Recombinant subtype A and B human respiratory syncytial virus clinical isolates co-infect the respiratory tract of cotton rats

Abstract

Human respiratory syncytial virus (HRSV) is an important respiratory pathogen causing a spectrum of illness, from common cold-like symptoms, to bronchiolitis and pneumonia requiring hospitalization in infants, the immunocompromised and the elderly. HRSV exists as two antigenic subtypes, A and B, which typically cycle biannually in separate seasons. There are many unresolved questions in HRSV biology regarding the interactions and interplay of the two subtypes. Therefore, we generated a reverse genetics system for a subtype A HRSV from the 2011 season (A11) to complement our existing subtype B reverse genetics system. We obtained the sequence (HRSVA11) directly from an unpassaged clinical sample and generated the recombinant (r) HRSVA11. A version of the virus expressing enhanced green fluorescent protein (EGFP) from an additional transcription unit in the fifth (5) position of the genome, rHRSVA11EGFP(5), was also generated. rHRSVA11 and rHRSVA11EGFP(5) grew comparably in cell culture. To facilitate animal co-infection studies, we derivatized our subtype B clinical isolate using reverse genetics toexpress the red fluorescent protein (dTom)-expressing rHRSVB05dTom(5). These viruses were then used to study simultaneous in vivo co-infection of the respiratory tract. Following intranasal infection, both rHRSVA11EGFP(5) and rHRSVB05dTom(5) infected cotton rats targeting the same cell populations and demonstrating that co-infection occurs in vivo. The implications of this finding on viral evolution are important since it shows that inter-subtype cooperativity and/or competition is feasible in vivo during the natural course of the infection.

Human Respiratory Syncytial Virus Subgroup A and B Infections in Nasal, Bronchial, Small-Airway, and Organoid-Derived Respiratory Cultures

“…In another study, rHRSV A11 expressing EGFP and rHRSV B05 expressing dTom were used in cotton rats and cotton rat cell lines. Occasional double-infected cells were observed, both in vitro and in vivo (27). Our data confirm these observations, with as added value that we use primary human epithelial cells.…”

“…rHRSV A2 [rHRSV A2 EGFP(5)] was commercially obtained from ViraTree (product no. R125), and previously rescued rHRSV A11 [rHRSV A11 EGFP(5), passage 5]; rHRSV B05 rHRSV B05 EGFP(5), passage 6; and rHRSV B05 dTom(5), passage 7 have been described before (the 5 between brackets refers to the position of the additional transcriptional unit in the genome) (27,28).…”

FeMV is a cathepsin-dependent unique morbillivirus infecting the kidneys of domestic cats

“…Once the FeMV US5 sequence was available from these amplicons, FeMV US5 -specific primers ( SI Appendix , Table S2 , U122 and US5 designations) were designed for cDNA synthesis, PCR, and sequencing. Rapid amplification of cDNA ends was used to generate amplicons containing leader and trailer sequences as previously described ( 79 ); these were sequenced to determine the authentic genomic termini of FeMV US5 . Sequences were aligned in DNAstar SeqMan Pro software (Lasergene), and contigs were generated corresponding to the consensus sequence.…”

Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants

“…The high-copy phagemid vector pBluescript has been previously reported to be a suitable vector for construction of fulllength cDNA clones for rhabdoviruses and paramyxoviruses (53)(54)(55) and also RSV strains belonging to the GA1 and BA genotypes (41,42). In contrast, we have found that use of pBluescript KS(+) as a vector backbone for full-length cDNA clones consistently results in full or partial deletions of the G gene of both RSV-A and -B strains.…”

“…Although these systems have proved invaluable in advancing our understanding of RSV molecular biology and pathogenesis, it is important to more rigorously examine well-documented RSV subtype and strain differences using panels of recombinant viruses based on currently circulating strains for which the consensus sequence of the parental virus of the patient has been determined. This approach has recently been used to construct reverse genetics systems for RSV-A and -B based on strains classified within the GA2 and BA genotypes, respectively, in which the pBluescript KS(+) phagemid vector was used as the vector backbone (41,42). The use of a high-copy vector backbone in these studies contrasts with earlier reverse genetics systems, and the extent to which this strategy can be generalized and applied to other RSV strains without concomitant instability of cloned viral sequences is unknown.…”