Molecular biology and structure of a novel penaeid shrimp densovirus elucidate convergent parvoviral host capsid evolution.

The giant tiger prawn (Penaeus monodon) is a decapod crustacean widely reared for human consumption. Currently, viruses of two distinct lineages of parvoviruses (PVs, family Parvoviridae; subfamily Hamaparvovirinae) infect penaeid shrimp. Here, a PV was isolated and cloned from Vietnamese P. monodon specimens, designated Penaeus monodon metallodensovirus (PmMDV). This is the first member of a third divergent lineage shown to infect penaeid decapods. PmMDV has a transcription strategy unique among invertebrate PVs, using extensive alternative splicing and incorporating transcription elements characteristic of vertebrate-infecting PVs. The PmMDV proteins have no significant sequence similarity with other PVs, except for an SF3 helicase domain in its nonstructural protein. Its capsid structure, determined by cryoelectron microscopy to 3-Å resolution, has a similar surface morphology to Penaeus stylirostris densovirus, despite the lack of significant capsid viral protein (VP) sequence similarity. Unlike other PVs, PmMDV folds its VP without incorporating a ßA strand and displayed unique multimer interactions, including the incorporation of a Ca2+ cation, attaching the N termini under the icosahedral fivefold symmetry axis, and forming a basket-like pentamer helix bundle. While the PmMDV VP sequence lacks a canonical phospholipase A2 domain, the structure of an EDTA-treated capsid, determined to 2.8-Å resolution, suggests an alternative membrane-penetrating cation-dependent mechanism in its N-terminal region. PmMDV is an observed example of convergent evolution among invertebrate PVs with respect to host-driven capsid structure and unique as a PV showing a cation-sensitive/dependent basket structure for an alternative endosomal egress.

Diversity of Sea Star-Associated Densoviruses and Transcribed Endogenous Viral Elements of Densovirus Origin.

A viral etiology of sea star wasting syndrome (SSWS) was originally explored with virus-sized material challenge experiments, field surveys, and metagenomics, leading to the conclusion that a densovirus is the predominant DNA virus associated with this syndrome and, thus, the most promising viral candidate pathogen. Single-stranded DNA viruses are, however, highly diverse and pervasive among eukaryotic organisms, which we hypothesize may confound the association between densoviruses and SSWS. To test this hypothesis and assess the association of densoviruses with SSWS, we compiled past metagenomic data with new metagenomic-derived viral genomes from sea stars collected from Antarctica, California, Washington, and Alaska. We used 179 publicly available sea star transcriptomes to complement our approaches for densovirus discovery. Lastly, we focus the study on sea star-associated densovirus (SSaDV), the first sea star densovirus discovered, by documenting its biogeography and putative tissue tropism. Transcriptomes contained only endogenized densovirus elements similar to the NS1 gene, while numerous extant densoviral genomes were recovered from viral metagenomes. SSaDV was associated with nearly all tested species from southern California to Alaska, and in contrast to previous work, we show that SSaDV is one genotype among a high diversity of densoviruses present in sea stars across the West Coast of the United States and globally that are commonly associated with grossly normal (i.e., healthy or asymptomatic) animals. The diversity and ubiquity of these viruses in sea stars confound the original hypothesis that one densovirus is the etiological agent of SSWS.IMPORTANCE The primary interest in sea star densoviruses, specifically SSaDV, has been their association with sea star wasting syndrome (SSWS), a disease that has decimated sea star populations across the West Coast of the United States since 2013. The association of SSaDV with SSWS was originally drawn from metagenomic analysis, which was further studied through field surveys using quantitative PCR (qPCR), with the conclusion that it was the most likely viral candidate in the metagenomic data based on its representation in symptomatic sea stars compared to asymptomatic sea stars. We reexamined the original metagenomic data with additional genomic data sets and found that SSaDV was 1 of 10 densoviruses present in the original data set and was no more represented in symptomatic sea stars than in asymptomatic sea stars. Instead, SSaDV appears to be a widespread, generalist virus that exists among a large diversity of densoviruses present in sea star populations.

Diaphorina citri densovirus is a persistently infecting virus with a hybrid genome organization and unique transcription strategy.

Diaphorina citri densovirus (DcDV) is an ambisense densovirus with a 5071 nt genome. Phylogenetic analysis places DcDV in an intermediate position between those in the Ambidensovirus and Iteradensovirus genera, a finding that is consistent with the observation that DcDV possesses an Iteradensoviris-like non-structural (NS) protein-gene cassette, but a capsid-protein (VP) gene cassette resembling those of other ambisense densoviruses. DcDV is maternally transmitted to 100 % of the progeny of infected female Diaphorina citri, and the progeny of infected females carry DcDV as a persistent infection without outward phenotypic effects. We were unable to infect naïve individuals by oral inoculation, however low levels of transient viral replication are detected following intrathoracic injection of DcDV virions into uninfected D. citri insects. Transcript mapping indicates that DcDV produces one transcript each from the NS and VP gene cassettes and that these transcripts are polyadenylated at internal sites to produce a ~2.2 kb transcript encoding the NS proteins and a ~2.4 kb transcript encoding the VP proteins. Additionally, we found that transcriptional readthrough leads to the production of longer non-canonical transcripts from both genomic strands.

Wolbachia modulates prevalence and viral load of Culex pipiens densoviruses in natural populations.

The inadequacy of standard mosquito control strategies calls for ecologically safe novel approaches, for example the use of biological agents such as the endosymbiotic α-proteobacteria Wolbachia or insect-specific viruses (ISVs). Understanding the ecological interactions between these "biocontrol endosymbionts" is thus a fundamental step. Wolbachia are transmitted vertically from mother to offspring and modify their hosts' phenotypes, including reproduction (e.g., cytoplasmic incompatibility) and survival (e.g., viral interference). In nature, Culex pipiens (sensu lato) mosquitoes are always found infected with genetically diverse Wolbachia called wPip that belong to five phylogenetic groups. In recent years, ISVs have also been discovered in these mosquito species, although their interactions with Wolbachia in nature are unknown. Here, we studied the interactions between a widely prevalent ISV, the Culex pipiens densovirus (CpDV, Densovirinae), and Wolbachia in northern Tunisian C. pipiens populations. We showed an influence of different Wolbachia groups on CpDV prevalence and a general positive correlation between Wolbachia and CpDV loads. By investigating the putative relationship between CpDV diversification and wPip groups in the different sites, we detected a signal linked to wPip groups in CpDV phylogeny in sites where all larvae were infected by the same wPip group. However, no such signal was detected where the wPip groups coexisted, suggesting CpDV horizontal transfer between hosts. Overall, our results provide good evidence for an ecological influence of Wolbachia on an ISV, CpDV, in natural populations and highlight the importance of integrating Wolbachia in our understanding of ISV ecology in nature.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus.

BACKGROUND: The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES: Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS: Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS: MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS: Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells.

BACKGROUND: Densoviruses (DVs) are highly pathogenic to their hosts. However, we previously reported a mutualistic DV (HaDV2). Very little was known about the characteristics of this virus, so herein we undertook a series of experiments to explore the molecular biology of HaDV2 further. RESULTS: Phylogenetic analysis showed that HaDV2 was similar to members of the genus Iteradensovirus. However, compared to current members of the genus Iteradensovirus, the sequence identity of HaDV2 is less than 44% at the nucleotide-level, and lower than 36, 28 and 19% at the amino-acid-level of VP, NS1 and NS2 proteins, respectively. Moreover, NS1 and NS2 proteins from HaDV2 were smaller than those from other iteradensoviruses due to their shorter N-terminal sequences. Two transcripts of about 2.2 kb coding for the NS proteins and the VP proteins were identified by Northern Blot and RACE analysis. Using specific anti-NS1 and anti-NS2 antibodies, Western Blot analysis revealed a 78 kDa and a 48 kDa protein, respectively. Finally, the localization of both NS1 and NS2 proteins within the cell nucleus was determined by using Green Fluorescent Protein (GFP) labelling. CONCLUSION: The genome organization, terminal hairpin structure, transcription and expression strategies as well as the mutualistic relationship with its host, suggested that HaDV2 was a novel member of the genus Iteradensovirus within the subfamily Densovirinae.

Densoviruses in oyster Crassostrea ariakensis.

Densoviruses have short ssDNA genomes and mainly infect arthropods. To characterize viral nucleic acid in shellfish, oysters (Crassostrea ariakensis) were analyzed using viral metagenomics. Two large de novo assembled contigs, CaaDV1 and CaaDV2, consisting of nearly complete densovirus genomes (5860 nucleotides (nt) and 4034 nt) with two major ambisense protein coding regions were identified. Several potential non-structural proteins and capsid proteins were encoded by these genomes, but these were divergent from the existing densoviral species. The NS1 protein of the two CaaDVs shared 43.3%~61.5% amino acid identities with the sea star-associated densovirus and cherax quadricarinatus densovirus, with the four species clustering by phylogenetic analysis. This is the first report of densovirus detection in shellfish, increasing the potential host range of densoviruses and the genetic diversity of the genus Ambidensovirus.

Structural proteins of Helicoverpa armigera densovirus 2 enhance transcription of viral genes through transactivation.

Herein, we report the identification of putative promoters for the non-structural proteins (NS) and capsid structural proteins (VP) of Helicoverpa armigera densovirus (HaDV2) as well as a potential mechanism for how these promoters might be regulated. For the first time, we report that VP is able to transactivate the VP promoter and, to a lesser degree, the NS promoter in densoviruses. In addition to this, another promoter-like sequence designated P2, when co-transfected with the VP gene, enhanced luciferase activity by approximately 35 times compared to a control. This suggests that there are two promoters for VP in HaDV2 and that the VP of parvoviruses might play a more important role in viral transcription than previously appreciated.

Prevalence, diversity and co-occurrence of the white spot syndrome virus, monodon baculovirus and Penaeus stylirostris densovirus in wild populations of Penaeus monodon in the Philippines.

The farming of the black tiger shrimp Penaeus monodon in the Philippines relies on wild broodstock. PCR was thus used to determine the prevalence of white spot syndrome virus (WSSV), monodon baculovirus (MBV) and Penaeus stylirostris densovirus (PstDV) in a total of 178 shrimp from 6 geographically disparate locations where broodstock are captured for use in hatcheries. PCR amplicons were also sequenced to identify phylogenetic relationships of the virus haplotypes detected. Shrimp from southeastern Luzon (Camarines Norte) had the highest prevalence of each of the 3 viruses and were frequently co-infected with 2 or more viruses. No viruses were detected in shrimp from northwestern Luzon (Pangasinan). MBV was most prevalent and PstDV strains displayed the most genetic diversity. WSSV was detected at 3 sites, and a VP28 gene sequence examined was invariant and consistent with strains found in many countries, including Thailand, China, Japan, Korea, Indonesia, Iran, Brazil and Mexico. WSSV open reading frame 94 gene sequence analysis identified location-specific repeat types. MBV sequences were dissimilar to haplotypes detected in India. PstDV sequences were diverse and included 2 lineages detected either in Australia or in the United States, Ecuador, Taiwan, China and Vietnam. The PCR data confirmed that WSSV, MBV and PstDV are endemic in P. monodon in the Philippines but that populations at some locations might remain free of infection.

Densovirus associated with sea-star wasting disease and mass mortality.

Populations of at least 20 asteroid species on the Northeast Pacific Coast have recently experienced an extensive outbreak of sea-star (asteroid) wasting disease (SSWD). The disease leads to behavioral changes, lesions, loss of turgor, limb autotomy, and death characterized by rapid degradation ("melting"). Here, we present evidence from experimental challenge studies and field observations that link the mass mortalities to a densovirus (Parvoviridae). Virus-sized material (i.e., <0.2 µm) from symptomatic tissues that was inoculated into asymptomatic asteroids consistently resulted in SSWD signs whereas animals receiving heat-killed (i.e., control) virus-sized inoculum remained asymptomatic. Viral metagenomic investigations revealed the sea star-associated densovirus (SSaDV) as the most likely candidate virus associated with tissues from symptomatic asteroids. Quantification of SSaDV during transmission trials indicated that progression of SSWD paralleled increased SSaDV load. In field surveys, SSaDV loads were more abundant in symptomatic than in asymptomatic asteroids. SSaDV could be detected in plankton, sediments and in nonasteroid echinoderms, providing a possible mechanism for viral spread. SSaDV was detected in museum specimens of asteroids from 1942, suggesting that it has been present on the North American Pacific Coast for at least 72 y. SSaDV is therefore the most promising candidate disease agent responsible for asteroid mass mortality.

Transcription of densovirus endogenous sequences in the Myzus persicae genome.

Integration of non-retroviral sequences in the genome of different organisms has been observed and, in some cases, a relationship of these integrations with immunity has been established. The genome of the green peach aphid, Myzus persicae (clone G006), was screened for densovirus-like sequence (DLS) integrations. A total of 21 DLSs localized on 10 scaffolds were retrieved that mostly shared sequence identity with two aphid-infecting viruses, Myzus persicae densovirus (MpDNV) and Dysaphis plantaginea densovirus (DplDNV). In some cases, uninterrupted potential ORFs corresponding to non-structural viral proteins or capsid proteins were found within DLSs identified in the aphid genome. In particular, one scaffold harboured a complete virus-like genome, while another scaffold contained two virus-like genomes in reverse orientation. Remarkably, transcription of some of these ORFs was observed in M. persicae, suggesting a biological effect of these viral integrations. In contrast to most of the other densoviruses identified so far that induce acute host infection, it has been reported previously that MpDNV has only a minor effect on M. persicae fitness, while DplDNV can even have a beneficial effect on its aphid host. This suggests that DLS integration in the M. persicae genome may be responsible for the latency of MpDNV infection in the aphid host.

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture.

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus that are involved in integration of the densovirus in insect cell chromosomes and a promoter/enhancer system that results in high levels of expression. The plasmid also contains two markers that permit selection of transformed insect cells by antibiotic resistance or by cell-sorting for fluorescent protein expression. Transformation of Bombyx mori Bm5 or Spodoptera frugiperda Sf9 cultured cells with the pDP9 vectors results in the integration of the pDP9 plasmid into genomic DNA of Bm5 and Sf9 cells. pDP9 contains a multiple cloning site (MCS) 3' of the densoviral P9 promoter and insertion of a protein coding sequence within the MCS results in high level expression by pDP9 transformed cells. P9 driven transcription in the pDP9 transformed Sf9 cells produced foreign gene transcript levels that were 30 fold higher than actin 3 driven transgenes and equivalent to hr5IE1 driven transgenes. The pDP9 vector transformation results in the efficient selection of clones for assessment of promoter activity.

Effects of recombination on densovirus phylogeny.

Densoviruses are a group of arthropod-infecting viruses with a small single-stranded linear DNA genome. These viruses constitute the subfamily Densovirinae of the family Parvoviridae. While recombination in between vertebrate-infecting parvoviruses has been investigated, to date, no systematic analysis of recombination has been carried out for densoviruses. The aim of the present work was to study possible recombination events in the evolutionary history of densoviruses and to assess possible effects of recombination on phylogenies inferred using amino acid sequences of nonstructural (NS) and capsid (viral protein, VP) proteins. For this purpose, the complete or nearly complete genome nucleotide sequences of 40 densoviruses from the GenBank database were used to construct a phylogenetic cladogram. The viruses under study clustered into five distinct groups corresponding to the five currently accepted genera. Recombination within each group was studied independently. The RDP4 software revealed three statistically highly credible recombination events, two of which involved viruses of the genus Ambidensovirus, and the other, viruses from the genus Iteradensovirus. These recombination events led to mismatches between phylogenetic trees constructed using comparison of amino acid sequences of proteins encoded by genome regions of recombinant and non-recombinant origin (regulatory NS1 and NS3 proteins and capsid VP protein).

A new densovirus in cerebrospinal fluid from a case of anti-NMDA-receptor encephalitis.

We characterized the genome of a densovirus, tentatively called human CSF-associated densovirus 1 (HuCSFDV1), in cerebrospinal fluid (CSF) from a human case of encephalitis with antibodies against the N-methyl D-aspartate receptor. The presence of the viral genome in CSF was independently confirmed. This virus, which is proposed to be a member of a new species in the genus Iteradensovirus of the subfamily Densovirinae, showed the typical two ORFs encoding nonstructural and structural proteins with low-level identities of 22 and 16 % to the closest known densovirus relative. No other eukaryotic viral sequences were detected using deep sequencing. The replication and pathogenicity in humans of this virus, which belongs to a viral subfamily whose members are only known to replicate in invertebrates, remain to be demonstrated. Alternative explanations for the detection of densovirus DNA in CSF are discussed.

A formulated double-stranded RNA diet for reducing Penaeus monodon densovirus infection in black tiger shrimp.

Penaeus monodon densovirus (PmDNV) is one of the major causes of stunted shrimp in the aquaculture industry in Thailand. Significant reductions in levels of PmDNV as assessed by PCR analysis of shrimp hepatopancreas were seen in both prophylactic and curative experiments after feeding shrimp with a formulated diet containing mixed inactivated bacteria harboring dsRNAs corresponding to the PmDNV ns1 and vp genes. Significant reductions of approximately 88% (prophylactic) and 64% (curative) of PmDNV were observed, suggesting that this diet has a high potential for application in commercial aquaculture for reducing PmDNV associated stunted growth of shrimp.

Genetic variability of Myzus persicae nicotianae densovirus based on partial NS and VP gene sequences.

We previously described a novel densovirus [Myzus persicae nicotianae densovirus (MpnDV)] infecting M. persicae nicotianae (Hemiptera: Aphididae) with 34% prevalence. This single-stranded DNA virus has a 5480-nucleotide ambisense genome and belongs to the Densovirinae subfamily within the family Parvoviridae. In the present study, we estimated the genetic diversity of MpnDV using partial nonstructural protein (NS) and capsid protein (VP) gene sequences from 10 locations in China. First, we identified MpnDV-positive samples by amplifying a 445-bp fragment with primers MpDVF/MpDVR. Subsequently, we amplified and sequenced COI genes with primers MpCOIF/ MpCOIR, and partial NS and VP sequences with primers MpnDVF1/MpnDVR1. The respective 655-, 1461-, and 423-bp COI, NS, and VP fragments were used to analyze the genetic diversity of MpnDV using MEGA 6.0 and DnaSP 5.0. The high level of identity shared by all COI sequences (>99%) suggested that the aphids sampled were of the same species, and indicated population homogeneity across the 10 locations investigated. The nucleotide diversity of MpnDV sequences (0.0020 ± 0.0025) was significantly higher than that of the COI genes (0.0002 ± 0.0005). The pairwise fixation index for MpnDV was 0.832, and the total gene flow was 0.05. Phylogenetic analysis revealed that the MpnDV haplotypes clustered according to geographical location, except for those from the Liaoning and Shanxi provinces. In conclusion, MpnDV demonstrated a low level of gene flow and high genetic diversity, suggesting that it is vertically transmitted, and implying that endosymbiotic viruses could be used as markers in studies of insect population genetics.

Characterization and Distribution Analysis of a Densovirus Infecting Myzus persicae nicotianae (Hemiptera: Aphididae).

Densoviruses (DVs) are a group of viruses that contain a linear single-stranded DNA genome between 4­6 kb in length. Herein, we report a DV with a 5,480-nt genome, isolated from tobacco aphid (Myzus persicae nicotianae Blackman), named MpnDV. Unlike the genome of M. persicae densovirus (MpDV), which possesses five open reading frames (ORFs), the genome of MpnDV contains four putative ORFs­the nonstructural protein 1 (NS1) and NS2 from MpnDV are 98- and 52-amino acids longer than those of MpDV, respectively, at the N-terminus, and the capsid proteins (VP) are 102 amino acids longer at the C-terminus than those of MpDV. Mapping of the MpnDV transcripts by RACE method indicated that the ORF of NS2 started at nt 340 and the right two putative ORFs were combined together by deleting two introns, one of 95 bp located at nt 2,932­3,026 and the other of 145 bp located at nt 4,715­4,859, suggesting transcript mapping was necessary for analyzing of genome organization. Alignment analysis indicated that MpnDV shows 97% sequence identity with MpDV, and that the shortened ORFs resulted from nucleotide indels, suggesting MpnDV and MpDV were two isolates of the same virus. Thus, MpnDV and MpDV clustered together in a tree-based analysis. The prevalence of MpnDV infection in wild populations of tobacco aphids differed among 29 locations; 34% of the 622 individuals sampled were positive. The genome organization, transcript strategy, and widespread distribution in wild populations suggest that MpnDV might possess a biological function different from that of MpDV.

[Expression of cDNA of the Gene for the Capsid Protein VP2 of German Cockroach Densovirus in the Transgenic Strain of Drosophila melanogaster].

Transgenic strains of Drosophila melanogaster capable of expressing a cDNA fragment corresponding to open reading frame (ORF) of the gene for the German cockroach densonucleosis virus capsid protein VP2 (ORF VP2) in specific tissues and at a certain stage of development depending on the type of chosen driver strains (GAL-UAS system) were obtained. The ORF VP2 transcription was examined at the imago stage after crossing the obtained transgenic Drosophila with the driver line expressing the inducer protein (GAL4) under control of actin promoter (the ORF VP2 expression is induced in all tissues of the first-generation Drosophila). It was demonstrated that the greater part of transcribed foreign RNA was represented by three spliced variants in which RNA fragments either between nucleotides 137 and 353 or between nucleotides 609 and 1925 were excised; the third spliced variant was represented by RNA lacking both introns. Using the next-generation sequencing (NGS) technique, the proportion of unspliced form relative to spliced variants of the analyzed RNA was assessed. It was shown that the ratio of unspliced form to the identified spliced variants of the analyzed RNA was approximately 1:6. It is suggested that splicing of viral RNA foreign to Drosophila can be a sort of defense mechanism preventing the large-scale production of the capsid protein, potentially hazardous to the host organism.

PHYLOGENETIC ANALYSIS REVEALS GENETIC VARIATIONS OF DENSOVIRUS ISOLATED FROM FIELD MOSQUITOES IN BANGKOK AND SURROUNDING REGIONS.

Screening for densoviruses (DNVs) from Aedes, Culex and Toxorhynchites mosquitoes collected in Bangkok and surrounding regions identified two clades of Aedes DNV; Ae. aegypti DNV (AaeDNV) and Ae. albopictus DNV (AalDNV) by PCR-restriction fragment length polymorphism (PCR-RFLP). From nucleotide sequencing and phylogenetic analysis of PCR amplicons of a fragment of DNV capsid gene, these DNVs were shown to be new DNV genetic variations similar to AaeDNV. Isolation and identification of densoviruses from indigenous field mosquitoes reside in natural habitat should be helpful in monitoring the distribution of DNVs in important mosquitoes, especially Ae. aegypti and Ae. albopictus, vector for dengue and yellow fever viruses.

[Mammalian cell culture as a model for studying the intracellular traffic of densovirus proteins].

The intracellular localization of the fusion protein composed of green fluorescent protein (GFP) and one of the capsid proteins (namely VP1) of the German cockroach densovirus BgDV1 was investigated using the HeLa human cell culture. The intracellular localization of GFP was analyzed in a series of control experiments. Histochemical analysis with GFP antibodies showed that the fusion protein is localized exclusively inside the nucleus of cells because of the transitory expression of the corresponding vector constructions, whereas the GFP is located both in the nucleus and the cytoplasm. We can conclude that the signal of the nuclear localization of the capsid protein of the German cockroach densovirus is functionally active, not only within the cells of this insect but within the human cell culture as well. This observation extends the experimental possibilities for studying the genetic control of intracellular traffic of densovirus proteins.