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During the COVID-19 pandemic, SARS-CoV-2 surveillance efforts integrated genome sequencing of clinical samples to identify emergent viral variants and to support rapid experimental examination of genome-informed vaccine and therapeutic designs. Given the broad range of methods applied to generate new viral genomes, it is critical that consensus and variant calling tools yield consistent results across disparate pipelines. Here we examine the impact of sequencing technologies (Illumina and Oxford Nanopore) and 7 different downstream bioinformatic protocols on SARS-CoV-2 variant calling as part of the NIH Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) Tracking Resistance and Coronavirus Evolution (TRACE) initiative, a public-private partnership established to address the COVID-19 outbreak. Our results indicate that bioinformatic workflows can yield consensus genomes with different single nucleotide polymorphisms, insertions, and/or deletions even when using the same raw sequence input datasets. We introduce the use of a specific suite of parameters and protocols that greatly improves the agreement among pipelines developed by diverse organizations. Such consistency among bioinformatic pipelines is fundamental to SARS-CoV-2 and future pathogen surveillance efforts. The application of analysis standards is necessary to more accurately document phylogenomic trends and support data-driven public health responses.

Introduction Zika virus disease received little attention until its recent explosive emergence around the globe. The devastating consequences of this pandemic include congenital Zika syndrome (CZS) and the neurological autoimmune disorder Guillain-Barre syndrome. These potential outcomes prompted massive efforts to understand the course of Zika infection and to develop therapeutic and prophylactic strategies for treatment and prevention of disease. Area covered Preclinical and clinical data demonstrate that a safe and efficacious vaccine for protection against Zika virus infection is possible in the near future. Nevertheless, significant knowledge gaps regarding the outcome of a mass vaccination strategy exist and must be addressed. Zika virus circulates in flavivirus-endemic regions, an ideal Zika vaccine should avoid the potential of antibody-dependent enhancement from exposure to dengue virus. Prevention of CZS is the primary goal for immunization, and the vaccine must provide protection against intrauterine transmission for use during pregnancy and in women of childbearing age. Ideally, a vaccine should also prevent sexual transmission of the virus through mucosal protection. Expert opinion This review describes current vaccine approaches against Zika virus with particular attention to the application of virus-like particle (VLP) technology as a strategy for solving the challenges of Zika virus immunization.

An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.

As SARS-CoV-2 variants emerge, there is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines. A reference reagent comprised of post-vaccination sera from recipients of different vaccines allows evaluation of in vitro variant neutralization, and provides a reference for comparing assay results across laboratories. We prepared and pooled >1 L serum from donors who received the SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), a replication-incompetent adenovirus type 26 vaccine (Ad26.COV2.S, Johnson and Johnson), or recombinant spike protein expressed by baculovirus incorporated into a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). Twice frozen sera were aliquoted and are available for distribution to the research community (BEI Resources). The calculated WHO titer of pooled sera to spike protein was 1,312, 1,447, 1,936, and 587 and the reciprocal RBD binding to ACE-2 IC90-titers were 60, 64, 118, and 46 for BNT162b2, mRNA1273, Ad26.CoV2373, and NVX-CoV2373 sera, respectively.

Powassan virus (POWV) is a tick-borne flavivirus circulating in North America and the Russian Far East that can cause severe neuroinvasive diseases, including encephalitis, meningitis, and meningoencephalitis. The reported neuroinvasive case fatality is about 10%, and approximately 50% of the survivors from the neuroinfection exhibit long-lasting or permanent neurological sequelae. Currently, treatment of POWV infection is supportive, and no FDA-approved vaccines or specific therapeutics are available. A novel Powassan vaccine candidate was created using virus-like particle technology (POW-VLP) and assembled with the viral structural proteins pre-Membrane (prM) and Envelope (E). Western blot immunoassay demonstrated high antigenicity of POW-VLP structural proteins. Transmission electron microscopy indicated that the POW-VLP exhibited icosahedral morphology typical of flaviviruses. A dose-escalation study in a murine model was performed to test immunogenicity and safety. Serum antibody was tested by ELISA, demonstrating that POW-VLP afforded 100% seroconversion to the E protein. Reporter viral-particle neutralization assay demonstrated high levels of neutralizing antibodies in the serum of immunized mice. Hybridomas expressing monoclonal antibodies were produced following POW-VLP immunization. The POW-VLP vaccine candidate created in this study provides a strategy for inducing protective antibodies against Powassan neuroinvasive infection.

The function of the SARS-CoV-2 accessory protein p6, encoded by ORF6, is not fully known. Based upon its similarity to p6 from SARS-CoV, it may play a similar role, namely as an antagonist of type I interferon (IFN) signaling. Here we report the sequencing of a SARS-CoV-2 strain passaged six times after original isolation from a clinical patient in Hong Kong. The genome sequence shows a 27 nt in-frame deletion (Δ27,264-27,290) within ORF6, predicted to result in a 9 aa deletion (ΔFKVSIWNLD) from the central portion of p6. This deletion is predicted to result in a dramatic alteration in the three-dimensional structure of the resultant protein (p6Δ22-30), possibly with significant functional implications. Analysis of the original clinical sample indicates that the deletion was not present, while sequencing of subsequent passages of the strain identifies the deletion as a majority variant. This suggests that the deletion originated ab initio during passaging and subsequently propagated into the majority, possibly due to the removal of selective pressure through the IFN-deficient Vero E6 cell line. The specific function of the SARS-CoV-2 p6 N-terminus, if any, has not yet been determined. However, this deletion is predicted to cause a shift from N-endo to N-ecto in the transmembrane localization of the SARS-CoV-2 p6Δ22-30 N-terminus, possibly leading to the ablation of its native function.

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with Congenital Zika Syndrome (CZS), reflecting a wide range of congenital abnormalities in fetuses and infants infected with ZIKV before birth. ZIKV infections have also been associated with the neurological autoimmune disorder known as Guillian-Barre syndrome (GBS). To date, no vaccines or antiviral strategies are licensed for ZIKV. We used rational design to develop a novel ZIKV vaccine candidate using a Woodchuck Hepatitis core Antigen (WHcAg) Virus-Like Particle (VLP) scaffold for displaying selected antigens from the ZIKV Envelope (E) protein. A Zika-VLP vaccine candidate containing the CD Loop sub-structural domain from ZIKV E protein Domain III (WHcAg CD Loop) elicited a strong immune response in a murine model. Analysis of serum immunoglobulins demonstrated induction of both Th1- and Th2- mediated immune response. No cross-reacting antibodies were detected between Zika, dengue and yellow fever virus, demonstrating a high level of specificity for the ZIKV CD Loop antigen. Immunization with the WHcAg CD Loop vaccine candidate demonstrated immunoprotection in a murine model of ZIKV infection, stimulating protective antibodies associated with antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. The WHcAg CD Loop candidate may represent a safer vaccine for preventing antibody dependent enhancement (ADE).

The recent emergence of Zika virus (ZIKV) has been concentrated in the Caribbean, Southeastern United States, and South- and Central America; resulting in travel-based cases being reported around the globe. As multi-disciplinary collaborations are combatting the ZIKV outbreak, the need to validate the sequence of existing strains has become apparent. Here, we report high-quality sequence data for multiple ZIKV strains made publicly available through the National Institutes of Health- (NIH) funded biorepository, BEI Resources (www.beiresources.org). Next-generation sequencing, 3′ rapid amplification of cDNA ends (RACE), and viral genome annotation pipelines generated GenBank sequence records for 16 BEI Resources strains. Minor variants, consensus mutations, and consensus insertions/deletions were identified within the viral stocks using next-generation sequencing (NGS) and consensus changes were confirmed with Sanger sequencing. Bioinformatics analyses of the sequencing results confirm that the virus stocks available to the scientific research community through BEI Resources adequately represent the viral population diversity of ZIKV.

We report here the complete genome of a Dezidougou virus (DEZV) isolated from a passaged culture of the Zika virus strain DAK AR 41524. The consensus DEZV sequence we recovered shows 99% nucleotide similarity using BLASTN to a previously reported DEZV (accession no. JQ675604.1). The current sequence has additional repeat regions as well as a deleted repeat region, which we confirmed by Sanger sequencing, that were not present in the originally published sequence, JQ675604.1.