Complete Genome Sequencing of Mosquito and Human Isolates of Ngari Virus

Ngari virus (NRIV) is a recently described, naturally occurring reassortant between two other orthobunyaviruses, Bunyamwera virus (BUNV) and Batai virus (BATV). Intriguingly, this reassortment was associated with the acquisition of heightened virulence, although the molecular basis for this is not understood. Here we report the first complete genome sequences of Ngari virus. We include five isolates from various geographical locations, as well as samples isolated from both mosquitos and human cases. Based on an analysis of these sequence data, NRIVs are clearly genetically distinct from all known BUNV and BATV strains but are very closely related to one another regardless of their source.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

In Vitro and In Vivo Isolation and Characterization of Duvenhage Virus

A fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization of DUVV in vitro and its passage in BALB/c mice. The virus proved to be neuroinvasive in both juvenile and adult mice, resulting in about 50% lethality upon peripheral infection. Clinical signs in infected mice were those of classical rabies. However, the distribution of viral antigen expression in the brain differed from that of classical rabies virus infection and neither inclusion bodies nor neuronal necrosis were observed. This is the first study to describe the in vitro and in vivo isolation and characterization of DUVV.     

In Vitro and In Vivo Observations on a Murine C-Type Virus

From 40 discrete mouse tissue culture cell lines examined by electron microscopy or complement fixation, or both, for the presence of detectable virus, one (NCTC 4705), initiated and maintained on chemically defined medium, was chosen for a more extensive study. Virus-like particles (100 to 110 mμ), morphologically similar to previously reported immature and mature C-type leukemia virus particles, were found budding from the plasma membrane and free in the intracellular spaces of cells in tissue culture and in fibrosarcomas resulting from intramuscular implants of these tissue cultures. Complement-fixation tests for group reactive murine leukemia antigens were positive, with titers consistently higher to a broadly reactive anti-serum than to anti-Friend, anti-Moloney, or anti-Rauscher sera. The 4705 virus was neutralized by Gross antiserum, but not by the F-M-R antisera. When injected into DD, BALB/c, or C3H/He newborn mice, the virus thus far has manifested no leukemogenicity, though virus from tumor extracts and tissue culture medium has been shown to be capable of infecting C3H and Swiss mouse embryo tissue cultures and successfully replicating in them. The role of the virus in accelerating or inducing neoplastic transformation in NCTC 4705 is still not known. When it was introduced into NCTC [ill], a non-neoplastic cell line in other respects similar to NCTC 4705, 4823 manifested no signs of neoplastic transformation after harboring the virus more than 300 days in vitro.     

New World Simian Foamy Virus Infections In Vivo and In Vitro

Foamy viruses (FV) are complex retroviruses that naturally infect all nonhuman primates (NHP) studied to date. Zoonotic transmission of Old World NHP simian foamy viruses (SFV) has been documented, leading to nonpathogenic persistent infections. To date, there have been no reports concerning zoonotic transmission of New World monkey (NWM) SFV to humans and resulting infection. In this study, we developed a Western blot assay to detect antibodies to NWM SFV, a nested PCR assay to detect NWM SFV DNA, and a β-galactosidase-containing indicator cell line to assay replication of NWM SFV. Using these tools, we analyzed the plasma and blood of 116 primatologists, of whom 69 had reported exposures to NWM. While 8 of the primatologists tested were seropositive for SFV from a NWM, the spider monkey, none had detectable levels of viral DNA in their blood. We found that SFV isolated from three different species of NWM replicated in some, but not all, human cell lines. From our data, we conclude that while humans exposed to NWM SFV produce antibodies, there is no evidence for long-term viral persistence.     

In Vitro and In Vivo Anti-influenza A Virus Activity of Antisense Oligonucleotides

Abstract

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. The liposomally encapsulated and the free antisense phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were tested for their abilities to inhibit virus-induced cytopathogenic effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODN complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. Therefore, the antiviral effects of S-ODN-PB2-AUG and PA-AUG were examined in a mouse model of influenza virus A infection. PB2-AUG oligomer treated i.v. significantly prolonged the mean survival time in day (MDS) and increased the survival rates with does dependent manner.     

Genome Analysis of Environmental and Clinical P. aeruginosa Isolates from Sequence Type-1146

The genomes of Pseudomonas aeruginosa isolates of the new sequence type ST-1146, three environmental (P37, P47 and P49) and one clinical (SD9) isolates, with differences in their antibiotic susceptibility profiles have been sequenced and analysed. The genomes were mapped against P. aeruginosa PAO1-UW and UCBPP-PA14. The allelic profiles showed that the highest number of differences were in “Related to phage, transposon or plasmid” and “Secreted factors” categories. The clinical isolate showed a number of exclusive alleles greater than that for the environmental isolates. The phage Pf1 region in isolate SD9 accumulated the highest number of nucleotide substitutions. The ORF analysis of the four genomes assembled de novo indicated that the number of isolate-specific genes was higher in isolate SD9 (132 genes) than in isolates P37 (24 genes), P47 (16 genes) and P49 (21 genes). CRISPR elements were found in all isolates and SD9 showed differences in the spacer region. Genes related to bacteriophages F116 and H66 were found only in isolate SD9. Genome comparisons indicated that the isolates of ST-1146 are close related, and most genes implicated in pathogenicity are highly conserved, suggesting a genetic potential for infectivity in the environmental isolates similar to the clinical one. Phage-related genes are responsible of the main differences among the genomes of ST-1146 isolates. The role of bacteriophages has to be considered in the adaptation processes of isolates to the host and in microevolution studies.     

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.     

Polyadenylate Sequences on Newcastle Disease Virus mRNA Synthesized In Vivo and In Vitro

Polyadenylate [poly(A)] sequences are associated with the 35 and 50S Newcastle disease virus (NDV)-specific RNAs as well as all six to seven of the 18-22S NDV-specific messenger RNAs extracted from infected chicken embryo cells. The poly(A) associated with the 18-22S RNA has an average size of 120 to 130 nucleotides. The 18-22S RNA synthesized in vitro by NDV's virion-bound polymerase contains six to seven species of the same size and relative proportions as its intracellular counterpart. This in vitro synthesized 18-22S RNA also contains covalently linked poly(A) sequences which, although variable in size, are usually larger and more heterogeneous than those from the infected cell. In vitro RNA synthesis is supported not only by magnesium (at an optimal concentration of mM) but by manganese (at an optimal concentration of 0.5 to 1.0 mM) as well. However, the major product made in the presence of manganese, although sedimenting at 18 to 22S, differs somewhat from the product made in the presence of magnesium.     

芜菁花叶病毒的3’末端序列克隆与CP基因序列分析

目的:克隆芜菁花叶病毒(Turnip mosaic virus,TuMV)的3'末端序列,并进行CP基因序列分析.方法:以TuMV杭州榨菜分离物(TuMv-HZZC)接种病叶为材料,利用病毒粒子吸附法制备病毒RNA模板,经RT-PCR扩增获得了TuMV-HZZC 3'末端序列,将其克隆到PMD 18-T质粒上进行序列分析.结果:TuMV-HZZC分离物3'末端序列包括部分的Nib基因、完整的TuMVCP基因和3'-UTR,CP基因为864bp,分别编码288个氨基酸,3'-UTR序列(不包括PolyA尾巴)为213bp.经过与其他TuMV分离物的CP基因核苷酸和氨基酸比较,同源性分别达到88.0%～97.6%和91.0%-96.5%.结论:TuMV的系统进化具有典型的地域和寄主关联性.     

Mechanism of Synthesis of Vaccinia Virus Double-Stranded Ribonucleic Acid In Vivo and In Vitro

The synthesis of vaccinia virus double-stranded ribonucleic acid (RNA) in infected HeLa cells was sensitive to actinomycin D, suggesting that a deoxyribonucleic acid dependent reaction is involved. Some double-stranded RNA was made in the presence of cytosine arabinoside in infected cells. Double-stranded and complementary RNA were synthesized in vitro by using vaccinia cores. These two observations indicate that some of the double-stranded RNA is read from "early" genes. The double-stranded RNA synthesized in vitro had the same properties as that made in vivo. At least 70% of the double-stranded RNA made in vivo was in ribonuclease-resistant form prior to sodium dodecyl sulfate-phenol extraction. In addition, there was a complementary RNA in infected cells which could be converted to double-stranded RNA by annealing.     

Inhibitory Activity of Honeysuckle Extracts against Influenza A Virus In Vitro and In Vivo

Virologica Sinica - Honeysuckle has been used in the treatment of influenza virus infection for thousands of years in China. However, its main active components and the functional mechanisms remain...     

Methyl Gallate from Galla rhois Successfully Controls Clinical Isolates of Salmonella Infection in Both In Vitro and In Vivo Systems

Galla rhois is a commonly used traditional medicine for the treatment of pathogenic bacteria in Korea as well as in other parts of Asia. Methyl gallate (MG), a major component of Galla Rhois, exhibits strong antibacterial activity, but its mechanism of action against Salmonella spp. is unclear. In the present study, we investigated the antibacterial actions of MG against Salmonella. The antibacterial activity determined by broth dilution method indicated that the antibacterial activity of MG against Salmonella strains ranged from 3.9 to 125 µg/ml. In vitro bacterial viability test indicated that MG significantly decreased the viability of Salmonella over 40% when combined with ATPase inhibitors. The time-kill curves showed that a combined MG and ATPase inhibitors (DCCD and NaN3) treatment reduced the bacterial counts dramatically after 24 h. Oral administration of MG showed a strong anti-bacterial activity against WS-5 infected BALB/c mice. In contrast to the untreated Salmonella infected control animals, MG treated groups showed no clinical symptoms of the disease, such as lethargy and liver damage. It was observed that MG treatment significantly increased the survival of animals from Salmonella infection, while in untreated groups all animal succumbed to disease by the sixth day post infection. Thus, the present study demonstrates the therapeutic ability of MG against Salmonella infections.     

中国19个狂犬病病毒街毒分离株N基因的序列分析

测定了30年来从不同动物中分离的19个中国狂犬病病毒街毒株N基因的部分核酸序列,并对其核苷酸差异做了比较分析.可将中国狂犬病病毒街毒株分为4个组群,各组间的同源性为83.45%～88.62%.除广西地区分离的狂犬病病毒街毒株彼此差异较大外,其余街毒株的地理分布与其N基因核酸序列差异的距离是密切相关的,基本上可按其地理分布分为东、西二大组.     

In Vivo and In Vitro Analysis of Baculovirus ie-2 Mutants

Upon transient expression in cell culture, the ie-2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) displays three functions: trans activation of viral promoters, direct or indirect stimulation of virus origin-specific DNA replication, and arrest of the cell cycle. The ability of IE2 to trans stimulate DNA replication and coupled late gene expression is observed in a cell line derived from Spodoptera frugiperda but not in a cell line derived from Trichoplusia ni. This finding suggested that IE-2 may exert cell line-specific or host-specific effects. To examine the role of ie-2 in the context of infection and its possible influence on the host range, we constructed recombinants of AcMNPV containing deletions of different functional regions within ie-2 and characterized them in cell lines and larvae of S. frugiperda and T. ni. The ie-2 mutant viruses exhibited delays in viral DNA synthesis, late gene expression, budded virus production, and occlusion body formation in SF-21 cells but not in TN-5B1-4 cells. In TN-5B1-4 cells, the ie-2 mutants produced more budded virus and fewer occlusion bodies but the infection proceeded without delay. Examination of the effects of ie-2 and the respective mutants on immediate-early viral promoters in transient expression assays revealed striking differences in the relative levels of expression and differences in responses to ie-2 and its mutant forms in different cell lines. In T. ni and S. frugiperda larvae, the infectivities of the occluded form of ie-2 mutant viruses by the normal oral route of infection was 100- and 1,000-fold lower, respectively, than that of wild-type AcMNPV. The reduction in oral infectivity was traced to the absence of virions within the occlusion bodies. The infectivity of the budded form of ie-2 mutants by hemocoelic injection was similar to that of wild-type virus in both species. Thus, ie-2 mutants are viable but exhibit cell line-specific effects on temporal regulation of the infection process. Due to its effect on virion occlusion, mutants of IE-2 were essentially noninfectious by the normal route of infection in both species tested. However, since budded viruses exhibited normal infectivity upon hemocoelic injection, we conclude that ie-2 does not affect host range per se. The possibility that IE-2 exerts tissue-specific effects has not been ruled out.