Recovery of Infectious Foot-and-Mouth Disease Virus from Suckling Mice after Direct Inoculation with In Vitro-Transcribed RNA

Abstract

We assayed the infectivity of naked foot-and-mouth disease virus (FMDV) RNA by direct inoculation of suckling mice. Our results demonstrate that transcripts generated from full-length cDNA clones were infectious, as was virion-extracted RNA. Interestingly, infectious virus could be recovered from a mutant transcript encoding amino acid substitution L-1473P in capsid protein VP1, known to be noninfectious for BHK-21 cells. The model described here provides a useful tool for virulence studies in vivo, bypassing possible selection of variants during viral replication in cell culture.Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious and economically important disease affecting wild and domestic cloven-hoofed animals (22,43,44). The virus belongs to the family Picornaviridae and consists of nonenveloped particles containing a positive-sense single-stranded RNA genome of about 8.5 kb. A unique open reading frame encodes all of the capsid and nonstructural viral proteins. FMDV virion RNA as well as full-length RNA transcripts derived from infectious cDNA clones have been widely reported to be infectious when transfected into susceptible cell lines (36,51).Because of the high mutation rates operating during genome replication, FMDV populations are genetically heterogeneous and exhibit an important potential for variation and adaptation (10, 11). Antigenic diversity of the FMDV populations has been widely described and still represents an important obstacle to disease control (10, 28). Other remarkable manifestations of the population dynamics of FMDV include modifications of receptor usage and host tropism (3,17,25), as well as the presence of a molecular memory that reflects the evolutionary history of the virus (9, 37).In FMDV virions, the Arg-Gly-Asp (RGD) triplet located at the G-H loop of capsid protein VP1 (1, 23) is essential for the interaction with RGD-dependent integrins (6, 13, 18-20, 26, 29, 34) and with neutralizing antibodies (12,15,27,48,49). The RGD triplet is highly conserved among natural isolates of FMDV (11, 27), probably reflecting constraints imposed by the interaction with integrin receptors in vivo (13, 31). However, RGD can become dispensable upon large-population passages of FMDV in cell culture, which are associated with amino acid substitutions at the capsid surface and the use of alternative receptors for cell entry (3,4,16,24,38,50).Recent evidence suggests that changes in receptor specificity may also occur during FMDV replication in vivo. Unusual amino acid replacements affecting the RGD motif (R-1413G) or positions ϩ1 and ϩ4 relative to RGD (L-1443P and L-1473P), known to be critical for FMDV binding to some RGD-dependent integrins (29, 34), have been reported in vivo, in viruses escaping an immune response to synthetic peptides (45,46), and in the process of adaptation of FMDV to guinea pigs (33). Moreover, engineered viruses based on a Chinese strain with a KGE sequence instead of RGD were able to cause disease in pigs (50).The structural constraints i...

Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity

“…RNA from pMT28 was used as a control. An amount of 100 ng RNA from pMT28 killed 4 of the 5 animals injected, a lethality value similar to that reported for other FMDV RNAs derived from full-length infectious clones (7). RNA from pMT28-Q44R was also lethal for suckling mice, as 100 ng of RNA killed 3 out of 4 injected animals, and sequencing of the 3A coding region of RNA extracted from dead mice showed the presence of replacement Q44R, confirming the capacity of the virus carrying this mutation to infect suckling mice (47).…”

“…The infectivity of the FMDV 3A mutants in suckling mice was tested as described previously (7). Briefly, in vitro-tran- …”

Ribavirin-Resistant Variants of Foot-and-Mouth Disease Virus: the Effect of Restricted Quasispecies Diversity on Viral Virulence

“…The animal experiments were approved by the Animal Ethics Committee of the Harbin Veterinary Research Institute, CAAS, China. Three-day-old BALB/c suckling mice were assigned to 18 groups (6 to 9 mice per group) and were inoculated cervicodorsally with 100 l of diluted virus (0.01 to 10 TCID 50 ) in a 10-fold dilution series, as previously described (37,38). The percent survival of the animals was recorded every 12 h until 7 days after inoculation.…”

Guinea Pig-Adapted Foot-and-Mouth Disease Virus with Altered Receptor Recognition Can Productively Infect a Natural Host

“…In no case were any signs of viral contamination observed. Plaque assays and virus titration in suckling mice were performed as described previously (7,29).…”