Towards dynamic monitoring of cell cultures using high throughput sequencing.

We used a combination of DOP-PCR with high throughput sequencing (HTS) to study infected cell cultures over time to assess the feasibility of using this technique to provide a read-out other than cytopathic effect in cell culture infectivity assays. Because DOP-PCR primers feature a short constant sequence at their 3' terminus, the procedure yields a reproducible representational library of products from a given PCR template, including viral nucleic acids. Using SV40- and MVM-infected cultures harvested at different times, we show that the number of viral matches among DOP-PCR products parallels the quantity of virus as shown by real-time PCR, and further show that HTS analysis of specific DOP-PCR products that increase in quantity over time could be used to identify the infecting virus with a sensitivity similar to that of typical cell-culture assays that rely on cytopathic effect.

Evaluation of cells and biological reagents for adventitious agents using degenerate primer PCR and massively parallel sequencing.

We employed a massively parallel sequencing (MPS)-based approach to test reagents and model cell substrates including Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), African green monkey kidney (Vero), and High Five insect cell lines for adventitious agents. RNA and DNA were extracted either directly from the samples or from viral capsid-enriched preparations, and then subjected to MPS-based non-specific virus detection with degenerate oligonucleotide primer (DOP) PCR. MPS by 454, Illumina MiSeq, and Illumina HiSeq was compared on independent samples.

Optimization of virus detection in cells using massively parallel sequencing.

Massively parallel sequencing (MPS)-based virus detection has potential regulatory applications. We studied the ability of one of these approaches, based on degenerate oligonucleotide primer (DOP)-polymerase chain reaction (PCR), to detect viral sequences in cell lines known to express viral genes or particles. DOP-PCR was highly sensitive for the detection of small quantities of isolated viral sequences.

Discovery of a bovine enterovirus in alpaca.

A cytopathic virus was isolated using Madin-Darby bovine kidney (MDBK) cells from lung tissue of alpaca that died of a severe respiratory infection. To identify the virus, the infected cell culture supernatant was enriched for virus particles and a generic, PCR-based method was used to amplify potential viral sequences. Genomic sequence data of the alpaca isolate was obtained and compared with sequences of known viruses.

Molecular and infectivity studies of porcine circovirus in vaccines.

This report describes FDA's laboratory response to the 2010 reports that porcine circovirus type 1 (PCV-1) DNA was present in U.S.-licensed rotavirus vaccines and in cells used to produce inactivated poliovirus vaccines.

Expression and self-assembly of virus-like particles from two genotypes of marine vesiviruses and development of an ELISA for the detection of antibodies.

Sequences encoding the major and minor capsid proteins (VP1 and VP2) from two marine vesivirus isolates (Steller sea lion viruses V810 and V1415) were engineered for expression of virus-like particles (VLPs) in the baculovirus system. The resulting VLPs were morphologically similar to native vesivirus virions. Purified VLPs were probed in immunoblots with pooled antisera specific for nine San Miguel sea lion virus (SMSV) types, and a predominant protein of approximately 60kDa was detected.

A capsid gene-based real-time reverse transcription polymerase chain reaction assay for the detection of marine vesiviruses in the Caliciviridae.

A real-time reverse transcription polymerase chain reaction (rtRT-PCR) assay was developed for the identification of marine vesiviruses. The primers were designed to target a 176-nucleotide fragment within a highly conserved region of the San Miguel sea lion viruses (SMSVs) capsid gene. The assay detected viral RNA from nine marine vesivirus serotypes described previously, including two serotypes (SMSV-8 and -12) not identified with presently available molecular assays, a highly related bovine vesivirus strain (Bos-1), a mink vesivirus strain (MCV), and two novel genotypes isolated recently from Steller sea lions (SSL V810 and V1415).

Genomic characterization of novel marine vesiviruses from Steller sea lions (Eumetopias jubatus) from Alaska.

Marine vesiviruses were isolated in cell culture from oral and rectal swabs and vesicular fluid from Alaskan Steller sea lions (SSL; Eumetopias jubatus). Further characterization by RT-PCR, complete genomic sequencing, and phylogenetic analyses indicated that these viruses are most closely related to the marine vesiviruses, but are distinct viruses and represent two novel genotypes. The complete genome of these two SSL isolates was sequenced after cloning their viral cDNA.