Strain V4 Research Articles

Authorizing of Immunogenicity of Concentrated and Purified Newcastle Disease Virus V4 Strain using Downstream Processing.

Newcastle disease virus (NVD) from the Paramyxoviridae family is a single-stranded negative-sense RNA virus. This infection can affect both domestic poultry and almost all other bird species. It has been considered a very severe difficulty for the poultry industry all over the world. Even though it remains a potential threat to poultry industries, this virus is a powerful oncolytic virus as well. In this study, a process was accomplished to achieve concentrated and highly purified NDV V4 strain particles. Downstream processing of Newcastle virus strain V4 was characterized by amplifying virus in embryonated chicken eggs. Through a sequence of steps, harvesting allantoic fluid, clarification by centrifuge, concentration by ultrafiltration, and size exclusion separation, the reduced volume and pure virus particles were considered for the amount of ovalbumin, hemagglutinin activity, transmission electron microscopy (TEM), electrophoresis, and additionally immunogenicity of prepared antigens. The results presented a high recovery of HA activity in concentrated and purified virus with the removal of ovalbumin and the typical morphology based on TEM. Sepharose CL-4B was determined as the best media among all used resins to purify the virus. Prepared formulations as vaccines demonstrated positive hemagglutinin inhibition for 6 months and stability for 2 years. Strong evidence from organized studies supports the effectiveness of this method in concentrating and purifying intact NDV, which could be valuable in vaccine research, antiserum preparation, or even as an alternative oncotic agent to traditional methods. Despite further studies being conducted, this method can be utilized particularly on a semi-industrial scale to produce various vaccine components.

Sphingobium sp. V4, a bacterium degrading multiple allelochemical phenolic acids

BackgroundContinuous cropping challenges constrain the development of agriculture. Three main obstacles limit continuous cropping: autotoxicity of plant allelochemicals, deterioration of physicochemical characteristics of soil, and microflora imbalance. Plant-derived phenolic acids can cause autotoxicity, which is considered the main factor mediating continuous cropping obstacles. Reducing the phenolic acids in continuous cropping soils can decrease the autotoxicity of phenolic acids and ameliorate continuous cropping obstacles. Therefore, it is important to study the microbial resources that degrade allelochemical phenolic acids. Thus, the bacterial strain V4 that can degrade phenolic acids was isolated, identified, and genomically analyzed.ResultsStrain V4 isolated from strawberry soil using vanillic acid-mineral agar was identified as a Gram-negative short rod bacterium. Subsequent 16S rRNA phylogenetic analysis revealed that V4 clustered with members of the genus Sphingobium. The most closely related species were Sphingobium lactosutens DS20T (99% similarity) and Sphingobium abikonense NBRC 16140T (97.5% similarity). V4 also shared > 95% sequence similarity with other members of Sphingobium, so Sphingobium sp. V4 was named accordingly. Biochemical tests revealed that the biochemical characteristics of Sphingobium sp. V4 were similar to its most similar strains except for some properties. Sphingobium sp. V4 effectively degraded vanillic acid, ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, and syringic acid. V4 grew best at the conditions of 30 °C, pH 6.0–7.0, and 0–0.05% NaCl. 500 mg/L vanillic acid was completely degraded by V4 within 24 h under the optimal conditions. Whole genome analysis showed that Sphingobium sp. V4 contained one chromosome and three plasmids. Two genes involved in vanillic acid degradation were found in the V4 genome: the gene encoding vanillate O-demethylase oxidoreductase VanB on the chromosome and the gene encoding vanillate monooxygenase on a large plasmid. The organization of vanillate catabolic genes differed from the adjacent organization of the genes, encoding vanillate o-demethylase VanA and VanB subunits, in Pseudomonas and Acinetobacter.ConclusionsThe isolated bacterium Sphingobium sp. V4 degraded multiple phenolic acids. Its properties and genome were further analyzed. The study provides support for further investigation and application of this phenolic acid-degrading microorganism to alleviate continuous cropping obstacles in agriculture.

C-terminal truncation of the hemagglutinin-neuraminidase (HN) protein enhances the virulence and immunogenicity of Newcastle disease virus (NDV) vaccine strain V4.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a multifunctional protein with receptor recognition ability that plays an important role in the infection of cells by NDV. An alignment of NDV HN protein sequences of different genotypes showed that vaccine strains of NDV, such as the LaSota strain, generally have an HN protein of 577 amino acids. In comparison, the HN protein of the V4 strain has 616 amino acids, with 39 more amino acids at the C-terminus. In this study, we generated a recombinant NDV (rNDV) with a 39-amino-acid truncation at the HN C-terminus based on the full-length cDNA clone of the V4 strain. This rNDV, named rV4-HN-tr, displayed thermostability similar to that of the parental V4 strain. However, growth kinetics and pathogenicity analysis suggested that rV4-HN-tr is more virulent than the V4 strain. Notably, the C-terminus of HN affected the ability of the virus to adsorb onto cells. Structural predictions further suggested that the C-terminus of HN may obstruct the sialic acid binding site. Immunization of chickens with rV4-HN-tr induced a 3.5-fold higher level of NDV-specific antibodies than that obtained with the V4 strain and provided 100% immune protection against NDV challenge. Our study suggests that rV4-HN-tr is a thermostable, safe, and highly efficient vaccine candidate against Newcastle disease.

Variability of the response of human vaginal Lactobacillus crispatus to 17\u03b2-estradiol

We previously showed that the physiological concentration of 17β-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatus is a heterogeneous species. Here, we investigated the effect of 17β-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17β-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17β-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17β-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

Draft Genome Sequence of Lactobacillus crispatus Strain V4, Isolated from a Vaginal Swab from a Young Healthy Nonmenopausal Woman.

Lactobacillus crispatus strain V4 was isolated from a vaginal swab from a healthy nonmenopausal 35-year-old French woman. We report here its draft genome sequence of 2,091,889 bp, with an average G+C content of 37.02%.

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph \u201cCandidatus Methylacidiphilum kamchatkense\u201d strain Kam1 and comparison with its closest relatives

BackgroundThe candidate genus “Methylacidiphilum” comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the “Ca. Methylacidiphilum” and the sister genus “Ca. Methylacidimicrobium” represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.ResultsHere we present the closed genome of “Ca. Methylacidiphilum kamchatkense” strain Kam1 and a comparison with the genomes of its two closest relatives “Ca. Methylacidiphilum fumariolicum” strain SolV and “Ca. Methylacidiphilum infernorum” strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of “Ca. Methylacidiphilum” is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between “Ca. M. kamchatkense” strain Kam1 and strains SolV and V4 is ≤95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.ConclusionsComparing three closed genomes of “Ca. Methylacidiphilum” spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.

Implications of SiO2 nanoparticles for in vitro biodegradation of low-density polyethylene with potential isolates of Bacillus, Pseudomonas, and their synergistic effect on Vigna mungo growth

In the natural environment, low-density polyethylene (LDPE) contamination is one of the major sources of pollution and creates the hazardous threat. Biodegradation is an eco-friendly approach to control such type of pollution and maintain the sustainable environment. The presence of nanoparticles influences the growth ability of polymer-degrading microbes. In the present study, SiO2 nanoparticles were employed to scrutinize their effect on polymer biodegradation efficiency. LDPE containing minimal salt medium supplemented with SiO2 nanoparticles (20 nm) at concentration of 0.01% w/v. Out of five bacterial strains, Bacillus sp. V8 and Pseudomonas sp. C 2 5 were found to be the most potential strains for polymer degradation. SiO2 nanoparticles improved growth profiling by shifting in lag phase, and it was also found effective to increase the biodegradation efficiency of bacterial strains by means of λ-max shifts, and Fourier transform infrared analysis revealed the formation or alteration of the chemical structure of the degraded polymer. The values of λ-max were shifted 217–227, 211–226, 209–224, 210–225, and 214–224 in the presence of Bacillus sp. strain V8, Pseudomonas sp. strain C 2 5, Pseudomonas sp. strain V1, Acinetobacter sp. strain V4, and Paracoccus sp. strain B1 4-, respectively. Fourier transform infrared analysis showed absorption frequencies of bacteria-treated polymers corresponding to 3049, 3275, 3527.8, 2196, and 3034 and confirmed the C–H stretching, O–H stretching, and C≡H stretching, respectively. The study signifies the bacteria–nanoparticles interactions that significantly influence the competence of polymer degradation.

Distribution and diversity of Verrucomicrobia methanotrophs in geothermal and acidic environments

Recently, methanotrophic members of the phylum Verrucomicrobia have been described, but little is known about their distribution in nature. We surveyed methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in samples covering a broad temperature range (22.5-81.6°C), but only in acidic conditions (pH 1.8-5.0) and only in geothermal environments, not in acidic bogs or fens. Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic Verrucomicrobia were observed. Those detected in high-temperature geothermal samples (44.1-81.6°C) grouped with known thermoacidiphilic 'Methylacidiphilum' isolates. A second group dominated in moderate-temperature geothermal samples (22.5-40.1°C) and a representative mesophilic methanotroph from this group was isolated (strain LP2A). Genome sequencing verified that strain LP2A possessed particulate methane monooxygenase, but its 16S rRNA gene sequence identity to 'Methylacidiphilum infernorum' strain V4 was only 90.6%. A third group clustered distantly with known methanotrophic Verrucomicrobia. Using pmoA-gene targeted quantitative polymerase chain reaction, two geothermal soil profiles showed a dominance of LP2A-like pmoA sequences in the cooler surface layers and 'Methylacidiphilum'-like pmoA sequences in deeper, hotter layers. Based on these results, there appears to be a thermophilic group and a mesophilic group of methanotrophic Verrucomicrobia. However, both were detected only in acidic geothermal environments.

Thermotolerant Newcastle disease vaccines for use in flocks of family chickens.

Abstract Family chickens provide nutrition and income for poor families in rural areas in developing countries. Newcastle disease is a major constraint limiting the productivity of these flocks. Imported commercial Newcastle disease vaccines, requiring foreign exchange and coming in large dose containers, have been little used in small village flocks. The major problem with these vaccines has been their heat lability. Appropriate vaccines for village use would be locally produced, cheap and to some extent heat tolerant, allowing some independence from the cold chain. Two vaccines derived from naturally thermotolerant Australian strains of Newcastle disease virus have been artificially selected for enhanced heat resistance. One vaccine, V4, is now produced by commercial companies and is used mainly in commercial chickens. Master seed for the other vaccine, 1-2, is held at the University of Queensland, Brisbane, Australia and is made available without cost to laboratories in developing countries. Vaccines based on variants of strain V4 have been available for more than 30 years. Challenge experiments in many countries have indicated that V4 vaccine is protective against a variety of challenge strains. Many of the early studies were part of the search for a village vaccine. The commercialization of V4 vaccine meant that countries wishing to use it in family flocks needed to purchase vaccine or produce and verify their own master seed. Of special interest is the efficacy of V4 vaccine when administered to chickens on suitable feeds. The V4 virus will spread from vaccinated chickens to contact chickens. 1-2 is a Newcastle disease virus similar to V4. Being in the public domain, 1-2 master seed is available for local production of thermotolerant vaccines for use in the national flocks of family chickens. Many countries have undertaken laboratory trials and demonstrated effective protection on vaccination by various means including vaccination with carrier feeds. Some countries progressed to field trials and to campaigns to control Newcastle disease in the village setting. Local production and distribution of an appropriate vaccine has been only part of the process. Innovative extension materials have been developed, contributors to all phases of the project have been trained (vaccinators, laboratory workers and administrators) and lines of communication between all parties have been established.

Effects of Probiotic Bacillus subtilis on Intestinal Microbial Diversity and Immunity of Orange Spotted Grouper Epinephelus coioides

Groupers are an important mariculture in Taiwan and Southeast Asian countries. The rapidly growing orange spotted grouper Epinephelus coioides has experienced relatively severe bacterial disease problems. The proliferation of pathogens in fish can be suppressed by commensal microbe. In this research, probiotic seem to offer an attractive alternative. Bacillus subtilis is a probiotic bacterium that is administered in diet to suppress proliferation of pathogens. In the present study, E. coioides were fed for 6 months with diets contain B. subtilis . Percent weight gain and feed efficiency of the fish fed diets containing 0.1% or 1% probiotics (w/w) were significantly better than the control. The innate cellular response, respiratory burst of the supplemental groups was significantly higher compared to the control on 10 or 20 days after feeding, and even more significant on 30 days. Probiotic B . subtilis increased the intestinal microbial diversity by stimulating the bacterial populations of Paenibacillus sp, Lactobacillus oeni strain 59 b, and Methilacidophilum infernorum strain V4 that are beneficial for E . coioides . The best dose of probiotic B . subtilis based on growth performances, innate cellular responses and microbial profile of fish intestines is 0.1%, which showed equal efficacy as the 1% diet .

정장제, 신생아 분변 및 병원에서 분리한 장구균의 병독성인자 비교

Three isolates, E. faecium P1, P2 and P3, from intestinal drugs of three phamaceutical companies, four clinical vancomycin resistant isolates, E. faecium V1, V2, V3 and E. faecalis V4, and three isolates, E. faecalis DW01, DW07 and DW14, from infant feces were tested for the presence of virulence genes, ace, agg, esp, efaA, gelE, sprE, vanA and vanB as well as fsrABC, regulatory genes of gelE and sprE, cylMBA, cytolysin activation genes and cpd, cob and ccf, pheromone genes by PCR and for their phenotype activities such as protease, biofilm formation, cell clumping and hemolysis. The genes encoding cell surface adherence proteins, ace, agg, esp and efaA, were predominantly amplified from the vancomycin resistant strain V4 and the fecal isolates DW01, DW07 and DW14. Both protease and biofilm formation activity were detected only from E. faecalis V4 from which the PCR products of gelE and spreE as well as fsrABC were amplified. The pheromone genes were amplified from the V4, DW01, DW07 and DW14 strains and these strains showed clumping activity. Biofilm formation was observed from the strains DW01, DW07 and DW14, all of which produced PCR products of pheromone, and V4 as well. Whole cytolysin regulator genes were amplified from DW01, DW07 and DW14 and β-hemolysis activity was detected from these strains. Any virulence genes or activities except the pheomone gene ccf were not detected from the pharmaceutical isolates, E. faecium P1, P2 and P3.

Sweet potato little leaf strain V4 phytoplasma associated with snake bean in the Northern Territory, Australia

Sweet potato little leaf strain V4 phytoplasma (SPLL-V4) was found to be associated with 12-week old snake bean plants (Vigna unguiculata ssp. sesquipedalis) from a block with 890 plants in Lambells Lagoon, Northern Territory. Symptoms included witches’ broom, big bud, phyllody and little leaf. There was a 19.9 % incidence of symptoms on 366 plants assessed. The same phytoplasma was also detected from a single plant of Phyllanthus amarus with witches’ broom symptoms growing near the snake bean plants. The SPLL-V4 phytoplasma was identified based upon the 16S ribosomal RNA gene and intergenic spacer region nucleotide sequence and restriction fragment length polymorphism.

Detection of autotrophic verrucomicrobial methanotrophs in a geothermal environment using stable isotope probing

Genomic analysis of the methanotrophic verrucomicrobium “Methylacidiphilum infernorum” strain V4 has shown that most pathways conferring its methanotrophic lifestyle are similar to those found in proteobacterial methanotrophs. However, due to the large sequence divergence of its methane monooxygenase-encoding genes (pmo), “universal” pmoA polymerase chain reaction (PCR) primers do not target these bacteria. Unlike proteobacterial methanotrophs, “Methylacidiphilum” fixes carbon autotrophically, and uses methane only for energy generation. As a result, techniques used to detect methanotrophs in the environment such as 13CH4-stable isotope probing (SIP) and pmoA-targeted PCR do not detect verrucomicrobial methanotrophs, and they may have been overlooked in previous environmental studies. We developed a modified SIP technique to identify active methanotrophic Verrucomicrobia in the environment by labeling with 13CO2 and 13CH4, individually and in combination. Testing the protocol in “M. infernorum” strain V4 resulted in assimilation of 13CO2 but not 13CH4, verifying its autotrophic lifestyle. To specifically detect methanotrophs (as opposed to other autotrophs) via 13CO2-SIP, a quantitative PCR (qPCR) assay specific for verrucomicrobial-pmoA genes was developed and used in combination with SIP. Incubation of an acidic, high-temperature geothermal soil with 13CH4 + 12CO2 caused little shift in the density distribution of verrucomicrobial-pmoA genes relative to controls. However, labeling with 13CO2 in combination with 12CH4 or 13CH4 induced a strong shift in the distribution of verrucomicrobial-pmoA genes towards the heavy DNA fractions. The modified SIP technique demonstrated that the primary methanotrophs active in the soil were autotrophs and belonged to the Verrucomicrobia. This is the first demonstration of autotrophic, non-proteobacterial methanotrophy in situ, and provides a tool to detect verrucomicrobial methanotrophs in other ecosystems.

English

Influences of carbon adaptation on antagonistic activities of three Pseudomonasaeruginosa strains V4, V7 and V10 against Fusarium oxysporum f. sp. melonis weredetermined in this study. Results from this study showed that the P. aeruginosastrains and their adapted strains significantly inhibited the growth of mycelium of F. oxysporum f. sp. Melonis, while in vitro inhibition of P. aeruginosa on the mycelial growth was unaffected by carbon adaptation. In general, the growth of strain V4 and its antagonistic ability was unaffected by carbon adaptation. However, the growth of the adapted strains V7-C and V10-C was superior to the corresponding parental strains when root exudates were used as a sole carbon source. In addition, the adapted strains V7-C and V10-C caused a more decrease in Fusarium infection of melon compared with the corresponding parental strains. Overall, this study revealed that adaptation culture of P. aeruginosa strains on carbon-limited media may play an important role in the inhibition of Fusarium wilt of melon seedlings although the effect of carbon adaptation may depend on the test strain.   Key words: Melon, biocontrol, Fusarium wilt, in vitro, in vivo.

Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia

BackgroundThe phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia.ResultsWe report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins.ConclusionThe results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria.ReviewersThis article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.

Determination of Organ Tropism of Newcastle Disease Virus (Strain I-2) by Virus Isolation and Reverse Transcription–Polymerase Chain Reaction

The vaccines I-2 and V4 are avirulent strains of Newcastle disease virus. Organ tropism of strain V4 has been determined and the virus has a predilection for the digestive tract. Tropism of strain I-2 has not yet been determined. The objective of this study was to determine the distribution of strain I-2 in various body organs and fluids following vaccination in comparison with V4. Four-week-old chickens were vaccinated by eye drop separately with these two avirulent strains. Virus isolation and the reverse transcription-polymerase chain reaction technique were employed to detect I-2 and V4 viruses in various tissues and body fluids for 7 days following vaccination. Tissues from the respiratory tract showed earlier positive signals than tissues from other organs for chickens vaccinated with strain I-2. Conversely, tissues from mainly digestive tract produced earlier positive signals than from respiratory tract and other organs from chickens vaccinated with strain V4. In early infection, strain I-2 had preferential predilection for the respiratory tract and strain V4 for the digestive tract. Later after vaccination, other organs showed positive results from chickens vaccinated with both I-2 and V4 strains. The differences in organ tropism observed in this study suggest that I-2 may perform better than V4 as a live vaccine strain.

Development of a Cell Culture Method for Quantal Assay of Strain I-2 of Newcastle Disease Virus

Repeated titrations of strains of Newcastle disease virus (NDV) are more conveniently undertaken in cell cultures rather than in embryonated eggs. This is relatively easy with mesogenic and velogenic strains that are cytopathic to various cell lines, but is difficult with avirulent Australian isolates that are poorly cytopathic. Strain V4 for example has been shown to be pathogenic iin vitro only to of chicken embryo liver cells. Strain I-2 was reported to produce cytopathic effect (CPE) on chicken embryo kidney (CEK) cells. The present studies confirmed this observation and developed a quantal assay. CEK cells infected with strain I-2 developed CPE characterized by degeneration, rounding, granularity and vacuolation, and the formation of synctia. End points were readily established by microscopic examination of fixed and stained cells. In virus infectivity studies on strain I-2, where multiple titrations are required and where large numbers of samples are used, titration using CEK cell grown in microtitre plates is recommended. Such studies may not be feasible in embryonated eggs.

Early Events Following Oral Administration of Newcastle Disease Virus Strain V4

Heat-resistant strains of Newcastle disease virus, such as strain V4, are being used as vaccines to protect flocks of rural chickens in developing countries. Sometimes these vaccines are administered on food, a procedure that has yet to be optimized. Experiments were undertaken to determine the anatomical sites of initial infection with orally administered V4 virus, and the sites of initial viral replication. During the first 24 hours after introduction of V4 into the mouth of 3-weeks-old chickens, virus was isolated by egg inoculation from oesophagus, crop and trachea and less frequently from proventriculus. V4 virus was not isolated from other parts of the digestive tract, nor from blood. Evidence for viral replication was sought by egg inoculation and by immunohistochemistry 4 to 10 days after V4 strain was placed in the crop. Virus was detected most frequently and at highest titre from jejunum, ileum and caecum 6 days after infection. There were also isolations from blood, 4 days after infection. Viral antigens were detected in epithelial cells in most parts of the digestive tract and in some cells, possibly lymphocytes or macrophages, in lamina propria. It is suggested that V4 vaccine virus, given orally, attaches to cells in the upper digestive tract. Viral replication occurs mainly in cells of the lower digestive tract, probably reaching these sites as the result of a viraemia.

Identification of sweet potato little leaf phytoplasma associated withVigna unguiculatavar.sesquipedalisandLycopersicon esculentum

Vigna unguiculata var. sesquipedalis (snakebean) and Lycopersicon esculentum (tomato) plants with phytoplasma-like symptoms were found in the horticultural region in Broome, Western Australia. The symptoms on snakebean included ‘witches’ broom’ and proliferation of small leaves, and on tomato they varied from phyllody and ‘big bud’ to ‘witches’ broom’ and proliferation of small leaves. Using universal primers for phytoplasmas, the complete 16S rRNA gene and intergenic spacer region, and part of the 23S rRNA gene were amplified by PCR. Sequence analysis identified that the agent associated with the symptoms was a strain of sweet potato little leaf strain V4 (SPLL-V4) phytoplasma (16SrII group, strain of ‘Candidatus Phytoplasma australiense’). SPLL phytoplasma has not been reported before in snakebean or tomato in this isolated agricultural region.

Use of Survival Analysis to Determine the Postincubation Time-to-Death of Papaya Due to Yellow Crinkle Disease in Australia.

The current management recommendation for papaya (Carica papaya) plants exhibiting symptoms of yellow crinkle disease in Australia is the practice of ratooning infected plants. Ratooning involves removing the main stem of diseased papaya plants and allowing a lateral stem (supposedly pathogen-free) to develop and replace the diseased stem. Using nonparametric and parametric methods of survival analysis, we tested different hypotheses regarding plant factors that may influence the postincubation period survival time of phytoplasma-infected papaya. The factors included plant age, the season (wet versus dry) when papaya plants first became symptomatic, and the two predominant phytoplasma strains causing papaya yellow crinkle: tomato big bud (TBB) or sweet potato little leaf strain V4 (SPLL-V4). Median survival time was estimated to be from 4 to 5 months. Therefore, we estimated that the infectious period (incubation period plus the period from postincubation to time-to-death period) of infected papaya ranges from 6 to 9 months. Using parametric accelerated failure modeling and nonparametric Cox proportional hazard modeling, no significant improvement from a null model (no covariates) was found when analyzing plant age, the season a plant was observed to be symptomatic, or phytoplasma strain. However, the season in which a papaya plant became symptomatic differed between the two phytoplasma strains, indicating that the TBB and SPLL-V4 strains may have different modes of insect acquisition and transmission. Because of the long infectious period and the rate of plantto-plant spread, we question the use of ratooning as the primary management tactic for managing papaya yellow crinkle.