Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model.

An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID50), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.

Serotyping of foot and mouth disease virus and Pasteurella multocida from Indian gaurs (Bos gaurus), concurrently infected with foot and mouth disease and haemorrhagic septicaemia.

We report the serotyping of foot-and-mouth disease virus (FMDV) and Pasteurella multocida from Indian gaurs which were concurrently infected with foot-and-mouth disease (FMD) and haemorrhagic septicaemia. Bannerghatta biological park (BBP), a national park located in the outskirts of Bengaluru city, Karnataka, India, is bordered by several villages. These villages witnessed massive outbreaks of FMD which spread rapidly to the herbivores at BBP. Post-mortem was conducted on carcasses of two Indian gaurs that died with symptoms of FMD. The salient gross findings included extensive vesicular lesions on the tongue, gums, cheeks, upper palate and hooves. Haemorrhagic tracheitis and ecchymotic haemorrhages on the heart were characteristic. The vesicular lesions of oral cavity were positive for 'O' type of FMD virus by sandwich enzyme-linked immuno sorbent assay (ELISA). The heart blood and spleen samples yielded growth of pure cultures of P. multocida. The isolates were typed as P. multocida type B using KTSP61 and KTT72 primers yielding specific amplicons of 620 bp. The phylogenetic analysis of the isolates was carried by sequencing of 1.4-Kbp nucleotides on the 16S ribosomal RNA (rRNA) gene of the isolates.

Ontogeny of anti-viral hemorrhagic septicemia virus (VHSV) immunity in developing Japanese flounder.

We examined the ability of developing Japanese flounder (Paralichthys olivaceus) to acquire protective immunity after exposure to viral hemorrhagic septicemia virus (VHSV). Juveniles measuring 9.8 cm average body length were not susceptible to infection with VHSV at 20 °C, while the smaller fish were susceptible. Mortality was not observed after secondary infection at 15 °C in the 9.8 cm cohort that had previously been exposed to the virus at 20 °C, while the smaller fish were susceptible to secondary infection. The expression of interferon (IFN)-related genes was shown to be better developed in larger fish upon virus infection and basal expression levels of the virus recognition proteins were higher in larger fish. Virus-specific antibody was detected in the larger fish, but not in smaller fish. These data indicate that the largest juvenile (9.8 cm) acquired immunity against VHSV infection at the first virus challenge, but smaller fish did not. The anti-viral immune system in the Japanese flounder matures when juveniles reach approximately 10 cm.

Viral hemorrhagic septicemia and infectious pancreatic necrosis viruses replicate differently in rainbow trout gonad and induce different chemokine transcription profiles.

Viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) are two rainbow trout (Oncorhynchus mykiss) pathogens. While IPNV is known to be vertically transmitted to the next generation through the oocyte, VHSV is known to replicate in the ovary and be transmitted horizontally through the ovarian fluid. In this work, we wanted to study whether these differences had an effect on the immune response triggered in the ovary, with a focus on the chemokine response. We have studied the kinetics of viral gene expression and the sites of replication, confirming that great differences exist between the replication of the two viruses in the gonad. Next, we studied the levels of expression of several CXC and CC chemokines in the ovary and found that while VHSV strongly triggered chemokine transcription, IPNV had almost no effect. This lack of immune response might be an advantage that permits its vertical transmission.

Conformation- and fusion-defective mutations in the hypothetical phospholipid-binding and fusion peptides of viral hemorrhagic septicemia salmonid rhabdovirus protein G.

Fourteen single and two double point mutants in the highly conserved region (positions 56 to 159) of the G gene of viral hemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were selected and obtained in plasmids by site-directed mutagenesis. Fish cell monolayers transfected with the mutant plasmids were then assayed for protein G (pG) expression, conformation-dependent monoclonal antibody (MAb) reactivity, and cell-cell fusion. Some mutations located in the phospholipid-binding p2 peptide (positions 82 to 110; mutants P86A, A96E, G98A, and R107A) abolished both MAb recognition and fusion activity, while others (P79A, L85S, and R103A) abolished MAb recognition but retained fusion at similar or lower pHs compared to those for the wild type. Phospholipid-binding assays of p2-derived synthetic peptides suggested that phosphatidylserine binding was not affected by the mutations studied. On the other hand, three (P79A, L85S, and T135E) of the four mutants retaining fusion activity mapped around two locations showing amino acid variation in 22 VHSV isolates and in neutralizing MAb-resistant mutants described previously. Mutations located in the hypothetical fusion peptide (positions 142 to 159; mutants F147K, P148K, and W154K) abolished both MAb recognition and fusion activity. The existence of mutants with altered conformation and defective fusion in both p2 and fusion peptides provides further evidence in favor of the participation of these and adjacent regions in some of the steps of the VHSV fusion processes, as suggested by previous studies. In addition, because the studied region induced strong immunological responses in trout, some of the mutants described here might be used to design attenuated VHSV vaccines.

Notes on the life-history of the rabbit flea Caenopsylla laptevi ibera Beaucournu & Marquez, 1987 (Siphonaptera: Ceratophyllidae) in eastern Spain.

The rabbit flea Caenopsylla laptevi ibera occurs in arid environments of central and eastern Spain. Although the fleas breed during the coolest, most humid part of the year, the larvae survive and grow in sand at only 50-60% relative humidity. At 22 degrees C and 80% relative humidity eggs hatch in six days and the cocoon stage is reached 10-11 days after hatching. Female fleas emerge from pupation at about 17 days after cocoon spinning; males emerge a little later at a mean of 20 days. Adult fleas are mainly found on the host Oryctolagus cuniculus. Measurements of burrow microclimate confirmed that in south-eastern Spain burrow humidity was adequate for the development of C. I. ibera larvae over most of the year. However, breeding may be restricted for at least part of the year, as the larvae of C. I. ibera apparently cannot complete development at 25 degrees C or above. In the laboratory, fleas can enter a prolonged quiescent period while in the cocoon. This is possibly a facultative, pre-pupal diapause and the likely mechanism that accounts for the disappearance of adult fleas from the field by spring and their reappearance each autumn.