Induction of B-Cell Lymphoma in BALB/c Nude Mice with an Ecotropic, B-Tropic Helper Virus Present in the Murine AIDS Virus Stock

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.     

A nanobeads amplified QCM immunosensor for the detection of avian influenza virus H5N1

As a potential pandemic threat to human health, there has been an urgent need for rapid detection of the highly pathogenic avian influenza (AI) H5N1 virus. In this study, magnetic nanobeads amplification based quartz crystal microbalance (QCM) immunosensor was developed as a new method and application for AI H5N1 virus detection. Polyclonal antibodies against AI H5N1 virus surface antigen HA (Hemagglutinin) were immobilized on the gold surface of the QCM crystal through self-assembled monolayer (SAM) of 16-mercaptohexadecanoic acid (MHDA). Target H5N1 viruses were then captured by the immobilized antibodies, resulting in a change in the frequency. Magnetic nanobeads (diameter, 30nm) coated with anti-H5 antibodies were used for further amplification of the binding reaction between antibody and antigen (virus). Both bindings of target H5N1 viruses and magnetic nanobeads onto the crystal surface were further confirmed by environmental scanning electron microscopy (ESEM). The QCM immunosensor could detect the H5N1 virus at a titer higher than 0.0128 HA unit within 2h. The nanobeads amplification resulted in much better detection signal for target virus with lower titers. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear correlation between the logarithmic number of H5N1 virus titers and frequency shift was found from 0.128 to 12.8 HA unit. No significant interference was observed from non-target subtypes such as AI subtypes H3N2, H2N2, and H4N8. The immunosensor was evaluated using chicken tracheal swab samples. This research demonstrated that the magnetic nanobeads amplification based QCM immunosensor has a great potential to be an alternative method for rapid, sensitive, and specific detection of AI virus H5N1 in agricultural, food, environmental and clinical samples.     

利用Cre-loxP自动敲除H5亚型禽流感病毒血凝素重组鸡痘病毒中的报告基因

研究去除重组鸡痘病毒中的报告基因,构建一株只含目的基因的重组毒。将H5亚型AIV的HA基因作为靶基因,两侧含loxP序列的GFP表达盒插入鸡痘病毒重组臂基因构建了转移质粒载体,将其与脂质体混合转染CEF细胞,获得了表达H5和GFP的鸡痘病毒重组体。通过二次转染,利用Cre酶自动敲除重组病毒中的GFP基因,最终获得了只含H5血凝素基因表达盒的重组鸡痘病毒。免疫荧光和病毒滴度测定结果表明,经过连续传代后重组病毒仍然稳定复制并表达H5血凝素。用10⁵PFU和2×10⁵PFU rFPV H5免疫SPF鸡,28d后,免疫组鸡抗体平均滴度(HI)分别达到4log 2和4.5log 2,结果表明,H5HA基因重组病毒能刺激鸡群产生较高特异抗体。     

In Vivo Antiviral Properties of Biologically Active Compounds: II. Studies with Influenza and Vaccinia Viruses

The in vivo anti-influenza virus and antivaccinia virus activity of 156 biologically active compounds was determined. One of two criteria was used for evaluating activity against the influenza virus. The criteria were increase in survivor number and mean survival time, and reduction in virus-induced lung consolidation in treated, infected Swiss mice. Increase in survivor number and mean survival time were the criteria for evaluation of antivaccinia virus activity. Several drug doses were tested against two virus concentrations to demonstrate antiviral activity more clearly. Two compounds were considered significantly active against the influenza virus: DL-noformicin (NSC 72942) and amantadine hydrochloride (NSC 83653). Eleven compounds had reproducible activity against vaccinia virus: isatin-beta-thiosemicarbazone (NSC 721), 6-azauracil (NSC 3425), 9-alpha-fluoro-2alpha-methylhydrocortisone 21-acetate (NSC 12601), 5-[bis(2-chloroethyl)amino]uracil (NSC 34462), 5-iodo-2'-deoxyuridine (NSC 39661), streptonigrin (NSC 45383), N-methylisatin beta-thiosemicarbazone (NSC 69811), cytovirin (NSC 91770), 9-beta-D-arabinofuranosyladenine (NSC 404241), and 5-(mercaptomethyl)uracil (NSC 529351).     

Postembedding immunocytochemical localization of paramyxovirus antigens by light and electron microscopy

A postembedding method is described to localize antigens specific for various paramyxoviruses in sections of cells and tissues that have been fixed and embedded in epoxy resins for conventional electron microscopy. Viral antigens were localized in CV-1 cell cultures infected with simian virus 5 (SV5), brains of suckling hamsters inoculated with either neuroadapted mumps virus or hamster-adapted measles virus, and brains of adult mice infected with Sendai (parainfluenza I) virus. Both 1-micrometer-thick and thin (gold) tissue sections were etched with alcoholic sodium hydroxide-solution and then treated following either the unlabeled antibody peroxidase-antiperoxidase or the biotinylated protein A:avidin peroxidase procedure. Primary reagents included immunoglobulin isolated from hyperimmune rabbit sera with specificity to the major viral components of SV5 or SV5 hemagglutinin-neuraminidase, to whole mumps virus or mumps virus nucleocapsids, and to whole Sendai virus. Crude rabbit anti-Sendai virus antiserum and whole human subacute sclerosing panencephalitis (SSPE) sera were used in parallel. The results indicate that tissues processed for conventional evaluation by electron microscopy may be suitable, within limits, for postembedding immunocytochemical staining of paramyxovirus antigens.     

Mutations within the 5'' nontranslated region of hepatitis A virus RNA which enhance replication in BS-C-1 cells.

Passage of human hepatitis A virus (HAV) in cell culture results in attenuation of the virus as well as progressive increases in the efficiency of virus replication in cell culture. Because the presence of identical mutations within the 5' nontranslated regions (5'NTRs) of several independently isolated cell culture-adapted HAV variants suggests that the 5'NTR may play a role in determining this change in virus host range, we constructed chimeric infectious cDNA clones in which portions of the 5'NTR of cell culture-adapted HM175/p35 virus were replaced with cDNA from either wild-type virus (HM175/wt) or a second independently isolated, but closely related cell culture-adapted virus (HM175/p16). Substitution of the complete 5'NTR of HM175/p35 with the 5'NTR of HM175/wt resulted in virus with very small replication foci in continuous African green monkey kidney (BS-C-1) cells, indicating that 5'NTR mutations in HM175/p35 virus are required for optimal growth in these cells. A chimera with the 5'NTR sequence of HM175/p16 retained the large foci of HM175/p35 virus, while the growth properties of other viruses having chimeric 5'NTR sequences indicated that mutations at bases 152 and/or 203 to 207 enhance replication in BS-C-1 cells. These findings were confirmed in one-step growth experiments, which also indicated that radioimmunofocus size is a valid measure of virus replication competence in cell culture. An additional mutation at base 687 of HM175/p16 had only a minor role in enhancing growth. In contrast to their effect in BS-C-1 cells, these 5'NTR mutations did not enhance replication in continuous fetal rhesus monkey kidney (FRhK-4) cells. Thus, mutations at bases 152 and/or 203 to 207 enhance the replication of HAV in a highly host cell-specific fashion.     

Characterization of monoclonal antibodies against Hokkaido strain tick-borne encephalitis virus

A tick-borne encephalitis (TBE) patient was found in Hokkaido in 1993, and TBE viruses were isolated from animals and ticks in our previous studies. To develop a diagnostic reagent to identify TBE viruses, monoclonal antibodies (Mabs) were produced against the TBE virus strain Hokkaido (Oshima 5-10). Seven Mabs were obtained which reacted with the envelope protein of the Oshima 5-10 strain. These Mabs were flavivirus genus-specific, TBE virus complex-specific or TBE virus type-specific. The Mabs are applicable for identification of TBE virus strains.     

High genetic stability of dengue virus propagated in MRC-5 cells as compared to the virus propagated in vero cells

This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-free and serum-containing cultures were found to yield comparable titers of these viruses. The cloned DNA-derived DEN-4 started genetically more homogeneous was used to investigate the genetic stability of the virus propagated in Vero cells and MRC-5 cells. Sequence analysis revealed that the DEN-4 propagated in MRC-5 cells maintained a high genetic stability, compared to the virus propagated in Vero cells. Amino acid substitutions of Gly(104)Cys and Phe(108)Ile were detected at 70%, 60%, respectively, in the envelope (E) protein of DEN-4 propagated in Vero cells, whereas a single mutation of Glu(345)Lys was detected at 50% in E of the virus propagated in MRC-5 cells. Sequencing of multiple clones of three separate DNA fragments spanning 40% of the genome also indicated that DEN-4 propagated in Vero cells contained a higher number of mutations than the virus growing in MRC-5 cells. Although Vero cells yielded a peak virus titer approximately 1 to 17 folds higher than MRC-5 cells, cloned DEN-4 from MRC-5 cells maintained a greater stability than the virus from Vero cells. Serum-free microcarrier cultures of MRC-5 cells offer a potentially valuable system for the large-scale production of live-attenuated DEN vaccines.     

5''-terminal nucleotide sequences of mammalian type C helper viruses are conserved in the genomes of replication-defective mammalian transforming viruses.

The RNAs of replication-defective murine and primate type C transforming viruses were analyzed for the presence of nucleotide sequences homologous to the genomes of their respective helper type C viruses by using DNAs complementary (cDNA) to either the 5'-terminal (cDNA5') or total (cDNAtotal) nucleotide sequences of the helper virus RNA. The defective viruses examined have previously been shown to vary in their ability to express helper viral gag gene proteins. With cDNAtotal as a probe, these transforming viruses were shown to vary in their representation of helper sequences (15 to 60% hybridization of cDNAtotal). In striking contrast, 5'-terminal-specific sequences of the helper virus were conserved in the RNAs of every transforming virus tested (is greater than 80% hybridization of cDNA5'). These findings suggest a critical role for these sequences in the life cycle of the defective transforming virus.     

RIG-I, MDA5 and TLR3 synergistically play an important role in restriction of dengue virus infection

Dengue virus (DV) infection is one of the most common mosquito-borne viral diseases in the world. The innate immune system is important for the early detection of virus and for mounting a cascade of defense measures which include the production of type 1 interferon (IFN). Hence, a thorough understanding of the innate immune response during DV infection would be essential for our understanding of the DV pathogenesis. A recent application of the microarray to dengue virus type 1 (DV1) infected lung carcinoma cells revealed the increased expression of both extracellular and cytoplasmic pattern recognition receptors; retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene-5 (MDA-5) and Toll-like receptor-3 (TLR3). These intracellular RNA sensors were previously reported to sense DV infection in different cells. In this study, we show that they are collectively involved in initiating an effective IFN production against DV. Cells silenced for these genes were highly susceptible to DV infection. RIG-I and MDA5 knockdown HUH-7 cells and TLR3 knockout macrophages were highly susceptible to DV infection. When cells were silenced for only RIG-I and MDA5 (but not TLR3), substantial production of IFN-β was observed upon virus infection and vice versa. High susceptibility to virus infection led to ER-stress induced apoptosis in HUH-7 cells. Collectively, our studies demonstrate that the intracellular RNA virus sensors (RIG-I, MDA5 and TLR3) are activated upon DV infection and are essential for host defense against the virus.     

Inhibition of dengue virus serotypes 1 to 4 in vero cell cultures with morpholino oligomers

Five dengue (DEN) virus-specific R5F2R4 peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 microM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3'-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log10, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5' cyclization sequence region had moderate activity, generating 1.6- and 1.8-log10 reductions. Two P4-PMO compounds, 5'SL and 3'CS (targeting the 5'-terminal nucleotides and the 3' cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log10 compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5'SL and 3'CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 microM 3'CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log10, in most cases to below detectable limits. The extent of 3'CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5'SL and 3'CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5'SL and 3'CS compounds as potential DEN virus therapeutics is warranted.     

Abrogation of resistance to severe mousepox in C57BL/6 mice infected with LP-BM5 murine leukemia viruses.

Strain C57BL/6 (B6) mice infected with LP-BM5 murine leukemia virus (MuLV) develop a disease which combines abnormal lymphoproliferation with profound immunosuppression and has many features in common with human acquired immunodeficiency syndrome induced by HTLV-III/LAV retroviruses. To determine whether this LP-BM5 MuLV infection would affect the innate resistance of B6 mice to a naturally occurring, highly virulent murine pathogen, mice were exposed to ectromelia virus at various times after treatment with LP-BM5 viruses. At week 4 after infection with LP-BM5, mice challenged with ectromelia virus were unable to generate a humoral immune response to this virus, and between weeks 8 and 10 after infection, challenged mice lost the ability to generate an ectromelia virus-specific cytotoxic-T-cell response. Loss of the cellular immune responses to ectromelia virus was associated with an increased susceptibility to the lethal effects of the virus.     

Stereospecific Synthesis and Antiviral Activities of β-L-2′,3′-Dideoxy-5-chloropyrimidine Nucleoside Derivatives

Abstract

Several 5-chlorouracil and 5-chlorocytosine β-L-dideoxynucleosides were stereospecifically synthesized and their activities against human immunodeficiency virus (HIV) and hepatitis B virus (HBV) were examinated in cell culture.     

Evaluating a Parainfluenza Virus 5-Based Vaccine in a Host with Pre-Existing Immunity against Parainfluenza Virus 5

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.     

高滴度感染力H5N1亚型禽流感病毒假病毒颗粒的制备

利用一个瞬时共转染系统,将H5N1亚型禽流感病毒的血凝素（Hemagglutinin,HA）蛋白与神经氨酸酶（Neuraminidase,NA）蛋白整合到鼠白血病病毒假病毒颗粒表面,包装成表达HA与NA的假病毒颗粒,通过透射电子显微镜形态学观察、感染滴度分析、血凝试验和中和试验研究其生物学特性。研究获得了高滴度感染力的H5N1假病毒颗粒（H5N1 Pseudotyped particle,H5N1Pp）,H5N1Pp的感染力滴度为1E8 Pp/mL,形态、血凝活性及中和特性均与野生H5N1病毒相似,结果为H5N1病毒受体、HA与NA的功能、中和抗体、抗病毒药物开发研究的开展建立了平台。     

狂犬病病毒糖蛋白重组人腺病毒的构建及免疫效果的初步研究

构建表达狂犬病病毒弱毒SRV9糖蛋白(GP)的重组人5型腺病毒,检测其对小鼠的免疫效果。将狂犬病病毒SRV9株GP基因的完整开放阅读框克隆到腺病毒表达系统中的穿梭质粒多克隆位点,构建重组穿梭质粒pac-Ad5CMV-Gs9,以罗氏转染液介导线性化骨架质粒和重组穿梭质粒共转染293AD细胞,细胞病变后取培养物进行PCR鉴定并电镜观察,在293AD细胞上测定病毒滴度。以106 TCID50重组腺病毒腹腔接种昆明小鼠,免疫后不同时段采尾静脉血通过荧光抗体病毒中和试验(FAVN)检测小鼠血清狂犬病中和抗体效价。正确构建重组穿梭质粒pacAd5CMV-Gs9;获得表达狂犬病病毒SRV9株GP蛋白的缺陷型重组人5型腺病毒;病毒滴度达到106 CFU/mL以上;腹腔接种小鼠14d后均产生了抗狂犬病中和抗体,有效保护率达90%。成功获得了表达狂犬病病毒GP基因的重组腺病毒,该腺病毒免疫小鼠可产生保护性中和抗体,为进一步开发新型兽用狂犬病疫苗奠定了物质基础。     

Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) cells. The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype. Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.     

Heterosubtypic antibody response elicited with seasonal influenza vaccine correlates partial protection against highly pathogenic H5N1 virus

Background

The spread of highly pathogenic avian influenza (HPAI) H5N1 virus in human remains a global health concern. Heterosubtypic antibody response between seasonal influenza vaccine and potential pandemic influenza virus has important implications for public health. Previous studies by Corti et al. and by Gioia et al. demonstrate that heterosubtypic neutralizing antibodies against the highly pathogenic H5N1 virus can be elicited with a seasonal influenza vaccine in humans. However, whether such response offers immune protection against highly pathogenic H5N1 virus remained to be determined.

Methodology/Principal Findings

In this study, using a sensitive influenza HA (hemagglutinin) and NA (neuraminidase) pseudotype-based neutralization (PN) assay we first confirmed that low levels of heterosubtypic neutralizing antibody response against H5N1 virus were indeed elicited with seasonal influenza vaccine in humans. We then immunized mice with the seasonal influenza vaccine and challenged them with lethal doses of highly pathogenic H5N1 virus. As controls, we immunized mice with homosubtypic H5N1 virus like particles (VLP) or PBS and challenged them with the same H5N1 virus. Here we show that low levels of heterosubtypic neutralizing antibody response were elicited with seasonal influenza vaccine in mice, which were significantly higher than those in PBS control. Among them 2 out of 27 whose immune sera exhibited similar levels of neutralizing antibody response as VLP controls actually survived from highly pathogenic H5N1 virus challenge.

Conclusions/Significance

Therefore, we conclude that low levels of heterosubtypic neutralizing antibody response are indeed elicited with seasonal influenza vaccine in humans and mice and at certain levels such response offers immune protection against severity of H5N1 virus infection.