Recombinant LaSota V.F. (rLaSota V.F.) virus was recovered entirely from a cDNA clone of rLaSota using reverse genetics procedures [12]. Sequencing of the RT-PCR product containing the F cleavage site of rLaSota V.F. confirmed the presence of the desired mutations. Thus, the sequence G-R-Q-G-R present at the F cleavage site of avirulent NDV strain LaSota was altered to R-R-Q-K-R, to be analogous to the F cleavage site of neurovirulent strain BC (Fig. 1). It was also found that rLaSota V.F. did not require exogenous protease for propagation in cell culture, indicating that the F protein was cleaved by intracellular proteases.Fig. 1

Genomes of rBC, rLaSota and rLaSota V.F. viruses are shown schematically. Amino acid sequence at the F protein cleavage site of rLaSota was modified by mutagenesis to that of rBC to obtain rLaSota V.F. virus. The trypsin requirement for in vitro propagation shown on the side.

The kinetics and the magnitude of replication of rBC, rLaSota and rLaSota V.F. were very similar in DF1 cells (Fig. 2A). The plaque sizes and morphologies of all three viruses were also very similar (Fig. 2B). The replication of rBC, rLaSota, and rLaSota V.F. viruses in the brains of 1-day-old chicks after intracerebral inoculation was measured (Fig. 3). The rBC and rLaSota V.F. viruses initiated new virus formation after one day of infection. However, the yields of rBC virus were 100-fold higher than those of rLaSota V.F. virus. By day 4, all birds inoculated with rBC were dead. The replication kinetics of rLaSota virus was delayed and the virus yields were lower than those of rBC and rLaSota V.F. viruses. These results suggested that intracellular cleavage of the F protein is important for replication of NDV in the brains of chicks, but, probably, other viral factors are necessary for efficient replication. The genetic stability of the mutations introduced into the genome of LaSota V.F. virus was determined by sequence analysis of the RT-PCR fragment that covered the region of the F protein cleavage site of the mutant virus that was passed five times in 9-day-old chicken embryos, and also from the mutant virus that was passaged once in chicken brains. All the introduced mutations were found to be stable.Fig. 2

(A) Multistep growth curve of rBC, rLaSota and rLaSota V.F. viruses in DF1 cells. Cell monolayers in 25 cm² flask were infected with 0.05 PFU/cell with three replicate flasks per virus. Supernatant samples were taken every 8 h for 56 h. The virus in the supernatant was titrated by plaque assay. The medium of cells infected with rLaSota contained 1 μg/ml of acetyl-trypsin. (B) Plaque size and morphology of rBC, rLaSota and rLaSota V.F. The viruses were serially diluted and 100 μl of each serial dilution was added per well to confluent DF1 cells in 12-well plates. Duplicate wells were used for each serial dilution of every virus sample. After 60 min adsorption, cells were overlaid with DMEM (containing 2% fetal bovine serum and 0.9% methyl cellulose) and incubated at 37 °C for 3–4 days. The cells were then fixed with ethanol and stained with crystal violet for observation of plaques.

To examine the role of growth temperature in replication and biological activities of rBC and rLaSota V.F. viruses, the viruses were grown in primary chicken neuronal cells at two different temperatures. It was found that the rBC virus grew faster and induced a more rapid cytopathic effect at 41.5 °C (body temperature of chickens) than at 37 °C (Fig. 4A). To prove that the growth temperature does play a role in replication kinetics, we tested the growth of neurovirulent rBC virus by a multi-step growth curve in Vero cells at 37 and 41.5 °C. The results showed that the rBC virus replicated to higher titers at 41.5 °C than at 37 °C (Fig. 4B). The rBC virus also produced more cytopathic effects in chicken neuronal cells at 41.5 °C than at 37 °C, as evidenced by the destruction of the cell monolayers (Fig. 5A). Our results showed that chicken neuronal cells are more susceptible to rBC than Vero cells. At 41.5 °C, rBC in chicken neuronal cells grew to higher titers at 24 h post-infection. At 48 h and 56 h post-infection the rBC titer in chicken neuronal cells was lower at 41.5 °C, because most of the cells were destroyed at this temperature and at these time points. Hemadsorption (HAd) studies revealed higher HAd of rBC and rLaSota V.F. viruses at 41.5 °C than at 37 °C. HAd activity of rBC at 41.5 °C was 128% when compared to its activity at 37 °C (100% HAd activity), while rLaSota V.F. had a HAd activity of 14% at 37 °C and 70% at 41.5 °C. Virus-binding studies with purified biotinylated virus in a streptavidin-colloidal gold amplification system indicated that the binding of chicken neuronal cells by rBC was significantly higher than rLaSota V.F. (Fig. 5B).Fig. 4

(A) Multi-step growth curve of rBC in chicken neuronal cells at 37 °C and 41.5 °C. The virus was inoculated into chicken neuronal cells at a m.o.i. of 0.05 and maintained at 37 and 41.5 °C. The virus titers at 24, 48, and 72 h were determined by a plaque assay on DF 1 cells. (B) Multi-step growth curve of rBC in Vero cells at 37 and 41.5 °C. The viruses were inoculated into Vero cells at a m.o.i. of 0.05 and maintained at 37 and 41.5 °C. Supernatant was collected at 8 h interval until 56 h P.I. The virus titers were determined by a plaque assay on DF 1 cells.

The virulence of rBC, rLaSota, and rLaSota V.F. viruses was evaluated by intracerebral pathogenicity index (ICPI) tests in 1-day-old chicks, and by intravenous pathogenicity index (IVPI) tests in 6-week-old chickens (Table 1). The ICPI results showed that the modification of the F protein cleavage site resulted in a dramatic increase in virulence from ICPI value of 0.00 for rLaSota to ICPI value of 1.12 (out of a maximum possible value of 2.0) for rLaSota V.F. However, the virulence of rLaSota V.F. was lower than that of rBC virus (ICPI value of 1.58). Surprisingly, the results obtained by IVPI tests showed a different pattern. The rBC virus had an IVPI value of 1.45 (out of a maximum possible value of 3.00), but the IVPI values of rLaSota V.F. and rLaSota viruses were 0.00. These results demonstrated that the efficiency of cleavage of the F protein plays an important role if the NDV is delivered directly into the brains of chicks, but there could be other viral factors that probably affect peripheral replication, viremia, or entry into the central nervous system. The efficiency of attachment may, in part, determine the virulence, as indicated by our virus binding studies. To compare the pathogenesis of rBC, rLaSota and rLaSota V.F. viruses, 3-week-old chickens were infected intranasally with 10³ plaque forming units (PFU) of virus. The virus distribution in different tissues of the inoculated birds are presented in Fig. 6 . The rBC virus first appeared by day 3 in the blood, lungs, spleen and brain, and by day 5, the virus was undetectable in the blood. In the lungs, rBC grew to higher titers (500-fold) on day 5, but the titer was significantly reduced on day 7. It is possible that the humoral antibody IgA, which appears on day 5 post-infection and plays a major role in virus clearance in the respiratory tract, may be responsible for the decrease in virus titer seen on day 7. The rLaSota V.F. virus appeared by day 5 in the lungs and spleen, and in the blood by day 7, but was never detected in the brain. The rLaSota virus was detected only in the lungs by day 5. Sections of brain were also evaluated for the presence of viral RNA by in situ hybridization, using an antisense NP-specific ³⁵S-labeled riboprobe (Fig. 7A). We found that only rBC virus exhibited focal areas of viral infection in the brain by days 5 and 7. These areas were mostly found in the cerebellum. Immunohistochemical studies using a cocktail of monoclonal antibodies to the HN protein corroborated the results of hybridization (Fig. 7B). These results indicated that viral factors other than the cleavage efficiency of the F protein may determine the neurovirulence of NDV isolates, and that entry into the brain is an important factor.Fig. 6

Virus titers (PFU/g tissue at PFU/mL blood) in representative organs. Three-week-old chickens were infected intranasally with rBC, rLaSota or rLaSota V.F. virus (10³PFU/chicken). Chickens were sacrificed at 1, 3, 5 and 7 days post-infection, tissues were removed, homogenized and titrated by plaque assay.

DF1 (a chicken embryo fibroblast cell line) cells were maintained in DMEM, and Vero and HEp2 cells were maintained in EMEM media. Chicken neuronal cells were prepared from 10-day-old specific pathogen free (SPF) embryonated chicken eggs. Cytosine arabinoside (Ara C) was added to the medium to inhibit the growth of non-neuronal cells [32]. The identity of the neuronal cells was confirmed by immunostaining with an antibody to human neurofilament 200 kDa (Research Diagnostic, Inc. NJ).

NDV strains LaSota and BC were received from the National Veterinary Services Laboratory, Ames, IA. Each virus was propagated in the allantoic cavity of embryonated chicken eggs and the virus was purified from the allantoic fluid. Briefly, the allantoic fluid was harvested two days post-infection and clarified by low-speed centrifugation at 1800g for 30 min. Virus was pelleted by ultracentrifugation at 35,000g for 18 h and the pelleted virus was resuspended in 4 ml sterile PBS. The virus was then layered on top of a discontinuous sucrose gradient made with 3 ml of 55% and 5 ml of 20% sucrose in PBS, after which the gradient was ultracentrifuged at 43,000g for 1 h. The virus band at the interface of the 20 and 55% sucrose gradients was collected and pelleted at 35,000g for 18 h. The virus pellet was resuspended in 500 μl of PBS and stored at 4 °C.

Construction of the full-length antigenomic cDNAs of NDV strains LaSota and BC, and recovery of the recombinant viruses are described previously [12], [33]. In this study, the full-length antigenomic cDNAs of LaSota and BC were designated as pLaSota and pBC, respectively. The F protein cleavage site of lentogenic and avirulent NDV strain LaSota was altered and made identical to the F protein cleavage site of mesogenic and neurovirulent NDV strain BC. The amino acid sequence G-R-Q-G-R present at the cleavage site of the F protein of avirulent NDV strain LaSota was altered to R-R-Q-K-R, to be analogous to the cleavage site of the F protein of neurovirulent strain BC (Fig. 1). This was done by subcloning the Sac II-Not I fragment from pLaSota into a plasmid vector, pGEM-7Z (+) (Promega, Madison, WI). This subclone was designated Sac II-Not I pLaSota 7Z. The F cleavage site of pLasota was then made identical to that of pBC by site-directed mutagenesis [34], with phosphorylated forward primer (5′-⁴⁸⁸⁶aaaCGCtTTATAGGCGCCATTATTGGCG⁴⁹¹³-3′) and phosphorylated reverse primer (5′-⁴⁸⁸⁵CTGTCTCCtCCCTCCAGATGTAGT⁴⁸⁶²-3′, lower case representing mutation), on Sac II-Not I pLaSota 7Z. The mutated fragment was digested with Sac II and Not I restriction enzymes and inserted back into pLaSota. The resultant mutated pLaSota was called pLaSota V.F. To confirm the presence of the desired mutations at the F cleavage site and the absence of any undesired mutations in pLaSota V.F., it was sequenced to its entirety. Using reverse genetics procedures [11], [33], the recombinant virus rLaSota V.F. was recovered from its cDNA clone, pLaSota V.F.

The recovered recombinant viruses, rLaSota, rBC and rLaSota V.F., were grown in the allantoic cavity of 9-day-old SPF embryonated chicken eggs. After five passages, the allantoic fluid was harvested and clarified, and the virus was purified as described above. Viral RNA was extracted from the recovered viruses using TRIzol (Invitrogen, USA), according to the manufacturer's instructions. Reverse transcription was done with the extracted RNA, using the Thermoscript RT kit (Invitrogen, USA) to synthesize the first strand cDNA. RT-PCR was done to amplify the region that covered the F protein cleavage site and the resulting PCR product was sequenced to confirm the presence of the mutations made at the F cleavage site of rLaSota V.F. Similarly, the rLaSota V.F. virus was re-isolated from the brains of 1-day-old chicks and from 3-week-old chickens, and was subjected to RT-PCR and sequencing analysis to confirm the presence of the introduced mutations.

The growth kinetics of rBC, rLaSota and rLaSota V.F. were performed by multi-step growth curves in DF1 cells. The viruses were inoculated to confluent monolayers of DF1 cells at a m.o.i. of 0.05. The medium of cells infected with rLaSota contained 1 μg/mL of acetyl trypsin. Supernatant was collected at 8 h intervals until 56 h post infection (P.I.) The virus titer in the cell culture supernatant was assessed by plaque assay on DF1 cells.

To observe the growth of virus at different temperatures, the viruses were inoculated into chicken neuronal cells and Vero cells at a m.o.i. of 0.05, and maintained at 37 and 41.5 °C. The virus titers at 24, 48, and 72 h were determined by a plaque assay on DF 1 cells.

The ability of NDV to bind chicken neuronal cells was determined using biotinylated virus. Briefly, purified NDV was labeled with biotin by using a protein biotinylation kit (Amersham). The virus pellet was resuspended in biotinylation buffer (40 mM Na₂CO3, pH 8.6) at a protein concentration of 1 mg/mL. After brief sonication, 40 μL biotin reagent was added per mg of NDV protein. The mixture was shaken for 1 h at 4 °C and the reaction was terminated by addition of Tris–HCl (pH 8.5) to a final concentration of 50 mM. Biotinylated virus was collected after purification on a Sephadex G-25 column and diluted in PBS at a concentration of 0.2 mg/mL. Biotinylated virions were stored at 4 °C.

Direct virus binding studies were carried out with biotinylated virus. The chicken neuronal cells were washed three times with cold PBS containing 0.2% BSA (PBSA) and incubated over ice for 10 min. Different concentrations of biotinylated NDV in PBSA were mixed with 2×10⁵ cells per well and incubated on ice for 60 min. The cells were then washed three times with cold PBSA and incubated on ice with 1:10 diluted streptavidin-colloidal gold conjugate (CytImmune Sciences, MD, USA) for 60 min. The cells were washed once and fixed with 1% formaldehyde in PBS at room temperature for 1 min. The cells were then washed once with PBS plus 2% fetal calf serum. The mean number of colloidal gold particles bound to each cell was then counted using a bright field microscope. Specificity of binding was demonstrated in a competition experiment, in which 2×10⁵ chicken neuronal cells were incubated with different amounts of unlabeled NDV before performing the virus binding assay as described above.

Virus was inoculated to confluent monolayers of Vero cells in six-well plates at a m.o.i. of 10. After 18–24 h P.I., the media was decanted and the cells were overlaid with guinea pig RBCs in PBS at a concentration of 1×10⁸ cells/ml. The plates were kept at 4 °C for 15 min. The unbound RBCs were removed by washing twice with PBS. The RBCs bound to the virus-infected cells were lysed with 0.05 M ammonium chloride and the released hemoglobin was measured at 549 nm in a spectrophotometer.

To test the pathogenicity of the recovered viruses in vivo, ICPI and IVPI tests were performed in accord with standard procedures [2]. For ICPI, 0.05 ml of 1:10 dilution of fresh infective allantoic fluid of each virus was inoculated into groups of 10 1-day-old SPF chicks via the intracerebral (i.c.) route. The inoculation was done using a 27-guage needle, attached to a 1 ml stepper syringe dispenser that was set to dispense 0.05 ml of inoculum per inoculation. The birds were inoculated by inserting the needle up to the hub into the right or left rear quadrant of the cranium. The birds were observed for clinical symptoms and mortality once in every 12 h for a period of 8 days. For IVPI, 0.1 ml 1:10 dilution of fresh infective allantoic fluid of each virus was inoculated into groups of 10 6-week old SPF chickens intranasally (i.n.). We modified the route of inoculation of the virus from intravenous (which is the typical route of inoculation for the IVPI test) to intranasal in order to follow the natural route of infection of NDV in the birds. The birds were observed for clinical symptoms and mortality once every 12 h for a period of 10 days. Equal numbers of chickens were used in each experiment. Each experiment had mock-inoculated controls that received a similar volume of sterile PBS by the respective routes. The ICPI and IVPI values were calculated as described by Alexander [2] (Table 1).Table 1

Pathogenicity of rBC, rLaSota, and rLaSota V.F. viruses in vivo

To study the growth kinetics and tissue distribution in vivo, groups of 1-day-old and 3-week-old SPF chickens were inoculated with 10³ PFU of virus/chicken by the i.c. and i.n. routes, respectively. Brains from the 1-day-old chicks were collected daily for 7 days. Tissues, such as brain, spleen and lung were collected from inoculated 3-week-old chickens on days 1, 3, 5 and 7 P.I. and snap frozen immediately. Blood was also collected from the birds on these same days and assayed for viremia. The tissues were homogenized, and the virus titers in tissues and blood were estimated by plaque assay on DF1 cells. To determine the sites of localization of these recombinant viruses in the brain, one-half of the brains collected from the 3-week-old chickens were embedded in paraffin and sectioned at 5 μm/section. These sections were subject to either an immunohistochemical assay [35] using a cocktail of NDV monoclonal antibodies [36] or an in situ hybridization assay [35] employing a ³⁵S-labeled riboprobe specific for NP gene of NDV.