Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the beta-galactosidase gene

Summary.  We have constructed a transfer vector (pAgGal) containing the β-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The β-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. β-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed β-galactosidase protein was also shown to be biologically active by a β-galactosidase assay. Received September 5, 1999 Accepted March 13, 2000     

Molecular diversity of ecologically distinct <Emphasis Type="Italic">Mal de Río Cuarto virus</Emphasis> isolates based on restriction fragment length polymorphism (RFLPs) and genome sequence analysis of segments 1, 7, 9 and 10

Summary Viruses of the species Mal de Río Cuarto virus (genus Fijivirus, family Reoviridae) cause significant economic losses in maize in Argentina. Genetic changes in the virus genome leading to better adaptation to diverse ecological conditions were postulated that would account for the increasing MRCV variability. The genomic differences between MRCV isolates from four ecologically different areas (Río Cuarto, RC; Pergamino, P; Jesús María, JM; and Tafí del Valle, TV) were studied. RT-PCR-amplified fragments comprising four genomic segments (Seg1, Seg7, Seg9 and Seg10) of MRCV isolates were compared by RFLPs and nucleotide sequences. The segments were chosen based on the proteins they encode: RNA-dependent-RNA polymerase, proteins putatively associated with tubular structures and viroplasm and the major outer capsid protein, respectively. Genetic comparison suggested that JM and TV isolates were genetically similar, but RC and P were different. Therefore, they were clustered in three genetic groups (JM = TV, RC and P). Together, nucleotide and amino acid sequence identities of the genomic segments were often above 96%. Seg1 was more variable (viral polymerase), whereas Seg7 (putative tubular structure) was the most conserved. Phylogeny analysis showed that MRCV isolates could be clustered in ‘mountain area’ and ‘high production area’ groups according to their geographical occurrence.     

Determination of 5′-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs

Summary.  We determined the untranslated 5′-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5′-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide a priori knowledge for mutational identification of virulence determinants in the 5′ nontranslated part of the PRRSV genome. Received October 12, 1998 Accepted January 8, 1999     

Production of the Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line in stirred reactor and airlift reactor

The velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), is one of the main plagues for soybean crops. Velvetbean caterpillar larvae are susceptible to be infected by occlusion bodies of the baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), a biological insecticide. The insect cell line saUFL-AG-286 produces very high yields of occlusion bodies of AgMNPV in suspension cultures done in the low-cost serum-free medium UNL-10 in shake-flasks. However, its ability to adapt to conditions of industrial production in bioreactors was unknown. The aim of this study was to characterize the growth of saUFL-AG-286 cell cultures in UNL-10 medium, as well as its capability to replicate AgMNPV in two different bio-reactors at laboratory scale. The cell line was able to adapt to conditions that can be used at industrial scale, both in an airlift reactor and a stirred reactor, although the former was better than the last to support the cell growth. The infection with AgMNPV in the airlift reactor produced a high yield of occlusion bodies, with very low production of budded virus, the progeny used as inoculums. On the other hand, infection in the stirred reactor yielded high titers of budded virus. These results suggest that a feasible strategy for scaling-up the production of AgMNPV might involve the use of airlift reactors for the scaling-up of cell suspension cultures and the final production of occlusion bodies, while the scaling-up of the viral inoculums being carried out under conditions as those existing in stirred reactors.     

Functional and structural characterisation of <Emphasis Type="Italic">Ag</Emphasis>MNPV <Emphasis Type="Italic">ie1</Emphasis>

We have located and cloned the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate 2D (AgMNPV-2D) genomic DNA fragment containing the immediate early 1 ORF and its flanking regions. Computer assisted analysis of the complete ie1 locus nucleotide sequence information was used to locate regulatory signals in the upstream region and conserved nucleotide and amino acid sequences. Comparative studies led to the identification of several characteristic protein motifs and to the conclusion that AgMNPV-2D is more closely related to Choristoneura fumiferana defective NPV than to other Group I nucleopolyhedrovirus. We have also shown that the AgMNPV IE1 protein was able to transactivate an early Autographa californica MNPV promoter and its own promoter in transient expression assays. In order to investigate the biological functionality of the ie1 promoter, the ie1 upstream activating region (UAR) was molecularly dissected and cloned upstream of the E. coli lacZ ORF. The results obtained, after transfection of UFL-AG-286 insect cells, leading us to find that the −492 and −357 versions contains sequence motifs important for the level of the lacZ reporter gene expression. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. The GenBank accession number of the sequence reported in this paper is AF368905.     

Distribution of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> serogroups,monoclonal antibody subgroups and DNA sequence types in recent clinical and environmental isolates from England and Wales (2000–2008)

Clinical isolates of Legionella pneumophila, obtained from 167 patients, who acquired their illness in the community in England and Wales between January 2000 and March 2008, were compared with 276 environmental isolates of L. pneumophila obtained over the same period as part of the routine sampling of ‘managed’ water systems. The 443 isolates were typed by monoclonal antibody (mAb) subgrouping and the internationally standardised, seven-gene loci, sequence-based typing (SBT) scheme of the European Working Group for Legionella Infections (EWGLI). Of the clinical isolates, 97.6% were L. pneumophila serogroup (sgp) 1, compared with only 55.8% of environmental isolates (P = 0.0002); 91.6% were subgrouped as mAb3/1+ve, compared with only 8.3% of environmental isolates (P < 0.0001). The isolates were very diverse, with SBT identifying 111 sequence types (STs) (index of diversity [IOD] 0.954). Among the clinical isolates, 42 ST were seen, with one (ST47) accounting for 25.7% and three (ST47, ST37 and ST62) accounting for 46.1% of all isolates. Eighty-two STs were identified among the environmental isolates, with two (ST1 and ST79) accounting for 34.1% of these. Comparison of the STs seen among clinical and environmental isolates showed that there was very little overlap between the two populations (P < 0.0001), with common clinical strains found in the environment very infrequently: 0.4, 0.7 and 0% (ST47, ST37 and ST62, respectively), and common environmental strains rarely causing disease: 4.8 and 1.2% (ST1 and ST79, respectively). Combining phenotypic and genotypic data identified 144 phenons (IOD 0.970); 52 among clinical isolates and 101 among environmental isolates. The most abundant clinical strain, mAb ‘Allentown’ ST47, accounted for 22.8% of cases, but was only found once in the environment. Conversely, mAb ‘Oxford/OLDA’ ST1 was the most common environmental strain (17.0%), but only caused two infections. A review of the published data shows that mAb ‘Allentown’ ST47 is also an important cause of infection in France and possibly in the Netherlands. However, it was not found in a large study of German clinical isolates. This study confirms previous work showing that just a few strains of L. pneumophila cause the majority of community-acquired Legionella infection in England and Wales, and that these clinically significant strains are only rarely found in managed water systems. These data suggest that knowing which particular strain is present in an environment might be at least as important as knowing the quantity in which legionellae are present.

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Isolation and characterization of an iridovirus from Hermann’s tortoises (Testudo hermanni)

Summary.  A virus was isolated from tissues of 2 diseased Hermann’s tortoises (Testudo hermanni) and preliminarily characterized as an iridovirus. This conclusion was based on the presence of inclusion bodies in the cytoplasm of infected cells, sensitivity to chloroform, inhibition of virus replication by 5-iodo-2′-desoxyuridine and the size and icosahedral morphology of viral particles. The virus was able to replicate in several reptilian, avian and mammalian cell lines at 28°C, but not at 37°C. Restriction enzyme analysis showed resistance of the ral DNA to digestion with HpaII due to methylation of the internal cytosine at CCGG sequences. Part of the genomic region encoding the major capsid protein was amplified by PCR and subjected to sequence analysis. Comparative analysis of the obtained nucleotide sequence revealed that the isolate is closely related to frog virus 3, the type species of the genus Ranavirus. Accepted May 14, 1999 Received January 27, 1999     

Nucleotide sequence evidence for three distinct sugarcane streak mastreviruses

Summary.  The complete sequences of four clones of sugarcane streak virus (SSV) isolates from Egypt and one SSV clone from Reunion island were determined. The four Egyptian genomes were highly similar to one another (97–99% nt identity) and were considered as variants of the same virus. The Egyptian SSV was genetically different from all other mastreviruses, the closest virus being SSV from South-Africa (60% nt identity), and defined as a new mastrevirus species named SSEV. The SSV clone from Reunion was highly related to the SSV from Mauritius and SSV from Nigeria, for which only partial sequences were available, indicating that the three sugarcane streak isolates from Mauritius, Reunion and Nigeria were strains of the same virus tentatively named SSMV. This work further confirms that SSMV is a distinct viral species compared to other mastreviruses, including the SSEV (59% nt identity) and SSV (66% nt identity). By comparing two clones from the Mascarene islands, we correlated substitutions in the C-terminal end of the coat protein with a different response to a monoclonal antibody, providing data on the mapping of a specific epitope. Agroinoculations experiments demonstrated that an SSEV clone induced more severe symptoms on maize than two clones from the Mascarene. Inside the African streak virus cluster, the sugarcane mastrevirus isolates were gathered in a sub-cluster of three viruses, SSEV, SSV and SSMV. The diversity of the SSVs is discussed in relation to its host, sugarcane, an imported crop in Africa. Received March 15, 1999/Accepted July 28, 1999     

The nucleotide sequence and genome organization of Japanese iris necrotic ring virus, a new species in the genus Carmovirus

Summary.  The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5′-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5′-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3′-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF. JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5′ end of the viral RNA is uncapped. The 3′ end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae. Accepted September 23, 1999     

Detection of type 2 herpes simplex virus in cells of spermatogenic epithelium in infected testes of guinea pigs

We developed a model of herpetic orchitis in guinea pigs. Intratesticular inoculation of type 2 herpes simplex virus suspension results in infection of the testicular spermatocytes and spermatides. The possibility of viral infection dissemination from infected into intact testis is proven. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 7, pp. 79–83, July, 2007     

Molecular and biologic characterization of Leishmania parasites implicated in an epidemic outbreak in northwestern Argentina

Leishmania (Viannia)braziliensis and its variants were implicated in the epidemic outbreak of mucocutaneous leishmaniasis that occurred in Salta, northwestern Argentina, in 1985. A total of 24 suspected, untreated cases were evaluated clinically and parasitologically. Four of five stable isolates were consistent with the reference strain of L. (V.)braziliensis as determined by monoclonal antibodies and indirect immunofluorescence or radioimmunobinding assays. Zymodeme analysis in agarose gels showed a close relationship with L. (V.)guyanensis and L. (V.)panamensis. All zymograms obtained with polyacrylamide gels belonged to the subgenus Viannia; the patterns were different from, but very closely related to, the reference strains of L. (V.)braziliensis as determined by dendrogram analysis. Hamsters infected with two isolates showed a pattern consistent with L. (V.)braziliensis. The pattern of development in the gut of Lutzomyia longipalpis was consistent with members of Viannia. Received: 6 October 1999 / Accepted: 22 October 1999     

Phenotypic and genotypic characterisation of Blastocystis hominis isolates implicates subtype 3 as a subtype with pathogenic potential

Despite frequent reports on the presence of Blastocystis hominis in human intestinal tract, its pathogenicity remains a matter of intense debate. These discrepancies may be due to the varying pathogenic potential or virulence of the isolates studied. The present study represents the first to investigate both phenotypic and genotypic characteristics of B. hominis obtained from symptomatic and asymptomatic individuals. Symptomatic isolates had a significantly greater size range and lower growth rate in Jones’ medium than asymptomatic isolates. The parasite cells of symptomatic isolates exhibited rougher surface topography and greater binding affinity to Canavalia ensiformis (ConA) and Helix pomatia (HPA). The present study also identifies further phenotypic characteristics, which aided in differentiating the pathogenic forms from the non-pathogenic forms of B. hominis. Blastocystis subtype 3 was found to be correlated well with the disease.     

Genetic diversity of avian infectious bronchitis virus California variants isolated between 1988 and 2001 based on the S1 subunit of the spike glycoprotein

Summary.  Twenty-nine isolates of avian infectious bronchitis virus (IBV) recovered from commercial chicken flocks in California between 1988 and 2001 and identified as California variants by serotype and direct automated cycle sequencing of the IBV spike glycoprotein S1 subunit, were further characterized phylogenetically and by nucleotide sequence comparison. California variants were grouped according to production type of chicken, by comparison with public access sequence databases (NCBI GenBank and EMBL), or based on phylogenetic analysis. Fisher’s Exact test was used to compare mutations per year, purifying and positive selection, predictive antigenicity, and a ≥ 6 bp deletion between California variant groups. A high number of mutations at the nucleotide level (p = 0.013) and a ≥ 6 bp deletion in the nucleotide sequence (p = 0.006) was significantly associated with broiler-type chickens. However, 88% of significant comparisons at the amino acid level such as purifying and positive selection were seen in layer-type chickens. A pronounced predictive antigenicity in the HVR2 region was also associated with layer-type chickens (p = 0.001). The study indicates that IBV in California is in a phase of slow evolution with different evolutionary patterns being associated with the production type of chicken. Received March 14, 2002; accepted August 9, 2002     

Five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from Portuguese grapevine varieties

The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure. This tool was used to assess the prevalence of each phylogenetic group among the infected grapevine varieties. Although most of the isolates harbour variants from groups 1 and 2, variants from the remaining three groups exist in a number of varieties, reinforcing the notion that they are genuine genomic variants and are not isolated, atypical cases.     

New variant groups identified from HGV isolates

Summary.  We have determined the primary sequence of the 5^′ noncoding region (5^′ NCR) and putative helicase regions (NS-3) of hepatitis G virus (HGV) and GB virus C (GBV-C) that were isolated in Japan from suspected cases of nonA-nonB and/or nonA-nonB-nonC viral hepatitis by using RT-PCR, and we compared the newly isolated sequences with three established isolates. The addition of a "G" residue was found at the 5^′ terminus of all 8 Japanese isolates. These isolates were more clearly distinguished from the prototype viruses by comparison with the 5^′ NCR sequence than by comparison with the NS-3 region. Our results suggested that at least three distinct genomic variants of HGV exist. Genotyping of HGV by using RT-PCR based on the sequence of the 5^′ NCR seems highly feasible. Accepted November 18, 1996 Received September 11, 1996     

Characterisation of two citrus apscaviroids isolated in Spain

Summary.  Sequence variability in the PCR amplified cDNAs from two citrus apscaviroid isolates CVd-Ia and CVd-IIId from Spain, was analysed. CVd-IIId sequence was shown to be identical to previously described CVd-III sequences and no important variability was encountered within the viroid population. Conversely, CVd-Ia displayed population heterogeneity as shown by SSCP analysis, Sal I restriction site polymorphism and sequences of 27 CVd-Ia cloned DNAs. The CVd-Ia genomic heterogeneity is characterised by two major subpopulations with the most divergent sequences, and by the presence of individual variants, making a sequence continuum between the two major groups. Most sequence variations are clustered in the left part of the viroid molecule. Received October 1, 1999/Accepted April 3, 2000     

Production of occlusion bodies of Anticarsia gemmatalis multiple nucleopolyhedrovirus in serum-free suspension cultures of the saUFL-AG-286 cell line: Influence of infection conditions and statistical optimization

The influence of the conditions of infection on the yield of occlusion bodies (OBs) of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), produced in serum-free suspension cultures of saUFL-AG-286 cells, was investigated by two 2² full factorial experiments with centre point. Each experiment tested the effects of the initial cell density and the multiplicity of infection at two levels, in the four possible combinations of levels and conditions, plus a further combination with each condition set at the middle of its extreme levels. The yield of occlusion bodies proved to be sensitive to the modification of infection conditions. Maximum yield as high as 3 × 10⁸ OBs mL⁻¹ was attained provided that the maximum density of viable cells was in the range between 4 and 8 × 10⁵ cells mL⁻¹. The optimum value of the maximum density of viable cells could be reached by the combination of several values of initial cell density and multiplicity of infection. A regression model was established and validated in order to optimize the infection conditions. These results demonstrate the importance of an adequate selection of infection conditions, and they could be useful in the development of a feasible in vitro process to produce the AgMNPV insecticide in a new serum-free medium.