Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses

Natural isolates and laboratory strains of West Nile virus (WNV) and Japanese encephalitis virus (JEV) were attenuated for neuroinvasiveness in mouse models for flavivirus encephalitis by serial passage in human adenocarcinoma (SW13) cells. The passage variants displayed a small-plaque phenotype, augmented affinity for heparin-Sepharose, and a marked increase in specific infectivity for SW13 cells relative to the respective parental viruses, while the specific infectivity for Vero cells was not altered. Therefore, host cell adaptation of passage variants was most likely a consequence of altered receptor usage for virus attachment-entry with the involvement of cell surface glycosaminoglycans (GAG) in this process. In vivo blood clearance kinetics of the passage variants was markedly faster and viremia was reduced relative to the parental viruses, suggesting that affinity for GAG (ubiquitously present on cell surfaces and extracellular matrices) is a key determinant for the neuroinvasiveness of encephalitic flaviviruses. A difference in pathogenesis between WNV and JEV, which was reflected in more efficient growth in the spleen and liver of the WNV parent and passage variants, accounted for a less pronounced loss of neuroinvasiveness of GAG binding variants of WNV than JEV. Single gain-of-net-positive-charge amino acid changes at E protein residue 49, 138, 306, or 389/390, putatively positioned in two clusters on the virion surface, define molecular determinants for GAG binding and concomitant virulence attenuation that are shared by the JEV serotype flaviviruses.

Thermostability profile of Newcastle disease virus (strain I-2) following serial passages without heat selection

I-2 is an avirulent strain of Newcastle disease virus. During establishment of the I-2 strain master vaccine seed, a series of selection procedures was carried out at 56 degrees C in order to enhance heat resistance. This master seed is used to produce a working seed, which is then employed to produce the vaccine. These two passages are done without further heat selection; however, it is not known how rapidly and to what extent thermostable variants would be lost during further passage. The study was therefore conducted to determine the effect of passage on thermostability of strain I-2. The virus was serially passaged and at various passage levels samples were subjected to heat treatment at 56 degrees C for 120 min. The inactivation rates for infectivity and haemagglutinin (HA) titres were assayed by use of chicken embryonated eggs and HA test, respectively. Thermostability of HA and infectivity of I-2 virus were reduced after 10 and 5 passages, respectively, without heat selection at 56 degrees C. These results suggest that 5 more passages could be carried out between the working seed and vaccine levels without excessive loss of thermostability. This would result in increased vaccine production from a single batch of a working seed.

Fast accumulation of few polyhedra mutants during passage of a Spodoptera frugiperda multicapsid nucleopolyhedrovirus (Baculoviridae) in Sf9 cell cultures

Baculoviruses are a group of viruses that infect invertebrates and that have been used worldwide as a biopesticide against several insect pests of the Order Lepidoptera. In Brazil, the baculovirus Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV, Baculoviridae) has been used experimentally to control S. frugiperda (Lepidoptera: Noctuidae), an important insect pest of corn (maize) fields and other crops. Baculoviruses can be produced either in insect larvae or in cell culture bioreactors. A major limitation to the in vitro production of baculoviruses is the rapid generation of mutants when the virus undergoes passages in cell culture. In order to evaluate the potential of in vitro methods of producing SfMNPV on a large-scale, we have multiplied a Brazilian isolate of this virus in cell culture. Extensive formation of few polyhedra mutants was observed after only two passages in Sf9 cells.

Year-round presence of dugongs in Pumicestone Passage, south-east Queensland, examined in relation to water temperature and seagrass distribution

Pumicestone Passage is a narrow waterway that lies to the north of and adjacent to Moreton Bay, and between mainland Queensland and Bribie Island, Australia. Anecdotal reports have suggested that the Passage is home to dugongs year-round despite winter water temperatures that are known to cause dugongs to migrate elsewhere. To examine the pattern of distribution and abundance of dugongs within the passage on a year-round basis, eight years of sightings data collected by a charter boat operator were examined. Dedicated aerial surveys of the passage were also conducted at two-monthly intervals over two years, and more intensively over a single winter. Dugong sightings were examined in relation to water temperatures and seagrass prevalence. The number of dugongs sighted in the area on any one survey varied from 0 to 13. Dugongs were seen in all months of the year and in each of the eight winters, indicating that Pumicestone Passage is used year-round despite winter water temperatures dropping to below 18 degrees C from June to August inclusive and below 16 degrees C in June. All dugong sightings occurred in the southern part of the passage, south of Tripcony Bight. Dugongs were associated with shallows that support Halophila and Halodule species of seagrass, food species that are favoured elsewhere in their range. The northern part of the passage also supports seagrasses that are eaten by dugongs and has water temperature ranges that are not appreciably different to those of the southern passage. However, the narrow channels and very shallow nature of the northern passage provides little to no deep-water refugia for dugongs and the seagrass beds are less extensive. This study suggests that southern Pumicestone Passage requires protection concomitant with it being a year-round refuge of the vulnerable dugong.