The 2017-19 activity at Mount Agung in Bali (Indonesia): Intense unrest, monitoring, crisis response, evacuation, and eruption.

After 53 years of quiescence, Mount Agung awoke in August 2017, with intense seismicity, measurable ground deformation, and thermal anomalies in the summit crater. Although the seismic unrest peaked in late September and early October, the volcano did not start erupting until 21 November. The most intense explosive eruptions with accompanying rapid lava effusion occurred between 25 and 29 November. Smaller infrequent explosions and extrusions continue through the present (June 2019). The delay between intense unrest and eruption caused considerable challenges to emergency responders, local and national governmental agencies, and the population of Bali near the volcano, including over 140,000 evacuees. This paper provides an overview of the volcanic activity at Mount Agung from the viewpoint of the volcano observatory and other scientists responding to the volcanic crisis. We discuss the volcanic activity as well as key data streams used to track it. We provide evidence that magma intruded into the mid-crust in early 2017, and again in August of that year, prior to intrusion of an inferred dike between Mount Agung and Batur Caldera that initiated an earthquake swarm in late September. We summarize efforts to forecast the behavior of the volcano, to quantify exclusion zones for evacuations, and to work with emergency responders and other government agencies to make decisions during a complex and tense volcanic crisis.

Experimental Hendra virus infection of dogs: virus replication, shedding and potential for transmission.

OBJECTIVE: Characterisation of experimental Hendra virus (HeV) infection in dogs and assessment of associated transmission risk. METHODS: Beagle dogs were exposed oronasally to Hendra virus/Australia/Horse/2008/Redlands or to blood collected from HeV-infected ferrets. Ferrets were exposed to oral fluids collected from dogs after canine exposure to HeV. Observations made and samples tested post-exposure were used to assess the clinical course and replication sites of HeV in dogs, the infectivity for ferrets of canine oral fluids and features of HeV infection in dogs following contact with infective blood. RESULTS: Dogs were reliably infected with HeV and were generally asymptomatic. HeV was re-isolated from the oral cavity and virus clearance was associated with development of virus neutralising antibody. Major sites of HeV replication in dogs were the tonsils, lower respiratory tract and associated lymph nodes. Virus replication was documented in canine kidney and spleen, confirming a viraemic phase for canine HeV infection and suggesting that urine may be a source of infectious virus. Infection was transmitted to ferrets via canine oral secretions, with copy numbers for the HeV N gene in canine oral swabs comparable to those reported for nasal swabs of experimentally infected horses. CONCLUSION: HeV is not highly pathogenic for dogs, but their oral secretions pose a potential transmission risk to people. The time-window for transmission risk is circumscribed and corresponds to the period of acute infection before establishment of an adaptive immune response. The likelihood of central nervous system involvement in canine HeV infection is unclear, as is any long-term consequence.

Isolation and characterisation of a novel Bohle-like virus from two frog species in the Darwin rural area, Australia.

Twelve captive magnificent tree frogs Litoria splendida and 2 green tree frogs L. caerulea on a property in the Darwin rural area (Northern Territory, Australia) either died or were euthanased after becoming lethargic or developing skin lesions. Samples from both species of frog were submitted for histopathology and virus isolation. An irido-like virus was cultured from tissue samples taken from both species and was characterised using electron microscopy, restriction enzyme digests and nucleic acid amplification and sequencing. The isolates were determined to belong to the genus Ranavirus, were indistinguishable from each other and shared a 98.62% nucleotide similarity and a 97.32% deduced amino acid homology with the Bohle iridovirus over a 1161 bp region of the major capsid gene. This is the first isolation of a ranavirus from amphibians in the Northern Territory and the first report of natural infection in these 2 species of native frog. The virus is tentatively named Mahaffey Road virus (MHRV).

Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses.

Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.

Identification of a Bohle iridovirus thymidine kinase gene and demonstration of activity using vaccinia virus.

In recent years interest in the family Iridoviridae has been renewed by the identification of a number of viruses, particularly from the genus Ranavirus, associated with disease in a range of poikilotherms. Ranaviruses have been isolated from amphibian, piscine and reptilian species. Here we describe an open reading frame (ORF) identified in the genome of Bohle iridovirus (BIV) which contains a nucleotide binding motif conserved within the thymidine kinase (TK) genes of iridoviruses from other genera (lymphocystis disease virus, LCDV, type species of the genus Lymphocystivirus; Chilo iridescent virus, CIV, type species of the genus Iridovirus). The ability of this putative gene to express a functional TK was confirmed by rescue of a TK negative mutant vaccinia virus in the presence of selective media, when expression was controlled by a vaccinia virus promoter. The sequence of the BIV TK was compared with the homologous sequences from epizootic haematopoietic necrosis virus (EHNV), a virus associated with disease in fish, from Wamena iridovirus (WIV) associated with systemic disease in green pythons, and from frog virus 3 (FV3) the ranavirus type species. Comparisons between these sequences and those available from other ranaviruses, other iridoviruses, other DNA viruses and cellular TKs are presented.

Promoter activity in the 5' flanking regions of the Bohle iridovirus ICP 18, ICP 46 and major capsid protein genes.

Bohle iridovirus (BIV) belongs to the genus Ranavirus, of which Frog virus 3 (FV-3) is the type species. We are developing BIV as a recombinant viral delivery vector, and as a first step we located specific BIV promoter sequences to drive foreign gene expression in the recombinant virus. By comparison with FV-3 sequences, the genes encoding ICP 18 and ICP 46 in BIV were identified and sequenced. Putative promoter regions of these two early genes and of the major capsid protein (MCP) gene were identified, cloned into pSFM21, and luciferase production was then used to assess the promoter activity of these regions.

A single gene encoding the fiber is responsible for variations in virulence in the fowl adenoviruses.

Intertypic recombinant fowl adenoviruses (FAVs) were generated to determine regions of the viral genome involved in virulence. Recombinants were produced with two serotype 8 FAVs, mildly virulent CFA 3 and hypervirulent CFA 40. Restriction endonuclease fragments from the genomes of the two FAVs were used to transfect primary chicken kidney cells. Virulence testing of these recombinants located the region responsible for differences in virulence to an 8.4-kb fragment of the genome located between kb 26.6 and 35.0. According to data available for a serotype 10 FAV that had been partially characterized in the laboratory, this segment of the genome contained three genes of known identity (100K, 33K, and pVIII) and a region between kb 31 and 35 with unknown coding potential (although this information subsequently became available for a serotype 1 FAV, CELO). Therefore, the region between kb 30.5 and 34.5 was sequenced. The results revealed that the unknown region encoded a fiber gene on the right strand and several small open reading frames of unknown identity on the left strand. Further recombinant viruses containing defined exchanges within the 4-kb fragment were constructed, and virulence testing of these viruses indicated that the fiber was responsible for differences in virulence for CFA 40 and CFA 3.

Comparison by restriction enzyme analysis of three fowl adenoviruses of varying pathogenicity.

Restriction enzyme studies have been used to divide the fowl adenoviruses (FAV) into 5 groups - A, B,C,D and E. More detailed restriction enzyme studies of a series of group E FAV field isolates showed that these methods could differentiate between mildly and hypervirulent FAV belonging to this group. We have mapped the genomes of the hypervirulent (CFA 40) and two of the mildly virulent FAV (CFA 44 and CFA 3), using 11 different restriction enzymes: HindIII, BglII, XbaI, NdeI, SpeI, DraI, NotI, StuI, NheI, SfiI, and AvrII. Comparison of the three maps showed that the CFA 3 genome was approximately 3.5 kb smaller than that of CFA 44 and CFA 40. This size difference was discounted as a likely cause of the reduced pathogenicity of CFA 3 as the other mildly virulent virus, CFA 44, was the same size as the hypervirulent CFA 40. Other variations between the three viruses occurred in the region of the hexon and 100 K genes, but further studies are required to determine the significance of these variations in pathogenicity.

Fractional moving blood volume: estimation with power Doppler US.

PURPOSE: To estimate the fraction of moving blood in tissue with power Doppler ultrasound (US). MATERIALS AND METHODS: Power Doppler US measurements of moving scatterers in a flow tube were made as a function of successive dilutions of the perfusate. Measurements were normalized relative to the maximum Doppler power in the center of the flow tube at the highest concentration and were used to calculate the fractional dilution of the perfusate for each run with each dilution used to represent increasing amounts of non-moving soft tissue in the sample volume. The technique was also applied to two clinical examples. RESULTS: Successive dilutions of the perfusate in the flow experiment showed a monotonic, linear decrease in the Doppler power as a function of dilution. CONCLUSION: The power Doppler US technique has the potential to more accurately estimate alterations in blood flow and has the advantage of being a continuous parameter that can be depth normalized.

Ultrasonic estimation of tissue perfusion: a stochastic approach.

Imaging of blood flow perfusion is an area of significant medical interest. Recently, the advantages of using the total integrated Doppler power spectrum as the parameter that is encoded in color has been shown to result in an approximately threefold increase in flow sensitivity, a relative insensitivity to acquisition angle and lack of aliasing. We have taken this mode a step further and demonstrated the potential for quantifying blood flow using correlation-based algorithms applied to the power signal. We show that phi(tau) = phi(0)e-VT, where phi(tau) is the two-time correlation of the fluctuation in the power signal, and v is the specific flow (reciprocal of mean transit time). Scans of a dog's blood, pumped at a constant rate through gum rubber tubing, were obtained using a Diasonics Spectra 10-MHz linear array transducer at standard range-gated spectral mode (PRF = 1400 Hz, wall filter = 50 Hz, sample gate = 1.5 mm). A fixed Doppler angle of 68 degrees was used. Five different flow rates were tested, and the velocities determined by power decorrelation were compared to the mean velocities calculated from the Doppler shifts by linear regression (R2 = 0.987). We believe the results are very encouraging for using power decorrelation in perfusion evaluation.

Immunological and molecular comparison of fowl adenovirus serotypes 4 and 10.

Some discussion has centered on whether fowl adenovirus (FAV) serotypes 4 and 10 are distinct serotypes or in fact should be reclassified as a single serotype. We have undertaken a detailed characterisation of representatives of both serotypes in order to determine if types 4 and 10 should be grouped together or retained as distinct serotypes. Examination at the genomic level has revealed considerable similarities and few differences between these 2 serotypes. DNA cross-hybridization failed to distinguish between them and restriction enzyme analysis demonstrated limited sequence differences. In vivo studies demonstrated the cross-protection afforded by a natural route vaccination with serotype 4 FAV when chickens were challenged with serotype 10 FAV. On the basis of these studies it is suggested that these FAV serotypes be combined in future FAV classification. Physical maps for both serotype 4 and 10 have been constructed using the restriction enzymes Hpa I, Dra I, Nde I, Xba I and Not I for both serotypes and in addition Eco RI, Sfi I, Sma I and BglII for serotype 10.

Cloning and sequencing of the chicken anaemia virus (CAV) ORF-3 gene, and the development of an ELISA for the detection of serum antibody to CAV.

Chicken anaemia virus (CAV) is a small, unclassified virus involved in anaemia and suspected of causing immunosuppression in young chickens. We have developed an ELISA for the detection of serum antibody to CAV based on cloned antigen. The gene for ORF-3 (the putative capsid protein) was cloned, sequenced and expressed in a bacterial expression system, pGEX. An ORF-3 fusion protein was used to produce an indirect ELISA.

Serological relationships within the group E fowl adenoviruses.

Antisera were raised in chickens to six group E fowl adenoviruses (FAV) which have been divided into a highly virulent (hypervirulent) and a mildly virulent subgroup using restriction endonuclease analysis. Virus neutralisations showed that these two distinct restriction endonuclease groups were distinguishable serologically, and indicated a possible vaccine candidate for use against the hypervirulent FAV. The suitability of this candidate was established in challenge experiments where vaccination with this virus protected against challenge from another hypervirulent virus as well as one of the mildly virulent FAV.

A molecular karyotype of Eimeria tenella as revealed by contour-clamped homogeneous electric field gel electrophoresis.

DNA from sporozoites of Eimeria tenella was resolved by pulsed field gel electrophoresis into nine chromosomal bands. Some bands of this molecular karyotype contained more than one chromosome as determined by the relative intensity of both staining with ethidium bromide and hybridisation to an E. tenella telomeric probe. Haploid forms of the parasite must be presumed to contain at least 12 chromosomes. The two smallest chromosomes were about 1.1 and 1.4 megabases. Most chromosomes were in excess of 3 Mb with the largest over 5 Mb as determined by comparison with the co-migration of chromosomes from Schizosaccharomyces pombe. A 5S ribosomal gene probe hybridised to a single chromosomal band.

Interferon release as a measure of the T-cell response to coccidial antigens in chickens.

Spleen cells from chickens which had been immunised with Eimeria tenella secreted interferon within 24 h of stimulation with as little as 10 microg antigen per millilitre from E. tenella oocysts. Secretion of interferon by spleen cells first occurred at the time immunity was developing but after antibody against coccidia was first detected. Interferon-secreting cells in the spleen diminished by 80 days after the primary infection. The secretion of interferon by lymphocytes from chickens which are immune to coccidial infection is a useful method of measuring T-cell immunity and a sensitive way of identifying antigenic epitopes recognised by T-cells. Furthermore interferon may be an important mediator in the expression of immunity to coccidial infection in poultry.