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 I-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 I-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 I-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 I-2 confirmed its avirulent nature and its Australian origin.

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

The complete genome sequence of the Australian I-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 I-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 I-2 NDV vaccine.

Genetic diversity of bluetongue viruses in south east Asia.

Bluetongue viruses (BTV) were isolated from sentinel cattle in Malaysia and at two sites in Indonesia. We identified eight serotypes some of which appeared to have a wide distribution throughout this region, while others were only isolated in Malaysia or Australia. Nearly half of the 24 known BTV serotypes have now been identified in Asia. Further, we investigated the genetic diversity of their RNA segments 3 and 10. Using partial nucleotide sequences of the RNA segment 3 (540 bp) which codes for the conserved core protein (VP3), the BTV isolates were found to be unique to the previously defined Australasian topotype and could be further subdivided into four distinct clades or genotypes. Certain of these genotypes appeared to be geographically restricted while others were distributed widely throughout the region. Similarly, the complete nucleotide sequences of the RNA segment 10 (822 bp), coding for the non-structural protein (NS3/3A), were also conserved and grouped into the five genotypes; the BTV isolates could be grouped into three Asian genotypes and two Nth American/Sth African genotypes.

An outbreak of highly pathogenic avian influenza in Australia in 1997 caused by an H7N4 virus.

In November of 1997 an outbreak of highly pathogenic avian influenza occurred near the town of Tamworth, in northern New South Wales, Australia. The viruses isolated from chickens on two commercial chicken farms were identified as H7N4 viruses, with hemagglutinin cleavage site amino acid sequences of RKRKRG and intravenous pathogenicity indices of 2.52 and 2.90, respectively. A virus with an identical nucleotide sequence, but with an intravenous pathogenicity index of 1.30, was also isolated from cloacal swabs collected from asymptomatic emus kept on a third property.

Increased permeability of psoriatic skin to the protein, plasminogen activator inhibitor 2.

The penetration and permeation of the recombinant protein plasminogen activator inhibitor type 2 (PAI-2) in two formulations, one containing a penetration enhancer, into the psoriatic and uninvolved skin of eight patients with plaque-type psoriasis were investigated. Penetration and permeation of PAI-2 were measured by gamma counting and imaging following radiolabelling of a fraction of the applied PAI-2 with (123)I. The feasibility of topical delivery of drug to psoriatic plaques was confirmed by the finding that the permeability of psoriatic plaques to radiolabelled PAI-2 (P=0.007) and free (123)I (P=0.001) was approximately tenfold higher than the permeability of uninvolved skin. The addition of a penetration enhancer improved the permeation of PAI-2 into psoriatic plaques from an average of 35% to 46% (P=0.005). Occlusion decreased the permeation amount of PAI-2 from 46% to 15% due to losses on the occlusive dressing (P=0.001).

Newcastle disease virus fusion and haemagglutinin-neuraminidase gene motifs as markers for viral lineage.

Reverse transcriptase polymerase chain reaction was used to generate sequence data for 91 Australian Newcastle disease viruses (NDV) isolated from 1932 to 2000 covering the cleavage site of the fusion (F) protein and the C-terminus of the haemagglutinin-neuraminidase (HN) protein. Comparison of sequences at these two sites indicates distinct evolutionary relationships between these viruses. Typically, HN gene relationships revealed by phylogenetic analyses were also maintained in comparisons between F gene cleavage sites; however, the former analyses appeared to give a clearer indication of the lineage of a virus isolate. This data supports and extends earlier observations in that there is no evidence for gene exchange by recombination but that different strains appear to have evolved through synonymous mutations. Inter-relationships, especially between Australian NDV isolates, appear to be associated with lineages having the same C-terminal HN extensions rather than associated with virulence of the virus. A proposed mechanism for this observation is discussed.

A sequential approach to radiological interpretation.

A case of large glandular odontogenic cyst of the mandible is presented in which the panoramic radiograph also demonstrated a soft tissue lesion consistent with carotid aneurysm. CT confirmed the bucco-lingual extent of the mandibular lesion and the presence of a soft tissue lesion consistent with an aneurysm of the internal carotid artery. CT angiography, MR angiography and US were used to rule out a carotid artery aneurysm prior to surgery of the mandibular lesion.

Avian paramyxoviruses and influenza viruses isolated from mallard ducks (Anas platyrhynchos) in New Zealand.

A comprehensive study using virological and serological approaches was carried out to determine the status of live healthy mallard ducks (Anas platyrhynchos) in New Zealand for infections with avian paramyxoviruses (APMV) and influenza viruses (AIV). Thirty-three viruses isolated from 321 tracheal and cloacal swabs were characterized as: 6 AIV (two H5N2 and four H4N6), 10 APMV-1 and 17 APMV-4. Of 335 sera samples tested for AIV antibodies, 109 (32.5%) sera were positive by nucleoprotein-blocking ELISA (NP-B-ELISA). Serum samples (315) were examined for antibody to APMV-1, -2, -3, -4, -6, -7, -8, -9 by the haemagglutination inhibition test. The largest number of reactions, with titres up to > or =1/64, was to APMV-1 (93.1%), followed by APMV-6 (85.1%), APMV-8 (56%), APMV-4 (51.7%), APMV-7 (47%), APMV-9 (15.9%), APMV-2 (13.3%) and APMV-3 (6.0%). All of the H5N2 isolates of AIV and the APMV-1 isolates from this and earlier New Zealand studies had low pathogenicity indices assessed by the Intravenous Pathogenicity Index (IVPI) with the result 0.00 and Intracerebral Pathogenicity Index (ICPI) with results 0.00-0.16. Partial genomic and antigenic analyses were also consistent with the isolates being non-pathogenic. Phylogenetic analysis of the 10 APMV-1 isolates showed 9 to be most similar to the reference APMV-1 strain D26/76 originally isolated in Japan and also to the Que/66 strain, which was isolated in Australia. The other isolate was very similar to a virus (MC 110/77) obtained from a shelduck in France.

First identification of a ranavirus from green pythons (Chondropython viridis).

Ten juvenile green pythons (Chondropython viridis) died or were euthanized shortly after having been illegally imported into Australia from Indonesia in 1998. Histologic examination of two of the three snakes that died revealed moderately severe chronic ulceration of the nasal mucosa and focal or periacinar degeneration and necrosis of the liver. In addition there was severe necrotizing inflammation of the pharyngeal submucosa accompanied by numerous macrophages, heterophils, and edema. An iridovirus was isolated in culture from several tissues and characterized by immunohistochemistry, electron microscopy, enzyme-linked immunosorbent Assay, polyacrylamide gel electrophoresis, polymerase chain reaction and sequence analysis, restriction endonuclease digestion, and DNA hybridization. This is the first report of a systemic ranavirus infection in any species of snake and is a new member of the genus, Ranavirus.

Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2.

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.

Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop.

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.

Virulent Newcastle disease in Australia: molecular epidemiological analysis of viruses isolated prior to and during the outbreaks of 1998-2000.

Gene sequence analysis of fusion (F) gene cleavage motifs and haemagglutinin-neuraminidase (HN) carboxyl-terminal extension sequences was used to analyse Newcastle disease viruses (NDV) associated with virulent outbreaks of the disease which occurred in New South Wales, Australia in 1998-2000. PCR fragments were amplified directly from diseased tissue or allantoic fluids and sequence analyses used for phylogenetic comparisons between these viruses and Australian reference NDV. F and HN gene sequence comparison showed a strong relationship to sequences derived from endemic Australian NDV rather than those of overseas viruses or wild bird isolates. Prior to notification of the 1998 outbreak, an NDV was isolated from chickens suffering respiratory disease that appeared to be the progenitor virus from which the virulent virus originated. In turn, these viruses are closely related to two previously isolated 'ancestor' viruses that have the same unique HN extension sequence.

Nipah virus: a recently emergent deadly paramyxovirus.

A paramyxovirus virus termed Nipah virus has been identified as the etiologic agent of an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore. The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 encephalitis cases, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. Electron microscopic, serologic, and genetic studies indicate that this virus belongs to the family Paramyxoviridae and is most closely related to the recently discovered Hendra virus. We suggest that these two viruses are representative of a new genus within the family Paramyxoviridae. Like Hendra virus, Nipah virus is unusual among the paramyxoviruses in its ability to infect and cause potentially fatal disease in a number of host species, including humans.

Recovery of ranavirus dsDNA from formalin-fixed archival material.

The extraction and amplification of nucleic acid from formalin-fixed and paraffin-embedded tissues has become an important exercise in the collection of retrospective epidemiological data. A protocol is described that enables the extraction and amplification of dsDNA from fixed tissues within paraffin blocks and from specimens stored in 10% (aq) formalin. The procedure can be used for the examination of ranavirus DNA within archival tissues thereby providing valuable data for identifying the origin and tracing the spread of ranaviruses.

Comparative studies of piscine and amphibian iridoviruses.

A total of 30 iridoviruses collected from Australia, South-East Asia, North America, South America and Europe were characterised. With the exception of the South-East Asian iridoviruses all viruses were found to belong to the genus Ranavirus. All viruses, except those originating from South-East Asia, cross-reacted with antisera against epizootic haematopoietic necrosis virus (EHNV). Viruses or virus-infected cells were examined using electron microscopy, SDS PAGE, restriction endonuclease (RE) digestion, DNA hybridisation, and DNA sequencing. Data from RE digestion of genomic DNA, and from the sequencing of PCR products indicated that the viruses generally grouped according to their geographic and taxonomic (i.e. amphibian or fish) origin. The one exception to this was the viruses from the United Kingdom that grouped with the North American ranaviruses. The differences between specified genomic regions were small. To assess the validity of the differences in sequence homology, similar studies were performed with different isolates from two viruses (EHNV and Guatopo virus (GV), collected from different animals at different locations and time). The sequence data showed complete homology for the isolates for any one virus over the 200 and 586 bp regions examined. Collectively, the data showed that the coding region for the major coat protein (MCP) is stable for any one species (e.g. EHNV).

Epidemic of blindness in kangaroos--evidence of a viral aetiology.

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.