Nucleotide sequence variation in salmonid alphaviruses from outbreaks of salmon pancreas disease and sleeping disease.

We compared 18 salmonid alphaviruses (SAV) including the reference F93-125 salmon pancreas disease virus (SPDV) and S49p sleeping disease virus (SDV) isolates by nucleotide sequence analyses of regions within the E1, nsP4 and nsP3 genes, and found these to comprise 3 distinct groups, which we have designated Subtypes 1, 2 and 3: Subtype 1, which comprised SAVs with sequences closely similar to the reference SPDV isolate, included SAVs from pancreas disease (PD) outbreaks in farmed salmon in Ireland and Scotland over a 10 yr period; viruses from recent outbreaks of sleeping disease (SD) in freshwater-reared trout farmed in England, Scotland and France were closely similar to and were grouped with the reference SDV isolate in Subtype 2; 3 viruses isolated from PD-affected salmon in Norway were genetically different from viruses belonging to Subtypes 1 and 2 and have been assigned to Subtype 3; 1 virus isolated from PD-affected salmon in the Western Isles, Scotland, in 2003 showed consistent nucleotide sequence differences from SAV Subtypes 1, 2 and 3, but was more closely related to the Subtype 1 SAVs. The occurrence of the different subtype SAVs appeared to have a geographical basis, which may prove useful in future molecular epidemiology studies of SAV-induced disease outbreaks.

Circovirus-infected geese studied by in situ hybridization.

It has now been established that circovirus infection is common in farmed geese, but little is known about the clinicopathological significance of such infections. Ten clinically diseased geese suspected of being infected by circovirus were studied by in situ hybridization using a goose circovirus DNA probe. Circovirus DNA was demonstrated in the bursa of Fabricius (BF), spleen, thymus, bone marrow, liver, kidney, lung and heart, indicating that infection can be multisystemic. In some birds, virus DNA was present in very large quantities, most notably in the BF, liver and small intestine. With the exception of BF and thymus, there were no histological findings that would have suggested the presence of such quantities of circovirus DNA. In view of the very large quantities of virus DNA labelling present in some tissues, and by analogy to porcine circovirus type 2 infection and psittacine beak and feather virus infections, which are known to cause severe disease, and which have similar virus distribution to that found in our geese, it seems probable that the circovirus was important in the disease manifestations shown by the infected geese.

Detection and antigenic characterization of salmonid alphavirus isolates from sera obtained from farmed Atlantic salmon, Salmo salar L., and farmed rainbow trout, Oncorhynchus mykiss (Walbaum).

A simple method of detecting the presence of the salmonid alphaviruses (SAVs), salmon pancreas disease virus (SPDV) and sleeping disease virus (SDV), from serum samples is described. Using a 96-well tissue-culture plate format, test sera are diluted in medium and added to chinook salmon embryo (CHSE-214) cells. After incubation for 3 days at 15 degrees C, plates are fixed and stained using a monoclonal antibody (mAb)-based immunoperoxidase (IPX) detection system, and virus-infected cells are observed microscopically by white light. Application of this screening test, which is now used routinely in our laboratory in conjunction with an IPX-based virus neutralization (IPX-VN) test for detecting antibodies to SAVs, has resulted in the recovery of 12 additional isolates from salmon sera and four additional isolates from trout sera. A low level of antigenic variation was detected when these SAV isolates were investigated by indirect immunofluorescence using a panel of mAbs raised to reference SPDV and SDV isolates.

Diagnosis of goose circovirus infection in Hungarian geese samples using polymerase chain reaction and dot blot hybridization tests.

A polymerase chain reaction (PCR) and dot blot hybridization (DBH) test have been developed for the diagnosis of infection by a novel circovirus of geese (GoCV). These tests were applied to samples of bursae of Fabricius from sick and dead birds from commercial goose farms in Hungary. In this second report of the occurrence of circovirus infection in diseased geese, 103 of 214 (48.1%) and 37 of 150 (24.6%) birds, and 49 of 76 (64.5%) and 18 of 76 (23.7%) flocks were positive by PCR and DBH respectively. The sensitivity of the PCR test was such that 0.10 fg of virus DNA was detectable. The DBH test was less sensitive, only detecting larger amounts (40 pg) of DNA, but was used as a semi-quantitative method for detecting the presence of virus. The incidence of infection was affected by factors such as the age of the birds and rearing methods.

Evaluation of polymerase chain reaction and dot blot hybridisation tests in the diagnosis of pigeon circovirus infections.

Polymerase chain reaction (PCR) and dot blot hybridisation (DBH) tests for detecting pigeon circovirus (PiCV) DNA were developed and evaluated using tissue samples obtained from diseased and clinically normal pigeons, which originated in Belgium and Northern Ireland. When PCR product was visually detected, the limit of detection of the PCR test was 31 fg, while that of the DBH was 1.6p g. For evaluation purposes, the results of the PCR and DBH tests, performed with DNAs extracted from samples of bursa of Fabricius (BF), were compared with those of in situ hybridisation (ISH) and histology. In 32 samples tested by all four tests, 27 (84%) were positive by PCR, 24 (75%) were positive by ISH, 20 (63%) were positive by DBH, and 13 (41%) were positive by histology. Additional PCR testing showed that in some disease-affected birds, PiCV DNA could be detected in a range of tissues including thymus, spleen, liver, kidney and brain. The PCR detection of PiCV DNA in BF samples from clinically normal birds indicated that PCR can detect infections in the absence of disease, a finding that mitigates against its use as a disease diagnostic. In addition, nucleotide sequence determinations indicated that PCR test performance was adversely affected by the sequence diversity exhibited by selected PiCVs. The application of the DBH test to dilutions of test samples indicated that the BF from some diseased pigeons contained very large amounts of virus DNA, as much as 10(13)genome copies/g tissue, and suggested that this test may be a convenient method of providing a semi-quantitative estimate of virus load.

Production and characterisation of monoclonal antibodies to salmon pancreas disease virus.

Six mouse monoclonal antibodies (mAbs) specific to salmon pancreas disease virus (SPDV) were produced following immunisation with purified virus preparations. These mAbs and 2 mAbs resulting from an earlier investigation were characterised. None of the mAbs possessed virus neutralising activity but all reacted with 4 geographically different SPDV isolates as determined by indirect immunofluorescence. Three mAbs produced positive immunostaining with Western blots of SPDV proteins. The 4H1 mAb reacted with the 53 kDa structural E1 glycoprotein present in virus-infected cells and in gradient-purified virus. Two mAbs, 5A5 and 7B2, which exhibited unusual immunofluorescence staining of the nuclear margin, reacted with a 35 kDa protein, which is present in gradient-purified virus and which is considered to be the capsid protein. A sandwich ELISA, based on the use of mAb 2D9 for capture and a biotinylated conjugate of mAb 7A2 for detection, detected SPDV antigen in virus-infected Chinook salmon embryo-214 cells and gradient-purified virus. These mAbs may be of use in pathogenesis studies and in diagnostic test development.

Genome sequence determinations and analyses of novel circoviruses from goose and pigeon.

The genomes of novel circoviruses from goose and pigeon, which were isolated using degenerate primer and inverse primer PCR methods, were cloned and sequenced. Comparative nucleotide (nt) sequence analyses showed that the goose circovirus (GCV) and pigeon circovirus (PiCV) possessed genomes which were 1821 and 2037 or 2036 nt, respectively, and which had features in common with the genomes of porcine circoviruses types 1 and 2 (PCV1, PCV2) and psittacine beak and feather disease virus (BFDV), such that they can now be assigned to the genus Circovirus of the family Circoviridae. Common features include the possession of (i) a potential stem-loop/nonanucleotide motif with which the initiation of rolling circle replication of the virus DNA is associated; (ii) two major ORFs, located on the virus (V1 ORF) and complementary (C1 ORF) strands, which encode the replication-associated protein (Rep) and capsid protein, respectively; (iii) high levels of amino acid identity (41.2--58.2%) shared with other circovirus Rep proteins; and (iv) direct/inverted repeat sequences within the putative intergenic region. On the basis of nt and amino acid sequence identities, GCV is substantially less closely related to BFDV than PiCV is to BFDV.

Characterization of the genome of avian encephalomyelitis virus with cloned cDNA fragments.

cDNA fragments were generated from RNA extracted from preparations of avian encephalomyelitis virus (AEV) by a reverse transcription-polymerase chain reaction (RT-PCR) strategy, which exploited the probability that AEV is a picornavirus. Rapid amplification of the 3' cDNA ends, which utilized an oligo d(T)-based primer that hybrizes to the putative Poly (A) tract at the 3' terminus of picornavirus RNA, produced a 3.8-kbp fragment (3.8-kbp 3' RACE fragment), from which a 2.5-kbp cDNA fragment specific to the extreme 3' terminal region of the AEV genome was cloned. Positive hybridization reactions between RNA from gradient-purified virus and radiolabeled probes confirmed that the cloned 2.5-kbp fragment was AEV specific. The success of the RT-PCR amplification strategy adopted and the results of northern blotting hybridization experiments indicated that the AEV genome is a polyadenylated, single-stranded RNA, approximately 7.5 kb in size. Sequence analysis of a 869-base region at the 3' terminal of the genome indicated that this region encoded a protein with close homologies to picornaviral RNA polymerase proteins. On the basis that the highest levels of protein homologies were observed with hepatitis A virus, it is likely that AEV will be reassigned to a genus other than the enterovirus genus within the virus family Picornaviridae. The AEV-specific cloned DNA fragments and nucleotide sequence information resulting from this investigation may facilitate the development of in situ hybridization and RT-PCR methods that will be useful in AEV diagnosis.

Salmon pancreas disease virus, an alphavirus infecting farmed Atlantic salmon, Salmo salar L.

A 5.2-kb region at the 3' terminus of the salmon pancreas disease virus (SPDV) RNA genome has been cloned and sequenced. The nucleotide and predicted amino acid sequences show that SPDV shares considerable organizational and sequence identity to members of the genus alphavirus within the family Togaviridae. The SPDV structural proteins encoded by the 5.2-kb region contain a number of unique features when compared to other sequenced alphaviruses. Based on cleavage site homologies, the predicted sizes of the SPDV envelope glycoproteins E2 (438 aa) and E1 (461 aa) are larger than those of other alphaviruses, while the predicted size of the alphavirus 6K protein is 3.2 K (32 aa) in SPDV. The E2 and E1 proteins each carry one putative N-linked glycosylation site, with the site in E1 being found at a unique position. From amino acid sequence comparisons of the SPDV structural region with sequenced alphaviruses overall homology is uniform, ranging from 32 to 33%. While nucleotide sequence analysis of the 26S RNA junction region shows that SPDV is similar to other alphaviruses, analysis of the 3'-nontranslated region reveals that SPDV shows divergence in this region.

Gas chromatographic determination of residue levels of the herbicides trifluralin, benefin, ethalfluralin, and isopropalin in soil with confirmation by mass selective detection.

A method is presented for the simultaneous or individual determination of the dinitroaniline herbicides trifluralin, benefin, ethalfluralin, and isopropalin in soil. The herbicides are extracted with acetonitrile-water (99 + 1), and the extracts are purified with small, disposable Florisil cartridges prior to analysis by gas chromatography using an electron capture detector or a mass selective detector. When electron capture detection is used as the primary detection system, confirmation with selective detection can be obtained using gas chromatography-mass spectrometry with a mass selective detector and a capillary column operated in the split mode. The limit of detection is 0.01 ppm, and recoveries averaged 95-112% for the 4 herbicides in several different soil types fortified at levels of 0.01-0.33 ppm.

Tolerance and absence of rebound vasodilation following topical ocular decongestant usage.

Commercial preparations of two topical ophthalmic vasoconstrictors were evaluated for whitening ability, duration of action, tolerance, and rebound vasodilation in 11 normal volunteers. Both 0.02% naphazoline HCI and 0.05% tetrahydrozoline HCI significantly reduced baseline redness after a single use (Part I); however, naphazoline produced significantly more whitening than did tetrahydrozoline. Only naphazoline retained its whitening ability after ten days (Part II). The level of redness remained significantly below baseline for eight hours after a single use of either vasoconstrictor and for six hours after multiple use of naphazoline. The diminished effectiveness of tetrahydrozoline after the ten-day test period may encourage its overuse. Neither vasoconstrictor produced rebound vasodilation after discontinuation of use.

Aspirin therapy in vernal conjunctivitis.

Prostaglandin D2 is a secondary mast cell mediator that causes redness, chemosis, mucous discharge, and eosinophil chemotaxis in the eye. It may play an important role in allergic ocular disease. Although histamine is a key mediator of allergic inflammation, antihistamine therapy provides only symptomatic relief. We added aspirin therapy to the treatment regimen of three patients with vernal conjunctivitis. Aspirin acetylates the enzyme cyclooxygenase, thereby preventing the formation of prostaglandin D2. Within two weeks after initiation of aspirin therapy, we noted dramatic improvement in conjunctival and episcleral redness and resolution of keratitis and limbal infiltration. We recommend a trial of oral aspirin as adjunctive therapy for intractable cases of vernal conjunctivitis.

Conjunctival eosinophils in allergic ocular disease.

Tarsal conjunctival scrapings of 317 patients with allergic ocular disease demonstrated that eosinophils were found infrequently in scrapings of patients with mild allergic conditions and were found in only 63% (17/27) of the patients with vernal conjunctivitis. In similar studies of a 12-year-old boy with vernal keratoconjunctivitis, no eosinophils were recovered in his scraping, even though 12 eosinophils were found in five high-power fields of his biopsy specimen. We conclude that eosinophils present in the deep and superficial conjunctival tissues may not be recovered in scrapings; their absence from scrapings should not preclude the diagnosis of allergic ocular disease.

Conjunctival eosinophils in compound 48/80 rabbit model.

Compound 48/80 (N-methyl-p-methoxyphenethylamine formaldehyde condensation product) was used to selectively degranulate mast cells to induce conjunctival eosinophilia in 12 rabbits. Biopsy specimens of bulbar conjunctiva showed that eosinophils were present in all treated eyes. With repeated treatments the number of eosinophils increased; these cells were concentrated in the subepithelial and epithelial zones by day 3. Eosinophils were not found on scrapings of the bulbar conjunctiva in 75% of the rabbits that received single or multiple treatments. We conclude that deep and superficial eosinophil infiltration may be present even when eosinophils are not seen on conjunctival scrapings. Therefore, the absence of eosinophils in scrapings should not rule out the diagnosis of ocular allergy.

Normal human tear pH by direct measurement.

Tear pH was measured in 44 normal subjects by immersing the lip of a microcombination glass pH probe in the tear fluid in the inferior cul-de-sac. The normal pH range was 6.5 to 7.6; the mean value was 7.0. This method provides a rapid and accurate measure of tear pH and may better our understanding of tear physiology and tear buffering capacity.

Alkaline tear pH in ocular rosacea.

We compared the tear pH values of 44 normal, healthy volunteers, 20 patients with ocular disorders other than rosacea, seven patients with untreated, active ocular rosacea, and five patients with tetracycline-treated ocular rosacea. The group with untreated, active ocular rosacea had significantly more alkaline tear pH values than the other groups tested. In patients with tetracycline-treated ocular rosacea, tear pH values were not significantly different from those of normal subjects.