, they can also cause infections in humans. In this study, we compared the molecular characteristics of clinical isolates of Shewanella transcription and β-lactamase activity compared with the carbapenem-susceptible isolates. To confirm the clinical significance and antimicrobial resistance mechanisms of the S. algae clade members, analysis involving more clinical isolates should be performed in the future.S. chilikensis , and S. carassii ); however, all but one of the 13 environmental isolates were identified as Shewanella species members outside the S. algae clade. Although all the S. algae clade isolates possessed an approximately 12,500-bp genetic region harboring bla OXA-55-like , only one isolate showed carbapenem resistance. The carbapenem-resistant isolate showed a high level of bla OXA-55-like transcription and β-lactamase activity compared with the carbapenem-susceptible isolates. To confirm the clinical significance and antimicrobial resistance mechanisms of the S. algae clade members, analysis involving more clinical isolates should be performed in the future.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name " Pteropus lylei -associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus -related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology. IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei -associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei , in Asia. This study expands the knowledge on bat-associated alphaherpesvirology., (Copyright © 2020 American Society for Microbiology.)

Although hypervirulent Klebsiella pneumoniae (hvKp) has been associated with severe community-acquired infections that occur among relatively healthy individuals, information about hvKp infections in health care settings remains limited. Here, we systematically analyzed the clinical and molecular characteristics of K. pneumoniae isolates causing bloodstream infections in a cross-sectional study. Clinical characteristics of K. pneumoniae bloodstream infections from hospitals across Japan were analyzed by a review of the medical records. Whole-genome sequencing of the causative isolates was performed. Bacterial species were confirmed and hvKp were identified using whole-genome sequencing data. Clinical characteristics of hvKp infections were compared with those of non-hvKp infections by bivariate analyses. Of 140 cases of K. pneumoniae bloodstream infections, 26 cases (18.6%) were caused by various clones of hvKp defined by the carriage of cardinal virulence genes. Molecular identification revealed that 24 (17.1%) and 14 (10%) cases were caused by Klebsiella variicola and Klebsiella quasipneumoniae , respectively. Patients with hvKp infections had higher proportions of diabetes mellitus (risk ratio [RR], 1.75; 95% confidence interval [CI], 1.05 to 2.94), and their infections had significantly higher propensity to involve pneumonia (RR, 5.85; 95% CI, 1.39 to 24.6), liver abscess (RR, 5.85; 95% CI, 1.39 to 24.6), and disseminated infections (RR, 6.58; 95% CI, 1.16 to 37.4) than infections by other isolates. More than one-half of hvKp infections were health care associated or hospital acquired, and a probable event of health care-associated transmission of hvKp was documented. hvKp isolates, which are significantly associated with severe and disseminated infections, are frequently involved in health care-associated and hospital-acquired infections in Japan., (Copyright © 2019 American Society for Microbiology.)

Acyclovir (ACV) resistance-associated mutations in two recombinant herpes simplex virus 1 (HSV-1) clones were compared. Recombinant HSV-1 lacking its thymidine kinase (TK) and expressing varicella-zoster virus (VZV) TK ectopically had no mutations in the VZV TK gene. In contrast, recombinant HSV-1 expressing HSV-1 TK ectopically harbored mutations in the HSV-1 TK gene. These results suggest that the relatively low frequency of ACV-resistant VZV is a consequence of the characteristics of the TK gene., (Copyright © 2019 American Society for Microbiology.)

Hypervirulent Klebsiella pneumoniae strains have higher potential to cause more severe and disseminated infections than classic K. pneumoniae strains. While initially reported from East Asian countries, cases have now been identified worldwide, sometimes in conjunction with extensive drug resistance. In this issue of the Journal of Clinical Microbiology , T. A. Russo et al. (J Clin Microbiol 56:e00776-18, 2018, https://doi.org/10.1128/JCM.00776-18) validated the diagnostic accuracy of biomarkers that differentiate hypervirulent K. pneumoniae strains from classic strains. This represents a major step forward in building a consensus definition and designing international studies aimed at elucidating the global epidemiology, clinical features, and outcome of this important pathogen., (Copyright © 2018 American Society for Microbiology.)

We report here the first complete genome sequences of genotype GI.3, GI.4, GI.6, GI.7, and GII.7 sapovirus strains, detected from fecal samples of acute gastroenteritis patients. Complete or nearly complete genome sequences of all 18 genotypes of human sapoviruses are now available for phylogenetic analysis and primer design., (Copyright © 2018 Oka et al.)

Although KPC enzymes are most common among carbapenemases produced by Enterobacter cloacae complex globally, the epidemiology varies from one country to another. While previous studies have suggested that IMP enzymes are most common in Japan, detailed analysis has been scarce thus far. Here, we carried out a molecular epidemiological study and plasmid analysis of IMP-1-producing E. cloacae complex isolates collected from three hospitals in central Tokyo using whole-genome sequencing. Seventy-one isolates were classified into several sequence types (STs), and 49 isolates were identified as Enterobacter hormaechei ST78. Isolates of ST78 were divided into three clades by core-genome single nucleotide polymorphism (SNP)-based phylogenetic analysis. Whereas isolates of clade 3 were isolated from only one hospital, isolates of clade 1 and 2 were identified from multiple hospitals. Ten of 12 clade 1 isolates and 1 of 4 clade 2 isolates carried bla IMP-1 on IncHI2 plasmids, with high similarity of genetic structures. In addition, these plasmids shared backbone structures with IncHI2 plasmids carrying bla IMP reported from other countries of the Asia-Pacific region. All isolates of clade 3 except one carried bla IMP-1 in In1426 on IncW plasmids. An isolate of clade 3, which lacked IncW plasmids, carried bla IMP-1 in In1426 on an IncFIB plasmid. These observations suggest that IMP-producing E. cloacae complex isolates with a diversity of host genomic backgrounds have spread in central Tokyo, and they indicate the possible contribution of IncHI2 plasmids toward this phenomenon., (Copyright © 2018 American Society for Microbiology.)

Unlabelled: Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8(+) cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8(+) cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)(hi)/phosphorylated AMP-activated protein kinase (AMPK)(lo) subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control., Importance: In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Interaction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection also were inhibited without the overexpression of GLUT1, indicating that the localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in intracellular compartments separate from that of Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition probably is due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env., Importance: The deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhances the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We document the household spread of extended-spectrum β-lactamase-producing Escherichia coli. One isolate belonged to sequence type 1193 and caused urinary tract infection in a 4-month-old female, and the other isolate belonged to sequence type 131 and colonized three family members, including the index patient. These isolates carried similar Inc-I1-Iγ plasmids, harboring blaCTX-M-15.

Analysis of five CTX-M-2-producing Proteus mirabilis isolates in Japan demonstrated that bla(CTX-M-2) was located on the chromosome in four isolates and on IncT plasmids in three isolates, including two isolates that also carried the gene on the chromosome. In all four isolates with chromosomal bla(CTX-M-2), ISEcp1 was responsible for the integration of the gene into the chromosome. Three different sites in the P. mirabilis genomic sequence were utilized as integration sites.

Tripropeptin C (TPPC) is a naturally occurring cyclic lipodepsipeptide antibiotic produced by a Lysobacter sp. TPPC exhibits potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant Streptococcus pneumoniae. This antibiotic also inhibits the incorporation of N-acetylglucosamine into the peptidoglycan of S. aureus at a 50% inhibitory concentration (IC(50)) of 0.7 μM, which is proportional to its MIC (0.87 μM; equivalent to 1.0 μg/ml). Treatment of exponential-phase S. aureus cells with TPPC resulted in accumulation of UDP-MurNAc-pentapeptide in the cytoplasm. The antimicrobial activity of TPPC was weakened by the addition of prenyl pyrophosphates but not by prenyl phosphates, UDP-linked sugars, or the pentapeptide of peptidoglycan. The direct interaction between TPPC and undecaprenyl pyrophosphate (C(55)-PP) was observed by mass spectrometry and thin-layer chromatography analysis, indicating that TPPC can potentially inhibit C(55)-PP phosphatase activity, which plays a crucial role in the lipid cycle of peptidoglycan synthesis. As expected, TPPC inhibits this enzymatic reaction at an IC(50) of 0.03 to 0.1 μM in vitro, as does bacitracin. From the analysis of accumulation of lipid carrier-related compounds, TPPC was found to cause the accumulation of C(55)-PP in situ, leading to the accumulation of a glycine-containing lipid intermediate. This suggested that the TPPC/C(55)-PP complex also inhibits the transglycosylation step or flippase activity, adding to the inhibition of C(55)-PP dephosphorylation. This mode of action is different from that of currently available drugs such as vancomycin, daptomycin, and bacitracin.

Capsule-forming Klebsiella pneumoniae K1 caused primary liver abscess in two household members of a family. The causative isolates had identical pulsed-field gel electrophoresis patterns and were determined to be sequence type 23. An additional member of the family was found to carry the same strain without clinical manifestation.

For 4 months from September 2008, 102 conjunctival swab specimens were collected for surveillance purposes from patients across Japan suspected of having epidemic keratoconjunctivitis (EKC). Human adenovirus (HAdV) DNA was detected in 61 samples by PCR, though the HAdV type for 6 of the PCR-positive samples could not be determined by phylogenetic analysis using a partial hexon gene sequence. Moreover, for 2 months from January 2009, HAdV strains with identical sequences were isolated from five conjunctival swab samples obtained from EKC patients in five different regions of Japan. For the analyses of the 11 samples mentioned above, we determined the nucleotide sequences of the entire penton base, hexon, and fiber genes and early 3 (E3) region, which are variable regions among HAdV types, and compared them to those of other HAdV species D strains. The nucleotide sequences of loops 1 and 2 in the hexons of all 11 samples showed high degrees of identity with those of the HAdV type 15 (HAdV-15) and HAdV-29 prototype strains. However, the fiber gene and E3 region sequences showed high degrees of identity with those of HAdV-9, and the penton base gene sequence showed a high degree of identity with the penton base gene sequences of HAdV-9 and -26. Moreover, the complete genome sequence of the 2307-S strain, which was isolated by viral culture from 1 of the 11 samples, was determined. The 2307-S strain was a recombinant HAdV between HAdV-9, -15, -26, -29, and/or another HAdV type; however, the recombination sites in the genome were not obvious. We propose that this virus is a novel intertypic recombinant, HAdV-15/29/H9, and may be an etiological agent of EKC.

Integrating conjugative elements (ICEs) are mobile genetic elements that can transfer from the chromosome of a host to the chromosome of a new host through the process of excision, conjugation, and integration. Although SXT/R391-related ICEs, originally demonstrated in Vibrio cholerae O139 isolates, have become prevalent among V. cholerae isolates in Asia, the prevalence of the ICEs among gram-negative bacteria other than Vibrio spp. remains unknown. In addition, SXT/R391-related ICEs carrying genes conferring resistance to extended-spectrum cephalosporins have never been described. Here we carried out a genetic analysis of a cefoxitin-resistant Proteus mirabilis clinical isolate, TUM4660, which revealed the presence of a novel SXT/R391-related ICE, ICEPmiJpn1. ICEPmiJpn1 had a core genetic structure showing high similarity to that of R391 and carried xis and int genes completely identical to those of R391, while an IS10-mediated composite transposon carrying bla(CMY-2) was integrated into the ICE. A nucleotide sequence identical to the 3' part of ISEcp1 was located upstream of the bla(CMY-2) gene, and other genes observed around bla(CMY-2) in earlier studies were also present. Furthermore, the nucleotide sequences of hot spot 2 and hot spot 4 in ICEPmiJpn1 showed high similarity to that of hot spot 2 in SXT(MO10) and with a part of the nucleotide sequence found in P. mirabilis ATCC 29906, respectively. ICEPmiJpn1 was successfully transferred to Escherichia coli, Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Citrobacter koseri in conjugation experiments. These observations suggest that ICEs may contribute to the dissemination of antimicrobial resistance genes among clinically relevant Enterobacteriaceae, which warrants careful observation of the prevalence of ICEs, including SXT/R391-related ICEs.

N-(4-Chlorophenyl)-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide (NBD-556) is a low-molecular-weight compound that reportedly blocks the interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and its receptor CD4. We investigated whether the enhancement of binding of anti-gp120 monoclonal antibodies (MAbs) toward envelope (Env) protein with NBD-556 are similar to those of soluble CD4 (sCD4) by comparing the binding profiles of the individual MAbs to Env-expressing cell surfaces. In flow cytometric analyses, the binding profiles of anti-CD4-induced epitope (CD4i) MAbs toward NBD-556-pretreated Env-expressing cell surfaces were similar to the binding profiles toward sCD4-pretreated cell surfaces. To investigate the binding position of NBD-556 on gp120, we induced HIV-1 variants that were resistant to NBD-556 and sCD4 in vitro. At passage 21 in the presence of 50 microM NBD-556, two amino acid substitutions (S375N in C3 and A433T in C4) were identified. On the other hand, in the selection with sCD4, seven mutations (E211G, P212L, V255E, N280K, S375N, G380R, and G431E) appeared during the passages. The profiles of the mutations after the selections with NBD-556 and sCD4 were very similar in their three-dimensional positions. Moreover, combinations of NBD-556 with anti-gp120 MAbs showed highly synergistic interactions against HIV-1. We further found that after enhancing the neutralizing activity by adding NBD-556, the contemporaneous virus became highly sensitive to antibodies in the patient's plasma. These findings suggest that small compounds such as NBDs may enhance the neutralizing activities of CD4i and anti-V3 antibodies in vivo.

We have recently reported that the attachment of a bulky metabolically stable tert-butyl group at the C-2 position of a quinoline ring in primaquine results in a tremendous improvement in the blood schizontocidal antimalarial activity of 8-quinolinamine. Because free heme released from hemoglobin catabolism in a malarial parasite is highly toxic, the parasite protects itself mainly by crystallization of heme into insoluble nontoxic hemozoin. We now demonstrate the ability of 2-tert-butylprimaquine to inhibit in vitro beta-hematin formation, to form a complex with heme with a stoichiometry of 1:1, and to enhance heme-induced hemolysis. The results described herein indicate that a major improvement in the blood-schizontocidal antimalarial activity of 2-tert-butylprimaquine might be due to a disturbance of heme catabolism pathway in the malarial parasite.

Current assays for screening new antimalarials need initiators of beta-hematin formation that require laborious preparation, special devices, and substrates. In this study, based on reduction of heme absorption in beta-hematin formation, we developed a simple colorimetric assay using Tween 20 as an initiator and a microplate reader for high-throughput screening of inhibitors of beta-hematin formation.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.

Immunoglobulin G (IgG) antibodies to Epstein-Barr virus (EBV) nuclear antigens 2 and 1 (EBNA-2 and EBNA-1, respectively) were studied using sera from healthy individuals of a population with a high incidence of asymptomatic primary EBV infections during infancy or childhood in Japan. Two CHO-K1 cell lines expressing EBNA-2 and EBNA-1 were used for anticomplement and indirect immunofluorescence assays. The positivity rate for EBNA-2 IgG rose in the 1- to 2-year age group, increased and remained at a plateau ( approximately 45%) between 3 and 29 years of age (3- to 4-, 5- to 9-, 10- to 14-, and 15- to 29-year age groups), and then reached 98% by age 40 (>/== 40-year age group). Both seropositivity for EBNA-1 and seropositivity for EBNAs in Raji cells (EBNA/Raji) were detected in the 1- to 2-year age group, remained high, and finally reached 100% by age 40. The geometric mean titer (GMT) of EBNA-2 IgG reached a plateau in the 5- to 9- and 10- to 14-year-old groups and remained elevated in the older age groups (15 to 29 and >/== 40 years). The GMT of EBNA-1 IgGs increased to a plateau in the 1- to 2-year-old group and remained unchanged in the older age groups. The GMT of EBNA/Raji IgGs also reached a plateau in the 1- to 2-year-old group, remained level throughout the 3- to 14-year age groups, and decreased in the 15- to 29-year-olds. EBNA-2 IgGs emerged earlier than EBNA-1 IgGs in 8 of 10 patients with infectious mononucleosis, who were between 1 and 27 years old, and declined with time in three of eight cases. These results suggest that EBNA-2 IgG antibodies evoked in young children by asymptomatic primary EBV infections remain elevated throughout life, probably because of reactivation of latent and/or exogenous EBV superinfection.

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His(6) tag was efficiently purified to homogeneity by Ni(2+) affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.

Human immunodeficiency virus type 1 (HIV-1) heterogeneity contributes to the emergence of drug-resistant virus, escape from host defense systems, and/or conversion of the cellular tropism. To establish an in vitro system to address a heterogeneous virus population, we constructed a library of HIV-1 molecular clones containing a set of random combinations of zero to 11 amino acid substitutions associated with resistance to protease inhibitors by the HIV-1 protease. The complexity (2.1 x 10(5)) of the HIV-1 library pNG-PRL was large enough to cover all of the possible combinations of zero to 11 amino acid substitutions (a total of 4,096 substitutions possible). The T-cell line MT-2 was infected with the HIV-1 library, and resistant viruses were selected after treatment by the protease inhibitor ritonavir (0.03 to 0.30 microM). The viruses that contained three to eight amino acid substitutions could be selected within 2 weeks. These results demonstrate that this HIV-1 library could serve as an alternative in vitro system to analyze the emergence of drug resistance and to evaluate the antiviral activity of novel compounds against multidrug-resistant viruses.

EBNA-LP-associated proteins were identified by sequencing proteins that immunoprecipitated with Flag epitope-tagged EBNA-LP (FLP) from lymphoblasts in which FLP was stably expressed. The association of EBNA-LP with Hsp70 (72/73) was confirmed, and sequences of DNA-PK catalytic subunit (DNA-PKcs), HA95, Hsp27, prolyl 4-hydroxylase alpha-1 subunit, alpha-tubulin, and beta-tubulin were identified. The fraction of total cellular HA95 that associated with FLP was very high, while progressively lower fractions of the total DNA-PKcs, Hsp70, Hsp 27, alpha-tubulin, and beta-tubulin specifically associated with EBNA-LP as determined by immunoblotting with antibodies to these proteins. EBNA-LP bound to two domains in the DNA-PKcs C terminus and DNA-PKcs associated with the EBNA-LP repeat domain. DNA-PKcs that was bound to EBNA-LP phosphorylated p53 or EBNA-LP in vitro, and the phosphorylation of EBNA-LP was inhibited by Wortmannin, a specific in vitro inhibitor of DNA-PKcs.

Previous genetic and biochemical analyses have indicated that the Epstein-Barr virus EBNA-2 amino terminus is important for primary B-lymphocyte growth transformation and may be involved in self-association. We now report that EBNA-2 has at least two domains, amino acids 1 to 60 and 96 to 210, which independently mediate homotypic association, 1 to 60 with 1 to 60 and 96 to 210 with 96 to 210. EBNA-2 self-association is likely to be critical to the ability of EBNA-2 to interact simultaneously with multiple cellular transcription factors, coactivators, and histone acetyltransferases through its RBPJkappa binding and acidic activating domains.

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is a transcriptional transactivator of cellular and viral gene expression and is essential for the transformation of resting human B lymphocytes into long-term lymphoblastoid cell lines (LCLs). Previous molecular genetic analyses identified three domains that are critical for transformation and showed that the rest of EBNA-2 is not critical. We now find that codons 231 to 280 that were part of one of the critical domains (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991) can be deleted with only a small effect on the ability of EBNA-2 to transactivate gene expression. In transient transfection assays, EBNA-2 deleted for codons 231 to 280 accumulated to higher levels and was similar to wild-type EBNA-2 in activation of the BamC promoter and in association with RBPJk, a cellular transcription factor that is important for EBNA-2 interaction with promoter regulatory elements. However, EBNA-2 d231-280 activated the viral latent membrane protein 1 (LMP1) promoter with only 60% of wild-type efficiency. Recombinant EBVs specifically deleted for EBNA-2 codons 231 to 280 were efficient in initiating the transformation of resting primary human B lymphocytes into LCLs. However, these LCLs grew less well than wild-type EBV-transformed LCLs, and 4- to 10-fold more cells were required for outgrowth following limit dilution. EBNA-2 d231-280 accumulated to unusually high levels in the recombinant transformed LCLs, and this was associated with somewhat higher EBNA-1 and lower LMP1 expression, consistent with the near-wild-type activation of the BamC EBNA promoter and the abnormally low activation of the LMP1 promoter in transient transfection assays. Thus, EBNA-2 d231-280 modestly perturbed the regulation of viral gene expression and resulted in less LMP1, while having surprisingly subtle effects on LCL outgrowth. Deletion of EBNA-2 codons 292 to 310, which are closer to the site that specifies interaction with RBPJk, was more disruptive of RBPJk association and of the ability to transform B lymphocytes.

The retroviral attachment (att) sites at viral DNA ends are cis-acting regions essential for proviral integration. To investigate the sequence features of att important for human immunodeficiency virus type 1 (HIV-1) integration in vivo, we generated a series of 25 att mutants of HIV-1 by mutagenesis of the U3, U5, or both boundaries of att. Our results indicated that the terminal 11 or 12 bp of viral DNA are sufficient for specific recognition by HIV-1 integrase (IN) and suggested that IN might recognize each att site independently in vivo.

CD8+ T lymphocytes of asymptomatic human immunodeficiency virus type 1 (HIV-1) carriers (AC) suppress HIV-1 replication in vitro. Failure of host defense mechanisms and increased virus proliferation are associated with disease progression. The exact mechanisms inducing these changes at the advanced stage of the disease are still obscure. In this study, we searched for experimental conditions favoring the abrogation of the suppression of viral replication in peripheral blood mononuclear cells (PBMC) of AC by using various pharmacological and biological probes modifying cell activation. Among such agents, staphylococcal enterotoxin B (SEB) and phorbol 12-myristate 13-acetate (PMA) markedly increased otherwise low levels of HIV-1 replication in cultures of phytohemagglutinin-stimulated AC PBMC following in vitro HIV-1 LAI infection. A similar but less pronounced virus induction was also observed in macrophage-tropic HIV-1. Individual pretreatment of CD4+ and CD8+ PBMC fractions with these agents caused a reduction in CD8+ cell proliferation and enhanced HIV-1 replication in CD4+ cells. SEB- and PMA-mediated augmentation of HIV-1 replication in AC PBMC was significantly blocked by neutralizing antibody to tumor necrosis factor-alpha (TNF-alpha), although recombinant TNF-alpha alone failed to reproduce the effects of SEB or PMA. Our results suggest that the induction of TNF-alpha may be one of the mechanisms that overcomes the CD8+-induced suppression of HIV-1 replication in AC and that it may induce HIV-1 replication.

Epstein-Barr virus (EBV) nuclear proteins EBNA-LP and EBNA-2 are the first two proteins expressed in latent infection of primary B lymphocytes. EBNA-2 is essential for lymphocyte transformation, and EBNA-LP is at least critical. While EBNA-2 activates specific viral and cellular promoters, EBNA-LP's role has been obscure. We now show that EBNA-LP stimulates EBNA-2 activation of the LMP1 promoter and of the LMP1/LMP2B bidirectional transcriptional regulatory element. EBNA-LP alone has only a negative effect. EBNA-LP also stimulates EBNA-2 activation of a multimerized regulatory element from the BamC EBNA promoter. Since both viral regulatory elements can bind the EBNA-2-associated cell protein RBPJ kappa, consensus RBPJ kappa binding sites were positioned upstream of the herpes simplex virus type 1 thymidine kinase promoter and were found to be sufficient for EBNA-LP and EBNA-2 coactivation. EBNA-LP strongly stimulated activation of an adenovirus E1b promoter with upstream Gal4 binding sites by a Gal4 DNA binding domain/ EBNA-2 acidic domain fusion protein, indicating that EBNA-LP coactivation requires only the EBNA-2 acidic domain to be localized near a promoter. The EBNA-LP stimulatory activity resides in the amino-terminal 66-amino-acid repeat domain. The carboxyl-terminal unique 45 amino acids appear to regulate EBNA-LP's effects. The first 11 amino acids of the 45 have a strong negative effect, while the last 10 are critical for the ability of the last 34 to relieve the negative effect. These results indicate that EBNA-LP's critical role in EBV-mediated cell growth transformation is in stimulating (and probably regulating) EBNA-2-mediated transcriptional activation.

Previous molecular genetic analyses of Epstein-Barr virus nuclear protein 2 (EBNA2) identified a negative effect of deletion of codons 19 to 33 on transformation and gene transactivation, while deletion of codons 19 to 110 was a null mutation for transformation and gene transactivation. We here report the surprising finding that codons 2 to 88, which encode the highly conserved unique N terminus (amino acids 1 to 58) and most of the polyproline repeat (amino acids 59 to 95), can be deleted with only minimal effects on transformation. Codons 97 to 122 can also be deleted with only minimal effects on transformation. However, deletion of 35 of the 37 prolines (amino acids 59 to 93) or deletion of codons 2 to 95 results in a null transforming phenotype. Although EBNA2 from which codons 59 to 93 were deleted was a null mutation for transformation, it was similar to some transforming mutants of EBNA2 in abundance, in interaction with RBPJK, and in transactivation of the LMP1 promoter in transient transfection assays. These data indicate that between three and seven prolines are critical for EBNA2 structure or for intermolecular interaction. Aside from these seven prolines, codons encoding the rest of the N-terminal half (amino acids 2 to 230) of EBNA2 are nonessential for primary B-lymphocyte growth transformation.

Since deletion of region 3 (amino acids [aa] 333 to 425) of Epstein-Barr virus nuclear protein 2 (EBNA-2) results in EBV recombinants which cannot transform primary B lymphocytes (J. I. Cohen, F. Wang, and E. Kieff, J. Virol. 65:2545-2554, 1991), the role of domains of region 3 was investigated. Deletion of the Arg-Gly repeat domain, R-337GQSRGRGRGRGRGRGKG354, results in EBV recombinants that transform primary B lymphocytes with modestly decreased activity. The transformed cells grow slowly and are difficult to expand. EBNA-2 deleted for the Arg-Gly domain does not associate with the nuclear chromatin fraction. The Arg-Gly repeat has an intrinsic ability to bind to histone H1, to other proteins, including EBNA-1, and to nucleic acids, especially poly(G). Two independent deletions of each part of the rest of region 3 (aa 359 to 383 and 385 to 430) have little effect on transformation, while deletion of the rest of region 3 (aa 361 to 425) as a single segment substantially reduces transformation efficiency. EBNA-2 deleted for all of region 3 can still transactivate the LMP1 promoter in transient expression assays but is less active than EBNA-2 in transactivating the BamHI-C promoter. EBNA-2 deleted for the Arg-Gly domain is better than EBNA-2 at transactivating the LMP1 promoter and is as active as EBNA-2 in transactivating the BamHI-C promoter. These data are most compatible with a model in which the Arg-Gly domain of region 3 is a modulator of EBNA-2 interactions and activities, while the rest of region 3 is important in positioning the region 2 J kappa binding domain relative to the region 4 acidic transactivating domain. Despite the null phenotype of the region 3 deletion, region 3 is unlikely to mediate essential interactions with other proteins.

A synthetic peptide, RPI 312, that specifically inhibits the protease of the human immunodeficiency virus type 1 (HIV-1) showed a potent inhibition on virus production, maturation, and infectivity. Treatment with this agent prevented the cleavage of Gag protein at the site between p17 and p24 in HIV-1 chronically infected MOLT-4 cells as well as in the released virus. Passage of HIV-1 in the presence of gradually increasing concentrations of this protease inhibitor resulted in emergence of a variant that could evade the drug effects. In the resistant variant the maturation of Gag proteins appeared normal, but its infectivity was reduced compared with that of the parent virus. The nucleotides coding the amino acids at and around the cleavage site between Gag proteins p17 and p24 were not changed. One point mutation (A-->G) at site 2082 of the pol gene that resulted in one amino acid change at site 84 of the protease from isoleucine to valine (I-84-->V) could be detected in the resistant variant. An HIV-1 infectious DNA clone with the I-84-->V mutation also showed reduced sensitivity to this protease inhibitor. The findings that the resistant variant had lower infectivity and was still affected by higher doses of the drug support the speculation that resistance to protease inhibitors may not be as problematic as other drug resistance.

Two new enzyme-linked immunosorbent assays (ELISAs) with chimeric fusion polypeptides for the detection of human antibodies specific to Epstein-Barr virus nuclear antigen 1 (EBNA-1) are described. One is an indirect ELISA with affinity-purified beta-galactosidase-EBNA-1 fusion protein as the antigen. The other is a "sandwich" assay based on the use of anti-beta-galactosidase antibody to capture beta-galactosidase-EBNA-1 fusion proteins in bacterial extracts. A good correlation was shown between antibody titers determined by the ELISA with the EBNA-1 fusion proteins and those determined by a conventional anticomplement immunofluorescence test which is being widely performed with Raji cells for the purpose of research and clinical diagnosis. The advantage of the ELISAs for seroepidemiologic studies on Epstein-Barr virus was demonstrated by sensitive detection of marginal immunoglobulin G antibody to the EBNA-1 domain in serum samples from patients with infectious mononucleosis.

The putative envelope protein of hepatitis C virus (HCV) was expressed in insect cells by using a baculovirus expression vector and in monkey COS cells under the control of exogenous promoters. The expressed envelope proteins, identified by immunoblot analysis using sera from patients with chronic HCV infection, were a series of glycoproteins of 35 to 24 kDa (gp35-24) in insect cells and a single species of glycoprotein of 35 kDa (gp35) in monkey cells. The size difference of these proteins was due to the different degrees of glycosylation. The envelope proteins expressed in these cells were produced by common specific cleavage from the precursor protein, and cleavage positions of the envelope protein were mapped at about amino acids 190 and 380. The gp35-24 proteins expressed in insect cells were used for detection of antibody against HCV envelope protein in patient sera. The results showed that (i) the antibody is detected in 2 to 17% of various patients with hepatitis C, (ii) three patients were apparently cured after acquiring the antienvelope antibody, and (iii) in sera of patients with more than a 20-year history of infection, the antibody sometimes coexisted with HCV. These results suggest that the antienvelope antibody is neutralizing only in limited number of patients with hepatitis C.

A structural protein of hepatitis C virus (HCV) was expressed in monkey COS cells under the control of an exogenous promoter, and a protein of 22 kDa was identified by immunoblot analysis. This protein (p22), which was produced by processing in COS cells, reacted specifically to sera of chronic hepatitis C patients, and its coding region was mapped at the most amino-terminal part of the HCV polyprotein. These results suggested that the p22 protein is the nucleocapsid (core) protein of HCV. Moreover, the assay detecting antibody to p22 was found to be useful for early diagnosis of HCV infection.

A long asymptomatic period is one of the characteristics of human immunodeficiency virus (HIV) infection, despite its fatal consequences. Antiviral defense in HIV-infected individuals controls viral replication during this period. In the present study, we demonstrate that peripheral blood leukocytes (PBL) of asymptomatic HIV-1 carriers, following exogenous HIV-1 infection in vitro, do not support viral replication. These cells do not produce detectable amounts of reverse transcriptase or accumulate unintegrated proviral DNA. This is a striking contrast to the behavior of HIV-1-infected PBL of seronegative individuals, which produce large amounts of RT and unintegrated DNA. Such resistance to HIV-1 replication is not seen in PBL of patients with advanced disease. Since the binding of HIV-1 to CD4 molecule is not impaired in PBL of asymptomatic carriers, the interference with HIV replication must occur after the stage of virus binding. PBL lose their resistance when CD8+ lymphocytes are removed. In addition, these PBL are not resistant to an exogenous infection with HIV-2. These observations suggest that certain populations of CD8+ lymphocytes of asymptomatic HIV-1 carriers operate on the target cells in PBL to block viral replication in an HIV-1-specific manner. Such CD8+ lymphocyte-mediated interference with HIV replication could play an important role in the maintenance of the period of disease latency.

Cells of human leukemic T-cell line Molt-4 which were cloned in soft agarose from individual colonies were analyzed for the cytopathic effect and viral antigen expression after human T-cell lymphotropic virus type III infection. The induction of cytopathic effect and viral antigens by the infection varied significantly among the different cell clones. These clones did not show significant differences in the amount of OKT-4 antigen, a possible surface receptor for acquired immunodeficiency syndrome virus. Moreover, the virus-producing ability and the susceptibility to the virus of each clone appeared to be separable.

MT-4 cells, which are a human T-cell lymphotropic virus type I (HTLV-I)-positive cell line highly permissive to HTLV-III infection, were used to detect the biologically active virus. For quantitation of the virus, induction of HTLV-III-specific antigen(s) and inhibition of DNA synthesis in infected MT-4 cells were assessed by indirect immunofluorescence and by a proliferation assay measuring [3H]thymidine uptake, respectively. HTLV-III was fully inactivated by treatment at 56 degrees C for 30 min. It was not inactivated by treatment with fresh anti-HTLV-III-negative serum. Thus, these assay systems with MT-4 cells would be useful in further studies on acquired immune deficiency syndrome.

Human T cell lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus is the etiologic agent of the acquired immune deficiency syndrome (AIDS) and AIDS-related complex. The effect of 3'-azido-3'-deoxythymidine (AZT) on the HTLV-III/lymphadenopathy-associated virus infection was quantitatively studied with HTLV type I-carrying MT-4 cells. The AZT compound inhibited HTLV-III-induced cytopathic effect and virus-specific antigen expression in MT-4 cells at concentrations of 5 and 10 microM. In addition, a plaque-forming assay was performed to assess the effect of AZT on virus replication in MT-4 cells freshly infected with HTLV-III and in continuous HTLV-III-producing Molt-4/HTLV-III cells. Results showed that AZT efficiently and effectively inhibited the replication of HTLV-III in infected MT-4 cells. AZT is a strong inhibitor of reverse transcriptase activity of HTLV-III as a triphosphate, to such a degree that even 1.0 pM azido-TTP inhibits 50% of reverse transcriptase activity. However, it did not show any effect in the HTLV-III-producing cell line Molt-4/HTLV-III. Thus, AZT has no effect on virus replication of an already integrated virus. When 5 microM AZT was added to HTLV-III-infected MT-4 within 20 h after infection, a striking suppressive effect was noticed. This concentration was much lower than that which inhibits the growth of MT-4 cells. These results confirm those found in a previous report (H. Mitsuya, K. J. Weinhold, P. S. Furman, H. S. Clair, S. N. Lehrman, R. C. Gallo, D. Bolognesi, D. W. Barry, and S. Broder, Proc. Natl. Acad. Sci. USA 82:7096-7100, 1985) and suggest that AZT might be used as an experimental antiviral agent for AIDS and AIDS-related complex.

Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.

Maridomycin, a new macrolide antibiotic, and tetrahydromaridomycin were acylated into their mono, di, and tri acyl derivatives. These derivatives were compared with the parent antibiotic, maridomycin, for their (i) in vitro antimicrobial activities, (ii) protective effect in mice infected with Staphylococcus aureus (oral administration), (iii) blood levels attained in rats, and (iv) acute toxicity in mice (intraperitoneal administration). All the derivatives showed either the same or less activity in vitro, but 9-acyl, 9, 2'-diacylmaridomycin and 9, 13, 2'-triacetyltetrahydromaridomycin demonstrated improved therapeutic effects together with higher blood levels and low toxicity. 9-Propionylmaridomycin showed the most favorable biological properties.