The sexually transmitted insect virus,Hz-2V

Hz-2V is one of only a very few sexually transmitted viruses currently known in insects. Replication of this insect pathogenic virus results in sterility of infected moths rather than mortality. The sterility of the infected host is a consequence of virus directed malformation of adult reproductive tissues, which in females results in cellular proliferation and hypertrophy of these tissues. Virus replication has additional ramifications in infected females. Infected females produce more mating pheromones and attract more mates than healthy females, ultimately facilitating virus transmission and enhancing viral fitness. The molecular mechanisms used by the virus to manipulate the host to enhance its fitness are yet to be determined. Unraveling the underlying principles of these mechanisms promises to enhance our understanding of insect reproductive physiology, as well as provide molecular tools for use in novel approaches in sterile insect control programs.     

In vivo propagation of a microsporidan pathogenic to insects

Equivalent numbers of spores were produced when the microsporidan Nosema necatrix was propagated in either Trichoplusia ni or Heliothis zea. Maximum spore production was obtained at an inoculum level of 1 × 10⁵ spores/ml. Larvae inoculated 5 days post-hatching contained 1.6 × 10⁹ spores/gram larva after an incubation period of 21 days. Temperature optima for the parasite are 21–26°C in both hosts.     

Pathology and ultrastructure of Hz-2V infection in the agonadal female corn earworm,Helicoverpa zea

The pathology and ultrastructure of the reproductive tract of Hz-2V-infected female corn earworm moths, Helicoverpa zea, were studied. The identity of malformed reproductive tissues found in virus-infected moths was determined by examining these tissues in moths that were infected with the virus at different life stages. Malformation of reproductive tissues in the progeny of virus-infected female moths was first observed by 3 days post-pupation (dpp), indicating that virus replication had altered the differentiation of these tissues very early on in their development. The ultrastructure of the grossly malformed agonadal reproductive tissues from insects aged 3-10dpp revealed the absence of the cuticular lining found in the oviducts of normal moths, and the proliferation of epithelial cells in these infected oviduct tissues. In addition, large quantities of virus were found aggregated into a large mass in the lumen of the malformed cervix bursa of 10dpp agonadal female pharate adult moths. Prior to eclosion, the virus in the cervix bursa was observed separated into spherical masses, which are thought to exude through the ductus bursa and collect over the vulva, forming a viral "waxy plug" that is likely to play an important role in virus transmission.     

Replication of the gonad-specific virus Hz-2V in Ld652Y cells mimics replication in vivo

A newly discovered, nonoccluded insect virus, known as gonad-specific virus or Hz-2V, was found to replicate differently in two insect cell lines derived from ovarian tissues (Tn-368 cells from Trichoplusia ni and Ld652Y from Lymantria dispar). Differences between these two cell lines were observed in virus plaque forming ability, rate of viral DNA replication, time course of infectious virus production, and the mechanism of virus release from infected cells. Replication of Hz-2V in Ld652Y cells was more productive and more closely resembled in vivo virus replication.     

Horizontal transmission of Hz-2V by virus infected Helicoverpa zea moths

Helicoverpa zea female moths productively infected with Hz-2V have malformed reproductive tissues and are sterile. Virus replication in infected females occurs primarily in the reproductive tissues and culminates with the accumulation of virus-filled vesicles, which form plugs of virus covering the reproductive openings of these insects. The location of this large concentration of virus particles at the terminal abdominal segment of infected females suggests that it may serve as a source of virus that can be transmitted horizontally between moths during mating. In mating experiments it was found that healthy males are attracted to and attempt to mate with infected females, and that these males are able transmit Hz-2V to healthy females during subsequent matings, giving rise to virus infected progeny.     

Acquisition, persistence, and species susceptibility of the Hz-2V virus

Hz-2V, formerly called gonad-specific virus, is known to infect the reproductive organs of both males and females of the corn earworm Helicoverpa zea, rendering them agonadal or sterile. The primary mode of transmission is through mating by asymptomatic carrier moths. In this report we show that Hz-2V can be acquired by first instar larvae, through feeding on virus laced diet, although the incidence of agonadal condition was significantly lower. In a laboratory study, the virus appeared to persist for no more than three generations, with the incidence of agonadal progeny decreasing with each generation. Although, Hz-2V has been reported only from H. zea, in our tests when nine species of insects were artificially infected, four of the Noctuid species showed some signs of agonadal condition. Out of the remaining five species, the diamondback moth Plutella xylostella and the German cockroach Blatella germanica, showed no evidence of the virus in progeny of adults that were injected with Hz-2V, even after using the very sensitive PCR based assay.     

Characterization of the DNA of a Nonoccluded Baculovirus, Hz-1V

The DNA of the nonoccluded baculovirus (Hz-1V) obtained from the IMC-Hz-1 cell line was characterized by physicochemical and restriction endonuclease techniques. Hz-1V DNA isolated from purified virus had buoyant densities of 1.58 and 1.54 g/ml in CsCl-ethidium bromide density gradients, which corresponded to supercoiled and to relaxed circular and linear DNA, respectively. Neutral CsCl equilibrium centrifugation indicated that the Hz-1V DNA had a buoyant density of 1.7024 g/ml, which corresponded to a guanine-plus-cytosine (G+C) content of 43%. Thermal denaturation indicated a high G+C domain(s) in the Hz-1V genomic DNA. The domain(s), which included about 11% of the total genomic DNA, exhibited a T(m) of 97 degrees C. The remaining portion (89%) of the DNA had a T(m) of 86.5 degrees C. The T(m)s corresponded to G+C contents of 42 and 67%, respectively. The mean genetic complexity of Hz-1V DNA determined by DNA reassociation kinetic analysis was found to be 152 x 10(6). A possible rapidly reassociating component comprising approximately 13% of the genome was observed. The mean molecular weights from restriction endonuclease digests were 159 x 10(6) for both HindIII and EcoRI. Genomic heterogeneity was found in both the wild-type Hz-1V stock and in two plaque isolates. Of 12 single-plaque isolates, 3 basic restriction endonuclease DNA fragment patterns were observed. The molecular size estimates from electron microscopic contour lengths of uncloned viral DNA ranged from 70 to 158 megadaltons, and the mode was the 130- to 140-megadalton class.     

In vivo genetic variability of the human immunodeficiency virus type 2 V3 region.

The principal neutralizing epitope of the human immunodeficiency virus type 1 (HIV-1) lies between two invariant cysteines in the third variable region (V3) of the viral envelope (gp120), and its amino acid sequence varies among different HIV-1 isolates. HIV-2 carries an analogous amino acid sequence between two cysteines of the V3 regions, but its functional similarity with the HIV-1 principal neutralizing epitope is uncertain. We studied the degree of genetic variation of the HIV-2 V3 region in fresh blood samples from 12 HIV-2-seropositive individuals from Guinea-Bissau. Polymerase chain reaction was used to amplify viral fragments of 465 bp containing the V3 region from cellular DNA. Nucleotide sequence analysis of the entire envelope fragment from each patient revealed that the degree of variation among field isolates of HIV-2 is comparable to that observed in the analogous region of HIV-1. Most of the HIV-2 isolates studied were highly related, suggesting the existence of a limited number of different viral strains in the cohort studied. Thus, the HIV-2 and HIV-1 V3 regions vary to a similar degree and may also have analogous functions.     

In vivo function of airway epithelial TLR2 in host defense against bacterial infection

Decreased Toll-like receptor 2 (TLR2) expression has been reported in patients with chronic obstructive pulmonary disease and in a murine asthma model, which may predispose the hosts to bacterial infections, leading to disease exacerbations. Since airway epithelial cells serve as the first line of respiratory mucosal defense, the present study aimed to reveal the role of airway epithelial TLR2 signaling to lung bacterial [i.e., Mycoplasma pneumoniae (Mp)] clearance. In vivo TLR2 gene transfer via intranasal inoculation of adenoviral vector was performed to reconstitute TLR2 expression in airway epithelium of TLR2(-/-) BALB/c mice, with or without ensuing Mp infection. TLR2 and lactotransferrin (LTF) expression in airway epithelial cells and lung Mp load were assessed. Adenovirus-mediated TLR2 gene transfer to airway epithelial cells of TLR2(-/-) mice reconstituted 30-40% TLR2 expression compared with TLR2(+/+) cells. Such airway epithelial TLR2 reconstitution in TLR2(-/-) mice significantly reduced lung Mp load (an appropriate 45% reduction), coupled with elevated LTF expression. LTF expression in mice was shown to be mainly dependent on TLR2 signaling in response to Mp infection. Exogenous human LTF protein dose-dependently decreased lung bacterial load in Mp-infected TLR2(-/-) mice. In addition, human LTF protein directly dose-dependently decreased Mp levels in vitro. These data indicate that reconstitution of airway epithelial TLR2 signaling in TLR2(-/-) mice significantly restores lung defense against bacteria (e.g., Mp) via increased lung antimicrobial protein LTF production. Our findings may offer a deliverable approach to attenuate bacterial infections in airways of asthma or chronic obstructive pulmonary disease patients with impaired TLR2 function.     

In vivo bioluminescence imaging for integrated studies of infection

Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection.     

In vivo impairment of neutrophil recruitment during lentivirus infection

Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro. We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade. In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h. When the tissue was superfused with 10 micro g/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats. At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils. In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata. In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8. However, FIV infection of neutrophils could not be detected. In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction. These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.     

Pathology and ultrastructure of the insect virus,Hz-2V,infecting agonadal male corn earworms,Helicoverpa zea

The pathology of the reproductive tract of Hz-2V-infected agonadal male corn earworm moths, Helicoverpa zea, was studied. The examination of the reproductive tissues of adult agonadal males infected with Hz-2V during different lifestages allowed us to positively correlate the grossly malformed tissues of typical agonadal male moths to the corresponding normal tissues in uninfected males. The reproductive tissues responsible for producing sperm, a pheromonostatic peptide (PSP), and the spermatophore in normal male moths were absent or grossly malformed in the agonadal male moths. Hz-2V was observed replicating in one area of these malformed reproductive tissues in pharate adult males as early as 7 days post-pupation. Interestingly, reproductive tissues essential for initiation of copulation and transfer of reproductive fluids into a female moth during mating appear to be intact and may be functional. These data suggest that agonadal adult males are able to mate with healthy female moths and transfer Hz-2V particles, without fertilizing female moths or altering their sexual receptivity to further mating with other male moths.     

In vitro and in vivo extrapolations of genotoxin exposures: consideration of factors which influence dose-response thresholds

The concept of a threshold of activity of a genotoxic agent is primarily based upon considerations of protective mechanisms and multiple cellular targets, which require inactivation before a toxic response is produced. In this paper, we have considered and evaluated the influences of compound metabolism, DNA lesion formation, mutation induction and sequence content, aneuploidy induction and the influence of repair enzymes upon genetic endpoints produced by both DNA reactive chemicals and by those chemicals which modify non-DNA cellular targets. Thresholds of activity have been evaluated by critical analysis of the published literature and original data analysing both the role of sequence context upon point mutation induction and DNA repair mechanisms upon the sensitivity of cultured cells to the induction of aneuploidy. In the case of DNA reactive chemicals, the presence of a threshold of chemical activity will be dependent upon cellular activities such as those of the Phase II enzymes reducing the activity of chemicals before lesion formation takes place and/or those of the DNA repair enzymes which reduce the proportion of DNA lesions which are processed into DNA sequence changes. Under such conditions, a given exposure of a DNA reactive chemical does not produce a linear or semi-linear increase in DNA lesions or in mutation frequency. However, even when these protective mechanisms are overwhelmed by the high exposures of genotoxic chemicals the biological effects of a genotoxin may be influenced by the sequence context of the gene under consideration. Here, we demonstrate that point mutations are detected at relatively higher frequencies in the non-coding introns compared with the coding exons. Many of the base changes detected in the exons do not produce amino acid changes in the proteins coded for by the genes being monitored for mutation induction. Both sequence context and the types of base changes induced may provide a "buffering" effect reducing the biological consequences of mutation induction. Spindle damaging chemicals, such as colcemid and vinblastine, induce aneuploidy by modifying the numbers of spindle fibres which regulate the segregation of chromosomes during mitosis and meiosis. The redundancy of spindle fibres in the dividing mammalian cell leads to the prediction that only chemical exposures which damage most, if not all, of the fibres will lead to the induction of polyploidy and/or aneuploidy. Such predicted thresholds of chemical activity can be observed when both chromosome loss and non-disjunction are measured in wild type cultures. However, we observed a substantial increase in sensitivity to aneugenic chemicals when measurements were made in primary cell cultures derived from xerodoma pigmentosum and trichothiodystrophy patients. Further studies are necessary to evaluate the consequences of the genetic background of tester strains upon the nature of the dose-response curve of aneugenic chemicals.     

In vivo immunomanipulation of V gamma 9V delta 2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model

Vgamma9Vdelta2(+) cells represent the major population of gammadelta T cells in primate blood and react in an MHC-unrestricted fashion to a set of low m.w. nonpeptide phosphoantigens. Two types of structurally related agonists have been discovered so far: the natural phosphoantigens (hydroxydimethyl allyl-pyrophosphate or isopentenyl-pyrophosphate (IPP)) acting directly on Vgamma9Vdelta2(+) TCR and aminobisphosphonates, which block the mevalonate pathway in target cells, leading to accumulation of natural phosphoantigens that in turn activate Vgamma9Vdelta2(+) cells. We demonstrate in the cynomolgus monkey that Vgamma9Vdelta2 can be manipulated in vivo with bromohydrin pyrophosphate (BrHPP)/Phosphostim, a potent synthetic agonist for which the mechanism of action is similar to natural phosphoantigens. Although of very short half-life, injection of BrHPP leads to strong activation of Vgamma9Vdelta2, inducing production of a high level of Th1 cytokines. Combination of BrHPP with low-dose rhIL-2 induces specific amplification of effector-memory peripheral Vgamma9Vdelta2 in blood in a dose-dependant manner. This transient response returns to baseline within 10-15 days. Successive infusions of BrHPP and rhIL-2 induce less vigorous expansions, suggesting a progressive exhaustion of the response. As no toxicity is detected with or without IL-2, this scheme represents a promising immunotherapeutic strategy for induction of systemic Th1 cytokines and massive expansion of gammadelta T cell subset with antitumor and anti-infectious properties.     

In vivo and in vitro effects of monoclonal antibody to Ly antigens on immunity to infection

Injection of CBA mice with Brucella abortus strain 19 leads to chronic infection during which both cell-mediated immunity (delayed hypersensitivity and macrophage activation) and antibody production occur. Protection was efficiently transferred to naive mice using spleen cells from mice infected 5 or 12 weeks earlier. Selective lysis in vitro of these cells by antibody to cell surface antigens showed that Thy-1⁺ Ly-1⁺2⁺ T lymphocytes were required for transfer. Treatment with anti-Ia serum neither suppressed nor enhanced adoptive transfer. Thus Ia⁺ B lymphocytes were not required, and Ia⁺ suppressor T cells were not active in the response. Three injections per week of anti-Ly-1 monoclonal antibody beginning 5 days before infection led to a 10-fold increase in bacterial numbers 25 days after infection when acquired immunity was well established in untreated mice. The delayed hypersensitivity response was unaffected. In addition cells from these in vivo treated mice were unable to transfer resistance. Beginning the treatment on the day of infection abolished the IgG antibody response without affecting bacterial numbers. The studies emphasize the unique role of Ly-1⁺2⁺ T cells in immunity to Brucella and indicate the usefulness of these techniques in dissecting out those components of the immune response which contribute to recovery from infection.     

In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection

Background  

The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum.     

杆状病毒在昆虫中的持续感染

杆状病毒（Baculovirus）是一类特异性感染节肢动物的环状双链DNA病毒,是野外控制害虫种群的重要生物因子,并已被开发为一种生物杀虫剂加以应用。杆状病毒感染昆虫宿主并不一定导致昆虫死亡,其持续感染（persistentinfection）在昆虫种群中普遍存在,且在某些刺激条件下,持续感染可被激活为增殖性感染并引发病毒流行病爆发。因此,杆状病毒持续感染对昆虫种群动力学以及病毒流行病学的研究具有重要意义。     

In vitro host range of the Hz-1 nonoccluded virus in insect cell lines

A total of 13 insect cell lines spanning 4 orders (Lepidoptera, Coleoptera, Diptera, and Homoptera) were tested for their ability to replicate the nonoccluded virus Hz-1. Only the Lepidopteran cell lines supported replication of the virus with TN-CL1 and BCIRL-HZ-AM1 producing the highest titers of 2.4 × 10⁸ tissue culture infective dose (TCID)₅₀/ml and 2.0 × 10⁸ TCID₅₀/ml, respectively. A codling moth cell line (CP-169) was the only Lepidopteran cell line that did not replicate the virus and transfection of this cell line with Hz-1 DNA failed to replicate the virus. Also, transfection with DNA from a recombinant baculovirus carrying the red fluorescent protein gene (AcMNPVhsp70 Red) was not expressed in CP-169 cells. The replication cycle of Hz-1 in BCIRL-HZ-AM1 cells showed that this virus replicated rapidly starting at 16 h postinoculation (p.i.) and reaching a peak titer of 1.0 × 10⁸ TCID₅₀/ml 56 h postinoculation. Hz-1 when compared with several other baculoviruses has the widest in vitro host spectrum.