Evaluation of white spot syndrome virus variable DNA loci as molecular markers of virus spread at intermediate spatiotemporal scales

Variable genomic loci have been employed in a number of molecular epidemiology studies of white spot syndrome virus (WSSV), but it is unknown which loci are suitable molecular markers for determining WSSV spread on different spatiotemporal scales. Although previous work suggests that multiple introductions of WSSV occurred in central Vietnam, it is largely uncertain how WSSV was introduced and subsequently spread. Here, we evaluate five variable WSSV DNA loci as markers of virus spread on an intermediate (i.e. regional) scale, and develop a detailed and statistically supported model for the spread of WSSV. The genotypes of 17 WSSV isolates from along the coast of Vietnam – nine of which were newly characterized in this study – were analysed to obtain sufficient samples on an intermediate scale and to allow statistical analysis. Only the ORF23/24 variable region is an appropriate marker on this scale, as geographically proximate isolates show similar deletion sizes. The ORF14/15 variable region and variable-number tandem repeat (VNTR) loci are not useful as markers on this scale. ORF14/15 may be suitable for studying larger spatiotemporal scales, whereas VNTR loci are probably suitable for smaller scales. For ORF23/24, there is a clear pattern in the spatial distribution of WSSV: the smallest genomic deletions are found in central Vietnam, and larger deletions are found in the south and the north. WSSV genomic deletions tend to increase over time with virus spread in cultured shrimp, and our data are therefore congruent with the hypothesis that WSSV was introduced in central Vietnam and then radiated ou

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells

East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five™ cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP[ratio]p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF

Adolf Mayer (1843-1942) en zijn betekenis voor de Virologie als wetenschap

In 2007 is het 125 jaar geleden dat Adolf Mayer voor het eerst zijn baanbrekend werk over het besmettelijk karakter van mozaiekziekte van tabak publiceerde (Mayer, 1882). Deze publicatie, in het Nederlands, in het Groningse Tijdschrift voor Landbouwkunde, markeert het begin van de virologie als wetenschapsgebied zowel nationaal als internationaal. Dit 125 jarig jubileum van de publicatie van Mayer valt samen met het vijftigjarig bestaan van de leerstoelgroep Virologie van Wageningen Universiteit. Tevens is dit een goede gelegenheid om te laten zien dat de activiteiten van de huidige leestoel Virologie voortbouwen op de bevindingen van Maye

Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus

Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% ß-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infectio

Effects of single and mixed infections with wild type and genetically modified Helicoverpa armigera nucleopolyhedrovirus on movement behaviour of cotton bollworm larvae

Naturally occurring insect viruses can modify the behaviour of infected insects and thereby modulate virus transmission. Modifications of the virus genome could alter these behavioural effects. We studied the distance moved and the position of virus-killed cadavers of fourth instars of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) infected with a wild-type genotype of H. armigera nucleopolyhedrovirus (HaSNPV) or with one of two recombinant genotypes of this virus on cotton plants. The behavioural effects of virus infection were examined both in larvae infected with a single virus genotype, and in larvae challenged with mixtures of the wild-type and one of the recombinant viruses. An egt-negative virus variant caused more rapid death and lower virus yield in fourth instars, but egt-deletion did not produce consistent behavioural effects over three experiments, two under controlled glasshouse conditions and one in field cages. A recombinant virus containing the AaIT-(Androctonus australis Hector) insect-selective toxin gene, which expresses a neurotoxin derived from a scorpion, caused faster death and cadavers were found lower down the plant than insects infected with unmodified virus. Larvae that died from mixed infections of the AaIT-expressing recombinant and the wild-type virus died at positions significantly lower, compared to infection with the pure wild-type viral strain. The results indicate that transmission of egt-negative variants of HaSNPV are likely to be affected by lower virus yield, but not by behavioural effects of egt gene deletion. By contrast, the AaIT recombinant will produce lower virus yields as well as modified behaviour, which together can contribute to reduced virus transmission under field conditions. In addition, larvae infected with both the wild-type virus and the toxin recombinant behaved as larvae infected with the toxin recombinant only, which might be a positive factor for the risk assessment of such toxin recombinants in the environment

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective conditio

Helicoverpa armigera nucleopolyhedrovirus occlusion-derived virus-associated protein, HA100, affects oral infectivity in vivo but not virus replication in vitro

ORF100 (ha100) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been reported as one of the unique genes of group II alphabaculoviruses encoding a protein located in the occlusion-derived virus (ODV) envelope and nucleocapsid. The protein consists of 510 aa with a predicted mass of 58.1 kDa and is a homologue of poly(ADP–ribose) glycohydrolase in eukaryotes. Western blot analysis detected a 60 kDa band in HearNPV-infected HzAM1 cells starting at 18 h post-infection. Transient expression of GFP-fused HA100 in HzAM1 cells resulted in cytoplasmic localization of the protein, but after superinfection with HearNPV, GFPfused HA100 was localized in the nucleus. To study the function of HA100 further, an ha100-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the ha100-null virus had similar budded virus production kinetics to that of the parental virus. Electron microscopy revealed that deletion of HA100 did not alter the morphology of ODVs or occlusion bodies (OBs). However, bioassays in larvae showed that the 50% lethal concentration (LC50) value of HA100-null OBs was significantly higher than that of parental OBs; the median lethal time (LT50) of ha100-null OBs was about 24 h later than control virus. These results indicate that HA100 is not essential for virus replication in vitro. However, it significantly affects the oral infectivity of OBs in host insects, suggesting that the association HA100 with the ODV contributes to the infectivity of OBs in vivo

Proteomic analysis of Glossina pallidipes salivary gland hypertrophy virus virions for immune intervention in tsetse fly colonies

Many species of tsetse flies (Diptera: Glossinidae) can be infected by a virus that causes salivary gland hypertrophy (SGH). The viruses isolated from Glossina pallidipes (GpSGHV) and Musca somestica (MdSGHV) have recently been sequenced. Tsetse flies with SGH have a reduced fecundity and fertility which cause a serious problem for mass rearing in the frame of sterile insect technique (SIT) programs to control and eradicate tsetse populations in the wild. A potential intervention strategy to mitigate viral infections in fly colonies is neutralizing of the GpSGHV infection with specific antibodies against virion proteins. Two major GpSGHV virion proteins of about 130 kDa and 50 kDa, respectively, were identified by Western analysis using polyclonal rabbit antibody raised against whole GpSHGV virions. The proteome of GpSGHV, containing the antigens responsible for the immune-response, was investigated by liquid chromatography tandem mass spectrometry (LC-MS/MS) and 61 virion proteins were identified by comparison with the genome sequence. Specific antibodies were produced in rabbits against seven candidate proteins including the ORF10 / C-terminal fragment, ORF47 and ORF96 as well as proteins involved in peroral infectivity PIF-1 (ORF102), PIF-2 (ORF53), PIF-3 (ORF76) and P74 (ORF1). Antiserum against ORF10 specifically reacted to the 130 kDa protein in a Western blot analysis and to the envelope of GpSGHV using immunogold-EM. This result suggests that immune intervention of viral infections in colonies of G. pallidipes is a realistic optio

The coefficient of correction of effectiveness with the account of natural factors

The existing methods of determination of effectiveness don’t give the opportunity to emphasize the factors to the full extent. By which we can achieve the effect: the level of development of technologies, exploitation of natural resources, i,e. the damage to the environment etc. The economic damage, caused to the environment as a result of exploitation of natural resources and ecological violations at the given moment, doesn’t have a precise definition at the profound level. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/1280

Characterization of a nucleopolyhedrovirus isolated from the laboratory rearing of the beet armyworm Spodoptera Exigua (HBN.) in Poland

A nucleopolyhedrovirus isolated from the beet armyworm Spodoptera exigua (Polish laboratory culture), SeMNPV (P), morphologically similar to the viral bioinsecticide virus Spod-XR, was characterized molecularly and biologically. Phylogenetic analysis based on three conserved baculovirus genes, polh, lef-8 and pif-2, showed the highest homology of SeMNPV (P) to Mamestra brassicae (Mb) MNPV and M. configurata (Maco) MNPV, and much less to SeMNPV (Spod-XR). These findings were confirmed by genomic DNA restriction profile analyses. Bioassays revealed that SeMNPV isolated from the commercial bioinsecticide Spod-XR was themost infectious for S. exigua, while the infectivity of SeMNPV (P) and MbMNPV was significantly lower. These data suggest that SeMNPV (P) is a variant of MbMNPV