Molecular subtyping of feline immunodeficiency virus from domestic cats in Australia

Objective To determine the prevalent subtypes of feline immunodeficiency virus (FIV) present in the domestic cat population of Australia. Method Blood samples were collected from 41 FIV antibody positive cats from four cities across Australia. Following DNA extraction, polymerase chain reaction (PCR) was performed to amplify the variable V3-V5 region of the envelope (env) gene. Genotypes were assessed by direct sequencing of PCR products and comparison with previously reported FIV sequences. Phylogenetic analysis allowed classification of the Australian sequences into the appropriate subtype. Results Of the 41 FIV samples, 40 were found to cluster with previously reported subtype A isolates, whilst the remaining sample grouped within subtype B. Conclusions Subtype A was found to be the predominant FIV subtype present in Australia, although subtype B was also found. These results broaden our knowledge of the genetic diversity of FIV and the associated implications for preventative, diagnostic and therapeutic approaches.

Deduced amino acid sequences surrounding the fusion glycoprotein cleavage site and of the carboxyl-terminus of haemagglutinin-neuraminidase protein of the avirulent thermostable vaccine strain I-2 of Newcastle disease virus

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F-0 cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain 1-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain 1-2 had a sequence Motif of (112)RKQGRLIG(119), consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain 1-2 had a 7-amino-acid extension (VEILKDGVREARSSR). This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain 1-2 confirmed its avirulent nature and its Australian origin.

Development of a rapid biological assay for determination of potency of Newcastle disease vaccine (strain I-2)

A rapid biological assay based on incubation time has been developed for determination of the potency of Newcastle disease virus strain I-2 vaccine. It is based on the observation that the interval between inoculation and the first detection of haemagglutinin (HA) depends on the titre of the vaccine inoculated. Chicken embryonated eggs were inoculated with different titres (10(9), 10(6) and 10(3) EID50/0.1 ml) of vaccine and incubated for 24 h. At hourly intervals, 5 eggs from each vaccine titre were tested for the presence of HA. The results showed that the HA activity was detected from 5, 11 and 15 h after inoculation with vaccine doses of 10(9), 10(6) and 10(3) EID50, respectively. On the basis of these results it is suggested that if there is no HA detected from 5 to 11 h after inoculation of eggs with the vaccine virus, the vaccine should not be used to vaccinate chickens as it might have an infectivity titre of less than 10(6) EID50/0.1 ml, which is equivalent to the recommended single chicken dose. It is concluded that measuring the time between inoculation of the vaccine virus and the onset of HA activity might provide an estimate of the titre of the vaccine within 24 h.

Determination of organ tropism of Newcastle disease virus (strain I-2) by virus isolation and reverse transcription-polymerase chain reaction

The vaccines 1-2 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 1-2 has not yet been determined. The objective of this study was to determine the distribution of strain 1-2 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 1-2 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 1-2. 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 1-2 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 1-2 and V4 strains. The differences in organ tropism observed in this study suggest that 1-2 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 1-2 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 1-2 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 1-2, 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.

Genorne sequence of the thermostable Newcastle disease virus (strain I-2) reveals a possible phenotypic locus

The complete genome sequence of the Australian 1-2 heat-tolerant Newcastle disease virus (NDV) vaccine (master seed stocks) was determined and compared to the sequence of the parent virus from which it had been derived after exposure of the parent stock at 56 degrees C for 30 min. Nucleotide changes were observed at a number of positions with synonymous mutations being greater than those observed for non-synonymous mutations. Sequence data for the HN gene of a parental culture of V4 and two heat-tolerant variants of V4 were obtained. These were compared with the data for the 1-2 viruses and with published sequences for parental V4 and for a number of ND vaccine strains. Sequence analyses did not reveal the ARG 303 deletion in the HN protein, previously claimed to be responsible for the thermostable phenotype. No consistent changes were detected that would indicate involvement of the HN protein in heat resistance. The majority of alterations were observed in the L protein of the virus and it is proposed that these alterations were responsible for the heat-tolerant phenotype of the 1-2 NDV vaccine. (c) 2005 Elsevier B.V. All rights reserved.

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression. (c) 2006 Elsevier B.V. All rights reserved.

Viruses: agents of coral disease?

The potential role of viruses in coral disease has only recently begun to receive attention. Here we describe our attempts to determine whether viruses are present in thermally stressed corals Pavona danai, Acropora formosa and Stylophora pistillata and zoanthids Zoanthus sp., and their zooxanthellae. Heat-shocked P. danai, A. formosa and Zoanthus sp. all produced numerous virus-like particles (VLPs) that were evident in the animal tissue, zooxanthellae and the surrounding seawater; VLPs were also seen around heat-shocked freshly isolated zooxanthellae (FIZ) from P. danai and S. pistillata. The most commonly seen VLPs were tail-less, hexagonal and about 40 to 50 nm in diameter, though a diverse range of other VLP morphotypes (e.g. rounded, rod-shaped, droplet-shaped, filamentous) were also present around corals. When VLPs around heat-shocked FIZ from S. pistillata were added to non-stressed FIZ from this coral, they resulted in cell lysis, suggesting that an infectious agent was present; however, analysis with transmission electron microscopy provided no clear evidence of viral infection. The release of diverse VLPs was again apparent when flow cytometry was used to enumerate release by heat-stressed A. formosa nubbins. Our data support the infection of reef corals by viruses, though we cannot yet determine the precise origin (i.e. coral, zooxanthellae and/or surface microbes) of the VLPs seen. Furthermore, genome sequence data are required to establish the presence of viruses unequivocally.

PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam

A total of 36 tonsil swab samples were collected from healthy swine prior to slaughter at the abattoirs in Can tho and Tien giang provinces of Southern Vietnam, The presence of Pasteurella multocida in these samples was detected by the combination of direct cultivation and isolation, mouse inoculation and the polymerase chain reaction (PM-PCR). P. multocida was detected in 16 samples by PCR, with 17 strains ultimately isolated. All samples were negative for serogroup B by HSB-PCR and conventional serotyping, with isolates identified as A:3, D:1 or D:3. In addition, all samples were determined to be negative for the P. multocida toxin (PMT). Characterisation of isolated P, multocida by REP-PCR and biotyping revealed nine distinct REP profiles and seven biotypes among the 17 isolates. Some correlation was seen with P. multocida isolated from a previous Australian outbreak of acute swine pasteurellosis, and those isolated from fowl cholera outbreaks in Vietnamese poultry. (C) 2000 Elsevier Science B.V. All rights reserved.

Polycystic kidney disease in Bull Terriers: an autosomal dominant inherited disorder

The prevalence, mode of inheritance and urinalysis findings in Bull Terriers with polycystic kidney disease were assessed by screening 150 clinically normal dogs. The disorder was diagnosed in 39 dogs on the basis of renal ultrasound results and family history of the disease. In equivocal cases confirmation required gross and histopathological renal examination. Necropsy was performed on nine affected dogs and the kidneys from another five affected animals were also examined. Renal cysts were usually bilateral, occurred in cortex and medulla and varied from less than 1 mm to over 2.5 cm in diameter. Cysts were lined by epithelial cells of nephron origin. Abnormal urine sediment and proteinuria were common in affected dogs. The disease appears to be inherited in a highly penetrant autosomal dominant manner.

Newly discovered viruses of flying foxes

Flying foxes have been the focus of research into three newly described viruses from the order Mononegavirales, namely Hendra virus (HeV), Menangle virus and Australian Bat Lyssavirus (ABL). Early investigations indicate that flying foxes are the reservoir host for these viruses. In 1994, two outbreaks of a new zoonotic disease affecting horses and humans occurred in Queensland. The virus which was found to be responsible was called equine morbillivirus (EMV) and has since been renamed HeV. Investigation into the reservoir of HeV has produced evidence that antibodies capable of neutralising HeV have only been detected in flying foxes. Over 20% of flying foxes in eastern Australia have been identified as being seropositive. Additionally six species of flying foxes in Papua New Guinea have tested positive for antibodies to HeV. In 1996 a virus from the family Paramyxoviridae was isolated from the uterine fluid of a female flying fox. Sequencing of 10 000 of the 18 000 base pairs (bp) has shown that the sequence is identical to the HeV sequence. As part of investigations into HeV, a virus was isolated from a juvenile flying fox which presented with neurological signs in 1996. This virus was characterised as belonging to the family Rhabdoviridae, and was named ABL. Since then four flying fox species and one insectivorous species have tested positive for ABL. The third virus to be detected in flying foxes is Menangle virus, belonging to the family Paramyxoviridae. This virus was responsible for a zoonotic disease affecting pigs and humans in New South Wales in 1997. Antibodies capable of neutralising Menangle virus, were detected in flying foxes. (C) 1999 Elsevier Science B.V. All rights reserved.