Parechovirus typing in clinical specimens by nested or semi-nested PCR coupled with sequencing

Nix, W. Allan; Maher, Kaija; Pallansch, Mark A.; Oberste, M. Steven

doi:10.1016/j.jcv.2010.04.007

Cited by 73 publications

(58 citation statements)

References 45 publications

(70 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Genus-specific, real-time PCR-positive specimens were confirmed by nested or seminested RT-PCR targeting a portion of the genome region encoding the VP1 capsid protein, followed by amplicon sequencing (8,12). Virus type identity was determined by comparison of the VP1 amplicon nucleotide and deduced amino acid sequences with the VP1 sequences of all the reference strains for each virus genus by script-driven sequential pairwise comparison using the program Gap (Wisconsin Sequence Analysis Package, version 11.0; Accelrys, Inc., San Diego, CA) as described previously (8).…”

Section: Methodsmentioning

confidence: 99%

Naturally Acquired Picornavirus Infections in Primates at the Dhaka Zoo

Oberste

Feeroz

Maher

et al. 2013

J Virol

Self Cite

View full text Add to dashboard Cite

Section: Methodsmentioning

confidence: 99%

Naturally Acquired Picornavirus Infections in Primates at the Dhaka Zoo

Oberste

Feeroz

Maher

et al. 2013

J Virol

Self Cite

View full text Add to dashboard Cite

“…Virus identifications of genus-specific real-time RT-PCR-positive specimens were determined by nested or seminested RT-PCR targeting a portion of the genome region encoding the VP1 capsid protein (Tables 3 and 4), followed by amplicon sequencing (32,36). An additional human PeV (HPeV) partial VP1 assay (does not amplify Ljungan virus species) was used on a small number of specimens that yielded an unreadable sequence with the complete VP1 assay (Tables 3 and 4).…”

Section: Studymentioning

confidence: 99%

“…An additional human PeV (HPeV) partial VP1 assay (does not amplify Ljungan virus species) was used on a small number of specimens that yielded an unreadable sequence with the complete VP1 assay (Tables 3 and 4). The cDNA reactions for these specimens were performed as described previously for the parechovirus complete VP1 assay (36). The PCR1 reaction differed slightly from the previously described parechovirus complete VP1 PCR1 assay in that the HPeV primers (AN486 and AN488) were used at 0.5 M in the final reaction volume of 50 l. Similarly, the HPeV PCR2 primers (AN268 and AN489) were used at 0.4 M in the final reaction volume of 50 l. Thermocycler profiles for the HPeV partial VP1 assay were as described for the EV partial VP1 assay (36).…”

Section: Studymentioning

confidence: 99%

“…The cDNA reactions for these specimens were performed as described previously for the parechovirus complete VP1 assay (36). The PCR1 reaction differed slightly from the previously described parechovirus complete VP1 PCR1 assay in that the HPeV primers (AN486 and AN488) were used at 0.5 M in the final reaction volume of 50 l. Similarly, the HPeV PCR2 primers (AN268 and AN489) were used at 0.4 M in the final reaction volume of 50 l. Thermocycler profiles for the HPeV partial VP1 assay were as described for the EV partial VP1 assay (36). Virus type identity was determined by comparison of the VP1 amplicon nucleotide and deduced amino acid sequences with the VP1 sequences of all the reference strains for each virus genus by script-driven sequential pairwise comparison using the program Gap (Wisconsin Sequence Analysis Package, version 11.0; Accelrys, Inc., San Diego, CA) as described previously (32).…”

Section: Studymentioning

confidence: 99%

See 1 more Smart Citation

Characterizing the Picornavirus Landscape among Synanthropic Nonhuman Primates in Bangladesh, 2007 to 2008

Oberste

Feeroz

Maher

et al. 2013

J Virol

Self Cite

View full text Add to dashboard Cite

The term synanthropic describes organisms that thrive in human-altered habitats. Where synanthropic nonhuman primates (NHP) share an ecological niche with humans, cross-species transmission of infectious agents can occur. In Bangladesh, synanthropic NHP are found in villages, densely populated cities, religious sites, and protected forest areas. NHP are also kept as performing monkeys and pets. To investigate possible transmission of enteric picornaviruses between humans and NHP, we collected fecal specimens from five NHP taxa at16 locations in Bangladesh during five field sessions, from January 2007 to June 2008. Specimens were screened using real-time PCR assays for the genera Enterovirus, Parechovirus, and Sapelovirus; PCR-positive samples were typed by VP1 sequencing. To compare picornavirus diversity between humans and NHP, the same assays were applied to 211 human stool specimens collected in Bangladesh in 2007 to 2008 for acute flaccid paralysis surveillance. Picornaviruses were detected in 78 of 677 (11.5%) NHP fecal samples. Twenty distinct human enterovirus (EV) serotypes, two bovine EV types, six human parechovirus serotypes, and one virus related to Ljungan virus were identified. Twenty-five additional enteroviruses and eight parechoviruses could not be typed. Comparison of the picornavirus serotypes detected in NHP specimens with those detected in human specimens revealed considerable overlap. Strikingly, no known simian enteroviruses were detected among these NHP populations. In conclusion, enteroviruses and parechoviruses may be transmitted between humans and synanthropic NHP in Bangladesh, but the directionality of transmission is unknown. These findings may have important implications for the health of both human and NHP populations.

show abstract

“…To determine the complete VP1 coding sequences, HPeVpositive specimens were amplified using the nested PCR assays described by Nix et al 12 The nucleotide sequences obtained were deposited in the EBI/DDBJ/GenBank databases under the accession numbers JQ229434-JQ229466. These sequences were aligned with those of prototype and clinical strains from GenBank, using the ClustalW program.…”

Section: Hpev Genotypingmentioning

confidence: 99%