Previous studies (Mertens et al., Virology 157, 375-386, 1987) have shown that removal of the outer capsid layer from bluetongue virus (BTV) significantly reduces (approximately x 10(-4)) the infectivity of the resultant core particle for mammalian cells (BHK 21 cells). In contrast, the studies reported here, using a cell line (KC cells) derived from a species of Culicoides that can act as a vector for BTV (Culicoides variipennis), demonstrated a much higher infectivity of core particles than that in mammalian cells (approximately x 10(3)). This increase resulted in a specific infectivity for cores that was only 20-fold less than that of purified disaggregated virus particles (stored in the presence of 0.1% sodium-N-lauroylsarcosine (NLS)). Removal of this detergent caused intact virus particle aggregation and (as previously reported) resulted in an approximately 1 log10 drop in the specific infectivity of those virus particles which remained in suspension. In consequence the specific infectivity of core particles for the KC cells was directly comparable to that of the intact but aggregated virus. These data are compared with the results from oral infectivity studies using two vector species (C. variipennis and Culicoides nubeculosus), which showed similar infection rates at comparable concentrations of purified cores, or of the intact but aggregated virus particles (NLS was toxic to adult flies). The role of the outer core proteins (VP7) in cell attachment and penetration, as an alternative route of initiation of infection, is discussed. Previous studies (Mertens et al., Virology 157, 375-386, 1987) also showed that the outer capsid layer of BTV can be modified by proteases (including trypsin or chymotrypsin), thereby generating infectious subviral particles (ISVP). The specific infectivity of ISVP for mammalian cells (BHK21 cells) was shown to be similar to that of disaggregated virus particles. In contrast, we report a significantly higher specific infectivity of ISVP but not of the intact virus (approximately x 100) for two insect cell lines (KC cells and C6/36 mosquito cells (derived from Aedes albopictus)). In oral infection studies with adults of the two vector species, ISVP produced the same infection rate at approximately 100-fold lower concentrations than either core particles or the intact but aggregated virus particles. The importance of mammalian host serum proteases, or insect gut proteases, in modification of the intact virus particle to form ISVP and their role in initiation of infection and the vector status of the insect is discussed.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are neurodegenerative lysosomal storage diseases predominantly affecting children. Single administration of brain-directed lentiviral or recombinant single-stranded adeno-associated virus 9 (ssAAV9) vectors expressing ovine CLN5 into six pre-clinically affected sheep with a naturally occurring CLN5 NCL resulted in long-term disease attenuation. Treatment efficacy was demonstrated by non-invasive longitudinal in vivo monitoring developed to align with assessments used in human medicine. The treated sheep retained neurological and cognitive function, and one ssAAV9-treated animal has been retained and is now 57 months old, almost triple the lifespan of untreated CLN5-affected sheep. The onset of visual deficits was much delayed. Computed tomography and MRI showed that brain structures and volumes remained stable. Because gene therapy in humans is more likely to begin after clinical diagnosis, self-complementary AAV9-CLN5 was injected into the brain ventricles of four 7-month-old affected sheep already showing early clinical signs in a second trial. This also halted disease progression beyond their natural lifespan. These findings demonstrate the efficacy of CLN5 gene therapy, using three different vector platforms, in a large animal model and, thus, the prognosis for human translation.

Culicoides variipennis sonorensis Wirth & Jones and C. nubeculosus (Meigen) were orally infected with African horse sickness virus (AHSV) type 9 and subsequently incubated at 10, 15, 20 and 25°C (R.H. 80%±10%). A time course of infection rates and virus titres was recorded by assaying flies individually or in pools, and survival rates of flies were also estimated. Survival rates at 10, 15 and 20°C were very similar and 80-90% of flies remained alive after 14 days; at 25°C after the same period survival was reduced to 40%. None of the C. nubeculosus became persistently infected with AHSV, but the virus took longer to clear as the incubation temperature dropped. At temperatures of 10, 15, 20 and 25°C virus was undetectable on days 12, 8, 5, and 4 days post infection (dpi), respectively. In C. v. sonorensis both the infection rate and rate of virogenesis were related to temperature. At 25°C a maximum mean titre of 10 43 TCID 50 /fly was reached by 9 dpi and the infection rate remained between 60 and 80%. At 20°C virogenesis was slower and a maximum mean titre of 10 43 TCID 50 /fly was reached only after 23 days; the infection rate was also reduced to 50-70%. At 15°C there was an overall decline in virus titre with time, although between 12 and 15 dpi some pools of flies contained 10 30 -10 40 TCID 50 /fly, demonstrating that virogenesis can occur. The infection rate at this temperature decreased dramatically to 0-15% after 9 dpi. At 10°C there was no detectable virogenesis and all pools tested at 13 dpi were negative. The apparent infection rate dropped to 0-5% between 13 and 55 days post infection. However, when surviving flies were then returned to 25 °C for 3 days the infection rate increased to 15.5%. It therefore appears that at low temperatures the virus does not replicate but infectious virus may persist at a level below that detectable by the usual assay systems. The implications of these findings for the epidemiology of AHS are discussed.

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IntroductionThe neuronal ceroid lipofuscinoses (NCLs; Batten disease) are a group of fatal neurodegenerative lysosomal storage diseases of children caused by various mutations in a range of genes. Forms associated with mutations in two of these, CLN5 and CLN6, are being investigated in well‐established sheep models. Brain atrophy leading to psychomotor degeneration is among the defining features, as is regional progressive ossification of the inner cranium. Ongoing viral‐mediated gene therapy trials in these sheep are yielding encouraging results. In vivo assessment of brain atrophy is integral to the longitudinal monitoring of individual animals and provides robust data for translation to treatments for humans.MethodsComputed tomography (CT)‐based three‐dimensional reconstruction of the intracranial volume (ICV) over time reflects the progression of cortical brain atrophy, verifying the use of ICV measurements as a surrogate measure for brain size in ovine NCL.ResultsICVs of NCL‐affected sheep increase for the first few months, but then decline progressively between 5 and 13 months in CLN5−/− sheep and 11–15 months in CLN6−/−sheep. Cerebral ventricular volumes are also increased in affected animals. To facilitate ICV measures, the radiodensities of ovine brain tissue and cerebrospinal fluid were identified. Ovine brain tissue exhibited a Hounsfield unit (HU) range of (24; 56) and cerebrospinal fluid a HU range of (−12; 23).ConclusionsComputed tomography scanning and reconstruction verify that brain atrophy ovine CLN5 NCL originates in the occipital lobes with subsequent propagation throughout the whole cortex and these regional differences are reflected in the ICV loss.