Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ?+ ?M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ?+ ?M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.     

Studies in chimpanzees of live, attenuated hepatitis A vaccine candidates

Human hepatitis A virus was attenuated in virulence for chimpanzees by passage in FRhK6 and human diploid lung fibroblast cell cultures. A number of variants were developed by passage in cell cultures which showed different levels of virulence/attenuation for chimpanzees. These results were compared to those obtained with marmosets and reported previously. In general, most variants behaved similarly in the two animal types. Two chimpanzees which gave vaccine-like responses following inoculation with HAV cell culture variants were challenged with virulent HAV. Both animals were immune to HAV infection. These findings provide further evidence for the feasibility of developing live, attenuated vaccines against human hepatitis A.     

Changes in virulence, M protein and IgG Fc receptor activity in a type 12 group A streptococcal strain during mouse passages

A type 12 group A strain (1800) was passaged serially through mice 25 times. The ability to servive in normal human blood dropped from a growth index of 52 after the first passage to 1 after four passages. After 14 passages the growth index increased again and stabilized above 30. The virulence for mice increased from a LD100 of 10(8) colony forming units (CFU) to 10-100 CFU after 7 passages and then remained constant. The Mqw antigen disappeared after 4 passages as tested by immunodiffusion, electroimmunoassay and indirect bactericidal tests. Three antisera, raised in rabbits against strains originally belonging to types M3, M12 and M46 but devoid of type antigens after mouse passages showed high bactericidal indices against the 1800 strain after 14 or more passages on mice. Anti-type M1 serum was also found bactericidal for the passaged strains. The IgG Fc-receptor activity of the strain isolated after each mouse passage was tested in hemagglutination experiments with human red blood cells coated with "incomplete" anti-Rh and hot hydrochloric acid extracts of the strains. The capacity to agglutinate "Ripley"-coated cells increased gradually during the first 12 passages and subsequently the titres of the extracts stabilized between 1:160 and 1:320. The HUN coat, useful for detection of the G3m (5) maraker gave titraes increasing with the number of passages while the titres for IgG1 coats kept at 1:4 or below. On background of these results, the possible role of the IgG Fc-receptor as a virulence factor is discussed.     

A single amino acid substitution in the murine norovirus capsid protein is sufficient for attenuation in vivo

Murine norovirus (MNV), a prevalent pathogen of laboratory mice, shares many characteristics with human noroviruses. Previous results indicated that passage of MNV1 in the macrophage cell line RAW 264.7 results in attenuation in STAT1-deficient mice (C. E. Wobus, S. M. Karst, L. B. Thackray, K. O. Chang, S. V. Sosnovtsev, G. Belliot, A. Krug, J. M. Mackenzie, K. Y. Green, and H. W. Virgin, PLoS. Biol. 2:e432, 2004). Sequence analysis revealed two amino acid differences between the virulent and attenuated viruses. Using an infectious cDNA clone of the attenuated virus, we demonstrated that a glutamate-to-lysine substitution at position 296 in the capsid protein (VP1) is sufficient to restore virulence in vivo, identifying, for the first time, a virus-encoded molecular determinant of norovirus virulence.     

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

Background

It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.

Methodology/Principal Findings

By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.

Conclusions/Significance

These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.     

The characteristics of the microflora isolated in bovine endometritis]

117 pure bacterial cultures of numerous species isolated by the authors at acute postnatal pyo-catarrhal cattle endometritis have been studied for their virulence and resistance to 18 antibiotics with the aim to determine their etiologic role in the development of the given pathology and to make a prediction estimation of possible therapeutic and preventive efficiency of the antibiotics. The experiments on white mice have revealed pathogenic properties and virulence in most studied strains of bacteria of different species. The studied microflora is, mainly, resistant practically to all 18 tested antibiotics. A direct dependence is established between virulence of the microflora, isolated in case of endometritis, and its resistance to antibiotics.     

Correlation between the virulence of streptococci for mice and their IgG FcR activity in in vivo experiments

The immunological analysis of 24 spontaneous Strr, Rifr and Kanr mutants of streptococcal strain IP, highly virulent for mice and capable of binding polyclonal human IgG (IgG FcR+), was made. The characteristic feature of all these mutants was decreased virulence, restored after their passage in vivo. 23 mutants were capable of binding polyclonal IgG; one Strr mutant had no such capacity, but acquired it, together with an increase in virulence, after its passage in vivo. When stored in meat-peptone agar without antibiotics, 5 out of 10 Strr mutants lost their capacity for binding polyclonal human IgG. After passage in vivo they regained this property simultaneously with virulence.     

A comparison of their virulence for mice and the enzymatic activity of Streptococcus pneumoniae strains

In most cases no correlation between the virulence of S. pneumoniae and their enzymatic activity was registered in 101 S. pneumoniae strains isolated in pneumococcal infections of different localization. Pneumococcal strains belonging to different serotypes and characterized by their low virulence for mice (LD50 = 10(6) colony-forming units) had the highest neuraminidase and protease-alzolase activity in comparison with highly virulent cultures of these bacteria. In pneumococcal cultures in the R-form avirulence for mice occurred mainly in combination with low enzymatic activity.     

Transmissive drug resistance and virulence of "epidemic" strains of S. typhimurium]

A total of 59 "epidemic" strains of S. typhimurium and 52 cultures isolated from the cases with sporadic diseases were studied with respect to the nature of their resistance to 10 antibiotics and virulence for albino mice under conditions of subcutaneous and oral inoculation. The virulence of the cultures isolated from the cases with sporadic diseases was higher in the strains sensitive to antibiotics. The "epidemic" strains were characterized by the lowest virulence for the mice and resistance to 8-10 antibiotics simultaneously with the transmissive nature of resistance to 1-5 drugs. The transmissive nature of resistance to antibiotics and its spectrum may serve an additional epidemiological marker of the strains.     

The virulence of Streptococcus pneumoniae strains--the causative agents of pneumococcal infection at different sites]

A total of 256 S. pneumoniae strains, the causative agents of infectious processes with different localization, were studied for their virulence (in experiments on mice), neuraminidase and aldolase-protease activity (APA). In pneumococcal strains isolated 18-20 hours after intraperitoneal infection their virulence for mice increased, on the average, 1,000-fold and the average level of extracellular and cellular neuraminidase and APA increased 2- to 5-fold in comparison with the initial values. Pneumococcal strains causing acute pneumococcal infections with different localization, or the aggravation of such infections, exhibited higher virulence for mice and higher levels of neuraminidase and APA, while the inflammatory process at the period of clinical remissions was mainly maintained by S. pneumoniae cultures with low virulence.     

Experimental Infection with Parent and L-Phase Variants of Neisseria meningitidis

Thirty-six strains of Neisseria meningitidis, including groups A, B, and C, produced L forms in vitro in the presence of an osmotic stabilizer and high concentrations of horse serum using penicillin as the transforming agent. Transformation to L growth occurred most readily among strains recently isolated from patients, and an unusually high rate of transformation was observed in 7 of the 36 strains. Revertant L strains developed diplococcal colonies on blood-agar and L colonies on sucrose-serum-penicillin-agar-always in a ratio of approximately 10 to 100 diplococcal colonies to 1 L colony. Using mucin as a host depressant, comparison was made between parent and revertant L strains of their initial pathogenicity and development of virulence by serial mouse passage. In general, revertant L strains showed the same pathogenic characteristics as the parent. Heart blood cultures from mice dying of infection with revertant L strains retained their ability to grow as L forms on penicillin media. Three stable L strains were completely avirulent for mice, although persistence of L forms could be demonstrated in peritoneal exudate for 6 days after inoculation.     

Actinomycins inhibit the production of the siderophore pyoverdines in the plant pathogen Pseudomonas cichorii SPC9018

ABSTRACT

Pyoverdines, a group of peptide siderophores produced by Pseudomonas species, function not only in iron acquisition, but also in their virulence in hosts. Thus, chemical inhibition of pyoverdine production may be an effective strategy to control Pseudomonas virulence. In the plant pathogen Pseudomonas cichorii SPC9018 (SPC9018), pyoverdine production is required for virulence on eggplant. We screened microbial culture extracts in a pyoverdine-production inhibition assay of SPC9018 and found Streptomyces sp. RM-32 as a candidate-producer. We isolated two active compounds from RM-32 cultures, and elucidated their structures to be actinomycins X₂ and D. Actinomycins X₂ and D inhibited pyoverdine production by SPC9018 with IC₅₀ values of 17.6 and 29.6 μM, respectively. Furthermore, pyoverdine production in other Pseudomonas bacteria, such as the mushroom pathogen P. tolaasii, was inhibited by the actinomycins. Therefore, these actinomycins may be useful as chemical tools to examine pyoverdine functions and as seed compounds for anti-Pseudomonas virulence agents.     

季节性流感病毒 H1N1 BALB / c 鼠肺适应株的建立及其分子机制简

目的 建立季节性流感病毒H1N1的鼠肺适应株,并对适应的分子机理进行研究.方法 以病毒滴鼻感染小鼠,通过在BALB/c小鼠肺组织中连续传代,观察小鼠存活情况及肺病理改变,来获得季节性流感病毒H1N1的鼠肺适应株.结果季节性流感H1N1 A/Brisbane/59/2007病毒野生型毒株,经过在小鼠体内进行8次传代后,毒力逐渐增强,从无致病力到致死率达到100%,对鼠肺适应株与野生型毒株进行基因比对,发现适应株HA基因发生了3个有义突变.结论 野生季节性低致病力H1N1流感病毒可经在小鼠中经过多次传代而获得高致病力H1N1鼠肺适应株,HA蛋白89位Thr至Ile的突变对毒力的增强起决定性作用.     

Characterization of immune responses during infection with Mycobacterium avium strains 100, 101 and the recently sequenced 104

Mycobacterium avium strain 104 was chosen as the M. avium isolate to sequence, as it is virulent to humans, stable and readily transfectable. As this strain has not been widely studied we sought to investigate the pattern of 104 infection in mice. Bacterial growth and the immune response generated were compared with infection with the low virulence M. avium strain 100, and the high virulence common laboratory strain, 101. Mycobacterium avium strains 104 and 101 grew progressively within mice, while strain 100 was gradually cleared. Strains 104 and 101 induced strong T cell activation and spleen cell cultures produced similar levels of IFN-gamma. In mice infected with strain 100 no significant T cell activation or IFN-gamma production was measured. Further, mice infected with strain 104 or 101 also displayed comparable inflammatory responses and similar granuloma formation, while only minimal inflammation was seen in mice infected with strain 100. Strains 101 and 104 also grew in a similar fashion in bone-marrow-derived macrophages and induced significant levels of TNF and nitric oxide. Thus infection with M. avium strain 104 induced an immunological response comparable to M. avium strain 101 and, with the availability of its sequence, should be a useful tool for designing new vaccines or drugs therapies to treat the increasing incidence of M. avium infection in humans.     

Biological properties of promastigotes of Leishmania having undergone lyophilization]

Materials are presented of a comparative study of morphological and cultural properties and virulence of liophylized cultures of Leishmania tropica major after their rehydration or after a long passage on the NNN medium. Results of investigations of two initial strains and three substrains obtained after the recovery of liophylized cultures have shown that liophylization does not reduce essentially the initial strain virulence. The strains undergone liophylization and passed repeatedly on the NNN medium have a tendency to a quicker virulence reduction rate as compared to initial ones. The culture of promastigotes recultivated after liophylization does not differ from the initial one in the ranges of 1-7 passages in its morphology, mobility and ability to grow on the NNN medium.     

Virulence of chick embryo fibroblast-passaged flury HEP rabies virus and its revertants in mice

Chick embryo fibroblast-passaged Flury high egg passage (HEP) rabies virus failed to kill nude mice or cyclophosphamide-treated mice when inoculated intracerebrally. The virus regained neurovirulence for adult mice after three passages in mouse neuroblastoma C1300 cells (NA cells). However, even after 20 passages in NA cells, the virulence could not be increased to the level shown by the virus passaged several times in suckling mice. Some physiological and biological properties of the virus showing and not showing mouse virulence after five serial passages and after one single passage in NA cells, respectively, were compared.     

Correlations Between Virulence and Other Characters of Clostridium perfringens Type A

The correlation analysis which has already proved its value in ecology has not yet been applied to the determination of virulence indicators. Its application to a group of Clostridium perfringens type A strains has brought out some characters that may be considered as virulence indicators. This study suggests that the toxicity of the culture supernatant fluids for mice is significantly correlated with the virulence for mice and guinea pigs. A significant correlation was found between the virulence of the fluid cultures for mice or guinea pigs and the coagulation of milk, production of gas (in deep agar), hydrogen sulfide production, and fermentation of glucose, sucrose, maltose, and levulose.     

Molecular determinants of Ebola virus virulence in mice

Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in humans and nonhuman primates, with fatality rates in humans of up to 90%. The molecular basis for the extreme virulence of ZEBOV remains elusive. While adult mice resist ZEBOV infection, the Mayinga strain of the virus has been adapted to cause lethal infection in these animals. To understand the pathogenesis underlying the extreme virulence of Ebola virus (EBOV), here we identified the mutations responsible for the acquisition of the high virulence of the adapted Mayinga strain in mice, by using reverse genetics. We found that mutations in viral protein 24 and in the nucleoprotein were primarily responsible for the acquisition of high virulence. Moreover, the role of these proteins in virulence correlated with their ability to evade type I interferon-stimulated antiviral responses. These findings suggest a critical role for overcoming the interferon-induced antiviral state in the pathogenicity of EBOV and offer new insights into the pathogenesis of EBOV infection.     

Brucella melitensis Rev. 1 living attenuated vaccine: stability of markers, residual virulence and immunogenicity in mice.

Five commercial Brucella melitensis Rev. 1 vaccines from different sources were compared to the original Elberg Rev. 1 strain, in vitro for classic markers and in vivo in mice, for residual virulence and immunogenicity. Because colonies of several morphology types (smooth, non-smooth) were isolated from the vaccines, representative substrains were purified to study their in vitro and in vivo activities either at once (16 strains), or after storage by subculture (12 strains) and by lyophilization (eight strains) or after passage in mice (six strains). After purification, five strains had the characteristic pattern of resistance to penicillin and streptomycin of the original strain while 11 differed by a two-fold dilution or more. A few modifications only occurred after storage or passage. Residual virulence--the time taken by 50% of the subcutaneously vaccinated mice to eradicate the strain from their spleen--or recovery time 50%, and immunogenicity--the ability of the vaccinated mice to restrict the spleen count 15 days after a virulent intraperitoneal challenge--were compared on eight strains after purification, subculture and lyophilization. After purification, one smooth strain out of five had the same activities as the original strain and three were as immunogenic but less virulent. One smooth strain and the three non-smooth were neither immunogenic nor virulent. Some strains which were typically non-smooth after purification recovered a smooth phase aspect after subculture, concomitantly with an increase in immunogenicity but not in virulence.(ABSTRACT TRUNCATED AT 250 WORDS)