Instituto de Medicina Tropical “Pedro Kourí”

Evidence for nonpoliovirus enterovirus multiplication in L20B cells

Lic. Luis Sarmiento Pérez,¹ Dr. Pedro Más Lago,² Téc. Rosa Palomera Puentes,³ Lic. Luis Morier Díaz,⁴ Lic. Magile Fonseca Quintana⁵ y Dra. Sonia Resik Aguirre⁶

Summary

Stool specimens collected from 1 515 healthy children following a mass vaccination campaign in Cuba were tested for poliovirus excretion using L20B cell lines. In spite of the selectivity of this cell line for polioviruses (117/129; 90.7 %) some other nonpolio enteroviruses (12/129; 9.3 %), such as coxsackie A virus types 4, 8 and 10, can grow in L20B cells.

Key words: Poliovirus, L20B cell line, coxsackievirus, eradication.

Although recent developments in molecular detection technology make it probable that enterovirus diagnosis/surveillance will increasingly be achieved by non-culture-based methods, culture of poliovirus from clinical samples is the gold-standard method for virological surveillance in the worldwide initiative to eradicate wild type poliovirus.¹

Since 1999, the WHO Global Polio Laboratory Network has been using L20B cell lines for the isolation of poliovirus for acute flaccid paralysis surveillance. L20B is a transgenic mouse cell line that expresses the human poliovirus receptor on the surface (CD155). The expression of the receptor at the cell surface renders L20B cells susceptible to infection with poliovirus. Furthermore, the CD155 receptor is not shared with other enteroviruses, thus making L20B cells highly specific for poliovirus isolation.^2,3

Previous studies suggested that L20B cells were highly selective for poliovirus detection, reporting that only coxsackievirus B4 and reovirus type 2, among the viruses other than poliovirus, produced late cytopathic effect (CPE) in this cell line.^4-6 More recently, studies from India reported that the L20B cell line is susceptible to productive infection by group A coxsackieviruses.⁷

Methods

Samples and virus isolation

Stool specimens were collected from 1 515 apparently healthy children following a mass vaccination campaign in Havana, Cuba . The samples were tested for poliovirus excretion using RD (passages 230 to 240) and L20B (passages 25 to 35) cells. Both cell lines were obtained from Dr. DJ Wood, NIBSC, Potters Bar, United Kingdom . Standard procedures described in the laboratory manual for the WHO Global Polio Laboratory Network were used for the propagation of cell lines and for virus culture.¹⁰ All virus isolates from RD cells were passaged in L20B cells. For samples showing CPE in L20B cells, poliovirus serotypes were identified by neutralization with poliovirus typing antisera.¹⁰ Viruses other than poliovirus were tested in reverse transcription-polymerase chain reaction (RT-PCR) assays using pan-enterovirus and pan-poliovirus primers.^11,12 Enterovirus isolates, i.e., pan-enterovirus RT-PCR-positive but pan-poliovirus RT-PCR-negative isolates, were taken for virus neutralization tests using Lim-Benyesh-Melnick serum pools A to H for the identification of enterovirus.^10,13

Molecular typing of enterovirus isolates

To identify the serotypes of the untypeable enterovirus-like viruses by a method other than the neutralization assay, the partial VP1 sequences of the untypeable strains was determined by the use of primer pairs 012 and 011 or 040 and 011 as described by Oberste et al.^14,15 The sequences were BLAST searched for closely similar sequences in the GenBank using online computer program (www.ncbi.nlm.nih.gov/BLAST/). A serotype was identified when sequence similarity of more than 75 % was found with a known enterovirus sequence in the GenBank.¹⁴ Amino acid sequence homology of more than 88% was used to identify the serotypes.¹⁴

Phylogenetic analysis

A phylogenetic tree based on the partial VP1 nucleotide sequence was constructed by the neighbor-joining method.¹⁶ The reliability of the neighbor-joining tree was estimated by bootstrap analysis with 1 000 pseudoreplicate data sets.

Remicroneutralization tests

According to results of enterovirus phylogenetic analysis, remicroneutralization tests using in house monospecific rabbit antiserum were performed to confirm the serotype of the untypeable strains.

Results

On primary inoculation of RD cells, a substantial proportion (512/1 515; 33.8 %) of isolates was obtained. After the passages onto L20B cells, a total of 129 isolates was obtained. Of these 117/129 (90.7 %) were identified as poliovirus by the neutralization test with poliovirus typing antisera. The remaining 12 isolates were not neutralized by poliovirus antisera. None of the 12 isolates was amplified by using RT-PCR with pan-poliovirus primers. However, all of these isolates gave a positive RT-PCR with pan-enterovirus primers and were therefore grouped as nonpolio enteroviruses (NPEV).

The identity of the 12 NPEV isolates could not be determined by using Lim-Benyesh-Melnick serum pools A to H. The nucleotide sequences of all untypeable isolates were 78 to 86 % identical to the sequences of their respective prototype strains, as identified by a BLAST search. The predicted partial VP1 amino acid sequences of all untypeable isolates were more than 95 % identical to that of the homologous prototype strain (table).

A phylogenetic tree based on the partial VP1 nucleotide sequences of the 12 enterovirus-like untypeable strains and 40 prototype human enterovirus strains available from the GenBank database was constructed. The untypeable strains were classified nearest to coxsackie A virus types 4, 8 and 10 (fig.).

PV: poliovirus species; HEV-A: human enterovirus A species; HEV-B: human enterovirus B species; HEV-C: human enterovirus species; HEV-D: human enterovirus species.

Fig. Phylogenetic relationships among 12 untypeable strains and 40 prototype human enterovirus strains, based on partial VP1 gene sequences available from the GenBank database.

The results obtained with the remicroneutralization tests using in-house monospecific rabbit antiserum against reference strains of coxsackie A virus, types 4, 8 and 10 agreed with the results obtained with partial VP1 nucleotide sequences (table).

Table. Correspondence between typing by sequence comparison and by neutralization

* Primary isolate in L20B cells; nt: nucleotide; aa: amino acid.

Discussion

The results of our studies show that L20B cells were highly sensitive and selective for the growth of polioviruses. Of special interest were the isolations of coxsackie A viruses in this cell line. Most of these isolations (10 of 12) were obtained after the passage from RD to L20B cells, indicating that a high multiplicity of infection is required. It is noteworthy that only two of the nonpolio strains produced CPE primarily in L20B cells. This may be due to the high initial titer of virus in these samples (data not shown).

Newborn mice were the preferred animal hosts for propagation of group A coxsackieviruses from clinical specimens for many years.¹⁷ As L20B cells are of murine origin, the growth of coxsackie A viruses on L20B cells is not surprising because of the ability of these viruses to use receptors on mouse cells.

L20B cells are not absolutely specific for poliovirus; however, the small number of NPEV that grow in L20B cells does not appear to affect the ability of the WHO Global Polio Laboratory Network to detect poliovirus. NPEV that can grow in L20B cells would not be expected to interfere with the detection of poliovirus. This assumption is based on the fact that the majority of coxsackie A virus isolated in L20B cells required a RD cells passage for initial amplification. L20B cells therefore are more sensitive for poliovirus than for coxsackie A virus.

The selectivity of L20B cells for poliovirus was confirmed by the failure of the vast majority of NPEV to grow in L20B cells. In fact, the use of this line simplified the work by reducing the number of isolates to be identified and considerably shortened the time required for reporting the results. These results consistently emphasize the importance of L20B as a powerful tool for the WHO Global Polio Laboratory Network. Nevertheless, it is worth noting that this cell line supports the growth of others NPEV, such as those that belong to coxsackievirus group A.

Evidencia para multiplicación de enterovirus nopoliovirus en células L20B

Resumen

Se estudió la excreción de poliovirus en muestras de heces fecales de 1 515 niños saludables, colectados después de la campaña de vacunación en Cuba con el empleo de células L20B. A pesar de la selectividad de esta línea celular para poliovirus (117/129; 90,7 %), algunos enterovirus nopoliovirus (12/129; 9,3 %), como los coxsackievirus A tipos 4, 8 y 10, pueden crecer en células L20B.

Palabras clave: Poliovirus, línea celular L20B, coxsackievirus, erradicación.

References

1. WHO. Manual for the virologic investigation of poliomyelitis. Geneva: World Health Organization; 2002.

2. Koike S, Itoshi H, Ise I, Okitsu A, Yoshida M, Iizuka N, et at. The poliovirus receptor is produced both as membrane-bound and secreted forms. EMBO J 1990;9:3217-24.

3. Mendelsohn C, Johnson B, Lionetti KA, Nobis S, Wimmer E, Racaniello V. Transformation of a human poliovirus receptor gene into mouse cells. Proc Natl Acad Sci USA 1986;83:7845-9.

4. Hovi T, Stenvik M. Selective isolation of poliovirus in recombination murine cell line expressing the human poliovirus receptor gene. J Clin Microbiol 1994;32:1366-8.

5. Pipkin PA, Wood DJ, Racaniello VR, Minor PD. Characterization of L cells expressing the human poliovirus receptor for the specific detection of poliovirus. J Virol Methods 1993;41:333-40.

6. Wood DJ, Hull B. L20B cells simplify culture of polioviruses from clinical samples. J Med Virol 1999;58:188-92.

7. Nadkarni SS, Deshpande JM. Recombinant murine L20B cell line supports multiplication of group A coxsackieviruses. J Med Virol 2003;70:81-5.

8. Más P, Caceres V, Galindo M, Gary H, Valcarcel M, Barrios J, et al. Persistence of vaccine-derived poliovirus following a mass vaccination campaign in Cuba: Implication for stopping polio vaccination after global eradication. Inter J Epidemiol 2001;30:1029-34.

9. Más P, Gary H, Sarmiento L, Caceres V, Barrios J, Palomera R, et al. Poliovirus detection in wastewater and stools following an immunization campaign in Havana. Inter J Epidemiol 2003;32:772-7.

10. World Health Organization. Polio laboratory manual. Department of Vaccines and Biologicals. Geneva:World Health Organization; 2001.

11. Yang CF, De L, Holloway BP, Pallansch MA, Kew OM. Detection and identification of vaccine-related polioviruses by polymerase chain reaction. Virus Res 1991;20:159-79.

12. Kilpatrick DR, Nottay B, Yang CF, Yang SJ, Mulders MN, Holloway BP, et al. Group-specific identification of polioviruses by PCR using primers containing mixed-based or deoxyinosine residues at positions of codon degeneracy. J Clin Microbiol 1996;34:2990-6.

13. Melnick JL, Rennick V, Hampil B. Lyophilized combination pools of enterovirus equine antisera: Preparation and test procedures for the identification of field strains of 42 enteroviruses. Bull World Health Organ 1973;48:263-8.

14. Oberste MS, Maher K, Pallansch MA. Molecular phylogeny of all human enterovirus serotypes based on comparison of sequences at the 5' end of the region encoding VP2. Virus Res 1998;58:35-43.

15. Oberste MS, Maher K, Kilpatrick DR, Flemister MR, Brown BA, Pallansch MA. Typing of human enteroviruses by partial sequencing of VP1. J Clin Microbiol 1999;37:1288-93.

16. Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-25.

17. Melnick JL, Wenner HA, Rosen L. The enteroviruses. En: Lennette EH, Schmidt NJ, eds. Diagnostic procedures for viral and rickettsial diseases. New York: American Public Health Association; 1964. p.194-245.

Recibido: 10 de mayo de 2007. Aprobado: 23 de mayo de 2007.
Lic. Luis Sarmiento Pérez. Instituto de Medicina Tropical “Pedro Kourí “Autopista Novia del Mediodía Km 6 ½, municipio La Lisa. Ciudad de La Habana, Cuba. Correo electrónico: sarmiento@ipk.sld.cu

¹ Máster en Virología. Licenciado en Microbiología. Investigador Agregado.
² Doctor en Ciencias Médicas. Especialista de II Grado en Microbiología. Investigador de Mérito.
³ Técnico en Microbiología.
⁴ Licenciado en Microbiología. Investigador Auxiliar.
⁵ Máster en Virología. Licenciado en Microbiología.
⁶ Máster en Virología. Especialista de II Grado en Microbiología. Investigador Auxiliar.