Mosquito densonucleosis viruses cause dramatically different infection phenotypes in the C6/36 Aedes albopictus cell line

Mosquito densoviruses generally establish persistent infections in mosquito cell lines including the C6/36 Aedes albopictus cell line. In contrast, the closely related Haemagogus equinus densovirus (HeDNV) causes dramatic cytopathic effects in the C6/36 Aedes albopictus cell line. Infection of C6/36 cells by HeDNV causes internucleosomal fragmentation of host chromosomal DNA, changes in cellular morphology (membrane budding, apoptotic bodies), caspase activation and exposure of phosphatidylserine on the cellular membrane. This is accompanied by a higher rate of infection and more vigorous production of virus in these cells. These observations are consistent with the induction of apoptosis during infection. In contrast, expression of AeDNV proteins in C6/36 cells does not cause obvious cytopathic effects although NS1 expression causes accumulation of cells in G2 phase. C6/36 cells persistently infected with AeDNV were not protected from superinfection with HeDNV. Thus, there does not seem to be an antiviral state induced by AeDNV persistent infection.     

Genetic, biochemical, and structural characterization of a new densovirus isolated from a chronically infected Aedes albopictus C6/36 cell line

We report the isolation, sequencing, biochemical, and structural characterization of a previously undescribed virus in a chronically infected Aedes albopictus C6/36 cell line. This virus is identified as a new densovirus under the Densovirinae subfamily of the Parvoviridae based on its biological and morphologic properties as well as sequence homologies, and is tentatively designated A. albopictus C6/36 cell densovirus (C6/36 DNV). Analysis of the 4094 nt of the C6/36 DNV genome revealed that the plus strand had three large open reading frames (ORFs): a left ORF, a right ORF, and a mid-ORF (within the left ORF), whose potential coding capacities are 91.0, 40.8, and 41.2 kDa, respectively. The left ORF likely encodes the nonstructural protein NS-1, which contains NTP-binding and helicase domains. The right ORF likely encodes structural proteins, VP1 and VP2. Our analyses revealed that C6/36 DNV has a similar genomic organization and shares very high homology in nucleotide sequence and amino acid sequences with Aedes aegypti densovirus (AaeDNV) and A. albopictus densovirus (AalDNV), members of the genus Brevidensovirus of the Densovirinae. Similar to other densoviruses, C6/36 DNV has a different genomic organization and no recognizable sequence homology with viruses in the Parvovirinae. The three-dimensional (3D) reconstruction of the C6/36 DNV at 15.6-A resolution by electron cryomicroscopy (cryoEM) revealed distinctive outer surface features not previously seen in other parvoviruses, indicating structural divergence of densoviruses, in addition to its genomic differences, while the inner surface of the C6/36 DNV capsid exhibits features that are conserved among parvoviruses.     

The truncated virus-like particles of C6/36 cell densovirus: implications for the assembly mechanism of brevidensovirus

The brevidensovirus is one of the smallest viruses in the world and the capsid of Aedes albopictus C6/36 cell densovirus (C6/36DNV) is the simplest and most compact capsid in brevidensovirus. To understand the assembly mechanism of icosahedral-virus capsid from this simplest model, we tried to express various lengths of virus proteins (VPs) of C6/36DNV in Bac-to-Bac system and evaluate their self-assembly capacities in insect Spodoptera frugiperda 9 (Sf9) cells. The result showed that the N-terminal GGSG sequence (residue 23-26), highly conserved glycine-rich region in Parvoviridae, and C-terminal GTGGVVTCMP (residue 344-353) were essential for capsid assembly, while the N-terminal nuclear localization signal, GTKRKR sequence (residue 15-20), was nonessential for the virus-like particles (VLPs) assembly, but did effect the formation of crystalline arrays in infected Sf9 cells. These information provided clues for how icosahedral-virus capsids formed and showed the potential of C6/36DNV-VLPs becoming a powerful nanoparticle vector.     

Transduction of Aedes aegypti mosquitoes with vectors derived from Aedes densovirus

Aedes densovirus (AeDNV)-based constructs that express green fluorescent protein (GFP) from either the P7 or the P61 promoter were made. The construct in which GFP protein was expressed as a fusion protein to the C-terminus of NS1 (NS1-GFP) showed the highest level of GFP expression. This hybrid NS1-GFP protein preserved the biological functions of the parental proteins: it showed GFP fluorescence, it stimulated expression from the virus promoters, and it facilitated rescue and replication of the cloned AeDNV genome. Similar to NS1, the hybrid NS1-GFP localized in the nucleus predominantly in a punctate pattern. Transducing virus particles carrying the NS1-GFP gene infected mosquito larvae. Expression of GFP was detected as early as 48 h postinfection and in larval and pupal stages. Midgut, hindgut, and Malpighian tubule cells expressed GFP soon after transduction. However, the anal papillae were the most commonly infected organ system. The anal papillae are syncytia and regulate ion concentration in the hemolymph of mosquito larvae, and they might be a novel route of mosquito larvae infection with densoviruses.     

贵州白纹伊蚊对登革病毒易感性的研究

目的 用细胞、分子生物学技术进行贵州省白纹伊蚊不同地理株对登革病毒(DEN)易感性的研究。方法 采集贵州省9个地(州)市共计15个县(区)白纹伊蚊幼虫标本，饲养为成蚊；取羽化后3～5日龄期的贵州不同地理株白纹伊蚊，用不同型别的DEN分别经口连续感染3d，于首次感染后的4、7、10、14d收集感染成蚊标本；制备蚊悬液，碘化钠法提取RNA，用DENNS1基因区通用引物经逆转录．聚合酶链反应(RT-PCR)检测DEN核酸；蚊悬液接种C6／36细胞进行病毒分离，制作细胞抗原片，经间接免疫荧光法检测DEN抗原；同时感染白纹伊蚊海南株作为对照。结果 DEN1-4型国际参考株感染白纹伊蚊贵州省不同地方株，其感染比率分别为12／15、12／15、8／15和13／15。结论 白纹伊蚊贵州省不同地方株对DEN1-4型国际参考株普遍易感，表明贵州省具备引起登革热流行的条件。     

Assessing the expression of chicken anemia virus proteins in plants

Chicken anemia virus (CAV) is an important pathogen of chicken worldwide, causing severe anemia and immunodeficiency. Its small single-stranded DNA genome (2.3kb) encodes three proteins: VP1, the only structural protein, VP2, a protein phosphatase, and VP3, also known as apoptin, which induces apoptosis. In this study, CAV proteins were expressed in plants as an alternative for recombinant protein production in animal cells. Additionally, the effect of VP3 expression was tested to evaluate possible involvement in programmed cell death in plants. The CAV genes were cloned in binary vectors with the Green fluorescent protein (GFP) as N terminal fusion, and into a Potato virus X (PVX) and Tobacco Mosaic Virus (TMV)-based vectors. Nicotiana benthamiana plants were inoculated with Agrobacterium tumefaciens containing the binary vector constructs or the PVX and TMV constructs. Upon transient expression GFP:VP1 and GFP:VP2 were observed throughout the nucleoplasm, whereas VP3 formed compact aggregates within the nucleus, indicating functional nuclear localization signals in all three proteins. An intense fluorescence was observed for VP2 and VP3 fusions, whereas GFP:VP1 fluorescence remained faint and was only detected in a limited number of cells. Co-expression of GFP:VP1 and VP2 had a marked alteration on the distribution of GFP:VP1, forming large VP1 aggregates throughout the nucleus, indicating an interaction of the two CAV proteins. No visible alteration on GFP pattern was detected upon co-expression of GFP:VP1 and VP3, or with GFP:VP2 and VP3. Plants infected with PVX or TMV-based vectors expressing VP3 displayed strong necrosis and wilting, however, a direct association with VP3 expression and programmed cell death in plants, could not be established. Overall, our results show that all CAV proteins can be expressed in plant cells, though expression level of VP1 needs to be further optimized before testing its potential as (edible) subunit vaccine.     

Heteroclite subgenomic RNAs are produced in porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) was shown to produce atypical subgenomic RNAs that contain open reading frame la nucleotides and are present under a wide variety of culture conditions, including high and low multiplicities of infection, in simian and porcine host cells, and during infection with cell-adapted and wild-type PRIRSV strains. Sequence analysis demonstrated that they are heterogeneous in 5-3' junction sequence and size and may code for different predicted fusion proteins. This is the first report of these novel RNA5 in arteriviruses and we have termed them heteroclite (meaning 'deviating from common forms or rules") subgenomic RNAs. The unique properties of these subgenomic RNAs include (a) apparent association with normal virus infection and stability during serial passage, (b) packaging of heteroclite RNAs into virus-like particles, (c) short, heterogeneous sequences which may mediate the generation of these RNAs, (d) a primary structure which consists of the two genomic termini with one large internal deletion, and (eJ little apparent interference with parental virus replication. These subgenomic RNA5 may be critical to, or a necessary side product of, viral replication. The expression of these novel RNA species support the template-switching model of similarity-assisted RNA recombination. In summary, PRRSV readily undergoes nonhomologous RNA recombination to generate heteroclite sub-genomic RNA5.     

A cosmid-based system for inserting mutations and foreign genes into the simian varicella virus genome

Simian varicella is a natural varicella-like disease of nonhuman primates. The etiologic agent, simian varicella virus (SVV), is genetically related to varicella-zoster virus (VZV) and SVV infection of nonhuman primates is a useful model to investigate VZV pathogenesis and latency. In this study, we report development of a cosmid-based genetic system to generate SVV mutant viruses. SVV subgenomic DNA fragments (32–38 kb) that span the viral genome were cloned into cosmid vectors. Co-transfection of Vero cells with four overlapping cosmid clones representing the entire SVV genome resulted in recombination and generation of infectious virus. SVV mutants were produced by manipulation of one cosmid and substitution into the genetic system. This genetic approach was used to insert a site-specific mutation within the SVV open reading frame 14 which encodes the nonessential glycoprotein C gene. In a subsequent experiment, the green fluorescent protein (GFP) gene was inserted into the SVV genome within ORF 14. These SVV mutants replicate as efficiently as wild-type SVV in cell culture. This cosmid-based genetic system will be useful to investigate the effect of viral mutations on SVV pathogenesis and latency and also to develop and evaluate recombinant varicella vaccines that express foreign antigens.     

Complete nucleotide sequence of a cell culture-adapted variant of hepatitis A virus: comparison with wild-type virus with restricted capacity for in vitro replication

To determine the molecular changes associated with adaptation of hepatitis A virus (HAV) to growth in cell culture, the genome of a cell culture-adapted variant of HM175 strain HAV (p16 HM175, 16th in vitro passage level) was molecularly cloned and the complete nucleotide sequence of the virus was determined. Compared with wild-type virus, p16 HM175 replicates efficiently in monkey kidney (BS-C-1) cells (approximately 58 RNA-containing particles per one infectious unit, compared with 2.4 x 10(5) for wild-type HM175). The nucleotide sequence of p16 HM175 revealed a total of 19 mutations from the wild-type genome, including 5 mutations in the 5' nontranslated region, 1 mutation in the 3' nontranslated region, and 13 mutations predicting 8 changes in the amino acid sequences of HAV proteins. Only one amino acid substitution occurred among the capsid proteins (VP2), while others involved proteins 2A, 2B, 2C, VPg, and 3Dpol. When the sequence of p16 virus was compared with that reported previously for an independently isolated, cell culture-adapted variant of HM175 virus (J.I. Cohen et al., (1987). Proc. Natl. Acad. Sci. USA 84, 2497-2501), there were three identical mutations in nontranslated regions of the RNA, and four mutations involving identical amino acids in proteins VP2, 2B, and 3Dpol. The distribution of these mutations within the genome suggests that changes in RNA replication may be of primary importance in adaptation of the virus to growth in vitro. These data are thus helpful in understanding the molecular basis of adaptation of HAV to cell culture and, since attenuation frequently accompanies adaptation of virus to growth in cell culture, may be of benefit in planning for attenuated vaccine development.     

Self-inactivating lentiviral vectors with U3 and U5 modifications.

Lentiviral vectors have gained much attention in recent years mainly because they integrate into nondividing host-cell genomes. For clinical applications, a safe and efficient lentiviral vector system is required. Previously, we have established a human immunodeficiency virus type 1 (HIV-1)-derived three-plasmid lentiviral vector system for viral vector production which includes a packaging vector pHP, a transducing vector pTV, and an envelope-encoding plasmid pHEF-VSVG. Cotransfection of these three plasmids into TE671 human rhabdomyosarcoma cells routinely yields 10(5)-10(6) infectious units per milliliter in 24 h. Here we have extensively modified long terminal repeats (LTRs) of pTV to generate a safer lentiviral vector system. The 5' U3 was replaced with a truncated cytomegalovirus (CMV) immediate early (IE) enhancer/TATA promoter and the 3' U3 (except for the integration attachment site) was also deleted. These modifications resulted in a vector with 80% wild-type vector efficiency. Further deletion of 3' U5 impaired vector function; however, this problem was solved by replacing the 3' U5 with bovine growth hormone polyadenylation (bGHpA) sequence. The pTV vector containing all these modifications including the 5' promoter substitution, the 3' U3 deletion, and the substitution of 3' U5 with bGHpA exhibited a self-inactivating (SIN) phenotype after transduction, transduced both dividing and nondividing cells at similar efficiencies, and produced vector titers twice as high as that of the wild-type construct. Thus, both safety and efficacy of the HP/TV vector have been improved by these LTR modifications. Further deletion of 5' U5 impaired vector efficiency, suggesting that the 5' U5 has critical roles in vector function.     

Engineering of Alfalfa mosaic virus RNA 3 into an expression vector

Summary.  RNA 3 of alfalfa mosaic virus (AMV) encodes the 5′-proximal movement protein (MP) gene and the 3′-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3′-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3. Received August 7, 2000 Accepted December 18, 2000     

Topology and immunoreactivity of capsid proteins in hepatitis A virus

Hepatitis A virus (HAV) was propagated in a hepatoma cell line and complete viral particles with a density of 1.34 g/ml were purified from cell extracts. The topography of the viral proteins (VPs) was studied by surface labelling with¹²⁵I and a solid-phase oxidant. The order of labelling intensity in complete particles was VP1?VP3>VP2; labelling of VP4 was undetectable. When the particles were denatured with sodium dodecyl sulfate at 100°C before iodination, the labelling efficiency was 6 times higher and the order of labelling intensity was VP3>VP2>VP1. After denaturation, the viral proteins no longer reacted with human anti-HAV antibody. The results suggest that (i) as with other picornaviruses, HAV exposes an essential part of VP1 at its surface whereas VP3 and especially VP2 are more hidden; (ii) naturally immunized individuals do not form detectable amounts of antibodies against the denatured capsid proteins. The apparent molecular weights of the VPs were 33 000, 29 000 and 28 000 daltons.     

Comparison of penaeid shrimp and insect parvoviruses suggests that viral transfers may occur between two distantly related arthropod groups

The DNA and putative amino acid sequences of representative insect and shrimp parvoviruses (subfamily Densovirinae) were analyzed using computer programs. Shrimp viruses included hepatopancreatic parvovirus (HPV) of Penaeus monodon (HPVmon) and P. chinensis (HPVchin), spawner-isolated mortality virus from P. monodon (SMVmon) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) from P. vannamei. Insect viruses included Aedes aegypti densovirus (AaeDNV), Aedes albopictus densovirus (AalDNV), Junonia coenia densovirus (JcDNV), Galleria mellonella densovirus (GmDNV), Bombyx mori densovirus 5 (BmDNV), Diatraea saccharalis densovirus (DsDNV) and Periplaneta fuliginosa densovirus (PfDNV). Virion size for all these viruses ranged between 18 and 30 nm diameter and ssDNA genome length was between 4 and 6 kb. Using BLAST or Clustal W with the sequence fragments available, no significant DNA homology was found except for 77% DNA identity between HPVmon and HPVchin. However, phylogenetic trees constructed by comparing DNA genome sequences for putative viral polypeptides, capsid proteins and nonstructural proteins placed the parvoviruses into two Clades: Clade 1 with SMVmon, PfDNV, DsDNV, GmDNV, JcDNV, and BmDNV; and Clade 2 with HPVmon, HPVchin, IHHNV, AalDNV and AaeDNV. The four shrimp parvoviruses fell into two different clades that grouped with different insect parvoviruses.     

Complete genome comparison of duck hepatitis virus type 1 parental and attenuated strains

Two complete duck hepatitis virus type 1 (DHV-1) genomes, strain SY5 and its chicken embryos passage descendent vaccine strain ZJ-A, were compared and analyzed in order to identify possible sites of attenuation. Of the 205 nucleotide changes, 22 resulted in sense mutations, 174 produced nonsense mutations. Besides, there are 7 consistent nucleotides substitutions in 5'UTR and 2 in 3'UTR. Three of these 22 sense mutations resided in VP0, 6 exists in VP1, one exists in VP3, 3 exists in 2A2, 3 exists in 2C, one was detected in 3B and 5 was in 3D. These results suggested that VP0, VP1, 3D, and 5'/3'UTR may contribute to the attenuation of DHV-1 in chicken/duck/embryos. The results provide a genetic basis for future manipulation of a DHV-1 infectious clone.     

Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death

The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover, NTRNS2 a chimeric virus equivalent to the previously reported FL-J6/JFH chimera, showed a 10-fold enhancement of virus titers compared to CNS2. NTRNS2 differs from CNS2 by three nucleotide differences residing in the 5' NTR and core coding sequence and all three nucleotide changes were necessary for increased virion production. Importantly, cells producing Cp7 virus showed increased apoptosis compared with JFH1, an effect correlating with virion production. These studies begin to unravel requirements for robust virus replication and the relationship between increased virion production and host cell viability.