Cyprinid herpesvirus 3 as a potential biological control agent for carp (Cyprinus carpio) in Australia: susceptibility of non-target species.

Carp (Cyprinus carpio L.) is a pest species in Australian waterways, and cyprinid herpesvirus 3 (CyHV-3) is being considered as a potential biological control (biocontrol) agent. An important consideration for any such agent is its target specificity. In this study, the susceptibility to CyHV-3 of a range of non-target species (NTS) was tested. The NTS were as follows: 13 native Australian, and one introduced, fish species; a lamprey species; a crustacean; two native amphibian species (tadpole and mature stages); two native reptilian species; chickens; and laboratory mice. Animals were exposed to 100-1000 times the approximate minimum amount of CyHV-3 required to cause disease in carp by intraperitoneal and/or bath challenge, and then examined clinically each day over the course of 28 days post-challenge. There were no clinical signs, mortalities or histological evidence consistent with a viral infection in a wide taxonomic range of NTS. Furthermore, there was no molecular evidence of infection with CyHV-3, and, in particular, all RT-PCRs for viral mRNA were negative. As a consequence, the results encourage further investigation of CyHV-3 as a potential biocontrol agent that is specific for carp.

Development and application of molecular methods (PCR) for detection of Tasmanian Atlantic salmon reovirus.

Molecular (PCR) diagnostic tests for the detection and identification of aquareovirus in general, and Tasmanian Atlantic salmon reovirus (TSRV) specifically, were developed, and their diagnostic sensitivity and specificity were determined and compared with virus isolation in cell culture. Intralaboratory and interlaboratory comparison of PCR (conventional hemi-nested RT-PCR & RT-qPCR) and virus isolation in cell culture using finfish cell lines, CHSE-214 and EPC, was carried out for the detection and identification of TSRV using field samples of farmed Atlantic salmon Salmo salar, L. from various aquaculture sites around Tasmania. The interlaboratory comparison of diagnostic methods was carried out between two laboratories, AAHL-CSIRO and DPIPWE-Tasmania. A total of 144 fish from nine sites (12-33 fish per site) were sampled from two regions of Tasmania (Tamar River estuary in the north and Huon River estuary in the south-east) during late spring to early summer of 2009, and the data were analysed using different statistical approaches. The prevalence of TSRV ranged from 6% to 22% in both regions. All the diagnostic methods (data from both laboratories) had high specificity, while the estimated sensitivity varied between tests with RT-qPCR being the most sensitive (95.2%) method followed by virus isolation and then conventional hemi-nested RT-PCR.

Isolation of bovine herpesvirus type 5 from the semen of a healthy bull in Australia.

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.

Isolation of a new foamy retrovirus from orangutans.

We have isolated a new foamy virus from blood samples taken from two apparently healthy orangutans (Pongo pygmaeus). The older orangutan has since died with encephalopathy after a brief acute illness, while the younger one, his grandson, remains well. These animals and 12 other orangutans had specific antibodies to foamy virus as measured by immunofluorescence. The new foamy virus and the antisera showed strong and specific neutralization, with only weak cross-reaction with other simian foamy virus strains. Southern blotting with gag and env probes of human foamy virus and PCR amplification showed that the new foamy virus, designated SFV-11, is related to, yet distinct from, previously characterized strains from humans, chimpanzees, and monkeys.

Effect of mutations in the V3 loop of HIV-1 gp120 on infectivity and susceptibility to proteolytic cleavage.

It has been suggested that the V3 domain of human immunodeficiency virus type 1 (HIV-1) isolates has to interact with a cell-surface-associated or endosomal proteinase during virus entry into susceptible cells. To investigate this hypothesis, we examined the effect of several mutations in the V3 loop on its susceptibility to proteolytic cleavage by thrombin and cathepsin E and compared it with the effect of these mutations on viral infectivity. The data obtained indicate that, if an interaction between the V3 loop and a proteinase is indeed crucial for viral entry, the substrate requirements for such a proteinase(s) would have to be very complex. In particular, it seems unlikely that a single enzyme with a unique specificity would be able to interact with all of the different HIV-1 and HIV-2/SIV strains isolated so far. Therefore, one would have to postulate the involvement of several cellular proteinases, or proteases with multiple specificities, in V3-based viral tropism.

A measles virus isolate from a child with Kawasaki disease: sequence comparison with contemporaneous isolates from 'classical' cases.

We examined the relationship between a measles virus isolate from a child with Kawasaki disease and two contemporaneous wild-type isolates from children with 'classical' measles and the Schwarz vaccine strain. Sequence analysis of 3118 bp from the nucleoprotein, matrix, fusion and haemagglutinin genes of each virus revealed that the isolate from the child with Kawasaki disease was not related to measles vaccine strains and did not contain any of the marked abnormalities previously found in subacute sclerosing panencephalitis isolates, but was more akin to wild-type isolates currently circulating in the U.K. A comparison of our sequences with those obtained from earlier wild-type U.K. isolates suggests significant evolution of measles virus in the U.K. over the last decade.

Virus genome detection by the polymerase chain reaction (PCR).

Since the polymerase chain reaction (PCR) technique was developed in 1986 (1), it has found wide application in molecular biology and is now regarded as an irreplaceable tool in many laboratories. This chapter will deal with its use for the detection of virus genomes for diagnostic and experimental purposes.

HTLV-1 envelope sequences from Brazil, the Caribbean, and Romania: clustering of sequences according to geographic origin and variability in an antibody epitope.

We sequenced the envelope genes of Human T-cell leukemia type I viruses (HTLV-I) derived from five Brazilian, two Caribbean, and one Romanian case of adult T-cell leukemia after amplification of the complete env gene by PCR. A comparison with previously reported HTLV-I sequences revealed that, although highly homologous, no two env sequences were identical. All envelope sequences differed from each other by 0.3-2.1% nucleotide differences. The five Brazilian sequences clustered together and were about as different from each other (0.5-0.75% nucleotide difference) as were three previously reported Japanese sequences (0.7-0.95%). In contrast, sequences of Caribbean origin were less homogeneous (0.5-1.9% nucleotide differences within this group). The Romanian sequence was not significantly more divergent than any of the others and was closest to our two Caribbean sequences. We observed two changes in a region (aa 176-209) which has previously been shown to contain a linear antibody epitope recognized by most human sera from seropositive individuals. One of these changes affects the binding of monoclonal antibodies to this epitope demonstrating the variability of an antibody epitope in the HTLV-I envelope.

Biological and molecular variability of human immunodeficiency virus type 2 isolates from The Gambia.

Seven new human immunodeficiency virus type 2 (HIV-2) isolates (CBL-20 to CBL-26) from The Gambia were characterized. Their cytopathogenicity and growth in vitro correlated with the severity of clinical disease. CBL-22 was highly sensitive to neutralization by HIV-2 sera and was cross-neutralized by some HIV-1 sera. These findings, the differing sizes of envelope glycoproteins of individual isolates, and the sequence analysis of amplified regions of the viral DNAs show that these HIV-2 isolates from one geographical region in West Africa exhibit biological and genome variability comparable to that observed for HIV-1.

PIP: Although the immunodeficiency diseases associated with human immunodeficiency virus (HIV) 1 and 2 are indistinguishable from each other, there is some evidence that HIV-2 isolates may have a longer incubation period. Thus, an investigation was conducted of the biological properties and molecular variability of the spectrum of HIV-2 isolates existing in The Gambia. Serum samples were obtained from 20 HIV-2-positive individuals attending a sexually transmitted diseases clinic in Fajara. Seven new HIV-2 isolates (CBL-20 to CBL-26) were identified. Each differed in terms of its growth rate, cytopathogenicity in vitro, and sensitivity to neutralizing antibodies in patient sera. In addition, there was a close association between the isolates' in vitro cytopathogenicity and the clinical cytopathogenic strains, were obtained from the two patients with acquired immunodeficiency syndrome (AIDS). In contrast, patients from whom CBL-25 and 26 were isolated have been asymptomatic for at least 3 years. CBL-22 was highly sensitive to neutralization by HIV-2 sera and was cross-neutralized by some HIV-1 sera. It is speculated that the observed differences in sensitivity to neutralization reflect differences in antigenic epitope expression or the number and presentation of envelope glycoprotein molecules on an infectious virion. Analysis of the molecular weight of the envelope precursor and the outer envelope protein of various HIV-2 isolates revealed that all the CBL isolated and SBL-6669 have smaller envelope proteins than the prototype strain, LAV-2 ROD. Also observed were differences in the amino acid sequences of these HIV-2 isolates.

A comparative study of the effects of ranitidine and cimetidine on carbohydrate tolerance, growth hormone secretion and the hypothalamic-pituitary-gonadal axis in man.

The effect of chronic oral administration of cimetidine (1 g per day) and ranitidine (300 mg per day) on plasma levels of prolactin (PRL), testosterone, dihydrotestosterone (DHT), luteinizing hormone (LH), follicle stimulating hormone (FSH), sex hormone binding globulin (SHBG) and human growth hormone was compared in 2 groups of male patients who presented with dyspeptic symptoms. Eight were treated with ranitidine and 9 with cimetidine for 4 weeks. The glucose and insulin response to a 100 g oral glucose load was also assessed. Cimetidine treatment resulted in a significant increase in plasma testosterone levels which was not found in the ranitidine group. No significant change occurred in PRL, LH, FSH, SHBG, DHT and growth hormone. There was no evidence of a significant alteration in carbohydrate metabolism.