David Safronetz

Zika viral dynamics and shedding in rhesus and cynomolgus macaques

Infection with Zika virus has been associated with serious neurological complications and fetal abnormalities. However, the dynamics of viral infection, replication and shedding are poorly understood. Here we show that both rhesus and cynomolgus macaques are highly susceptible to infection by lineages of Zika virus that are closely related to, or are currently circulating in, the Americas. After subcutaneous viral inoculation, viral RNA was detected in blood plasma as early as 1 d after infection. Viral RNA was also detected in saliva, urine, cerebrospinal fluid (CSF) and semen, but transiently in vaginal secretions. Although viral RNA during primary infection was cleared from blood plasma and urine within 10 d, viral RNA was detectable in saliva and seminal fluids until the end of the study, 3 weeks after the resolution of viremia in the blood. The control of primary Zika virus infection in the blood was correlated with rapid innate and adaptive immune responses. We also identified Zika RNA in tissues, including the brain and male and female reproductive tissues, during early and late stages of infection. Re-infection of six animals 45 d after primary infection with a heterologous strain resulted in complete protection, which suggests that primary Zika virus infection elicits protective immunity. Early invasion of Zika virus into the nervous system of healthy animals and the extent and duration of shedding in saliva and semen underscore possible concern for additional neurologic complications and nonarthropod-mediated transmission in humans.

Evaluation of Euroimmun Anti-Zika Virus IgM and IgG Enzyme-Linked Immunosorbent Assays for Zika Virus Serologic Testing

ABSTRACT With the emerging Zika virus (ZIKV) epidemic, serologic diagnosis relies on a labor-intensive IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and confirmation by a plaque reduction neutralization test (PRNT). To streamline serologic testing, several commercial assays have been developed. Our aim was to compare the commercial Euroimmun anti-ZIKV IgM and IgG assays to the reference MAC-ELISA and PRNT currently in use. Serum specimens submitted to Public Health Ontario Laboratory, Canada, were tested for IgM and IgG using the Euroimmun assays and the results were compared with those from MAC-ELISA. The PRNT was performed on positive or equivocal specimens using either MAC-ELISA or Euroimmun assays, MAC-ELISA-inconclusive specimens, and a convenience sample of specimens negative by both assays (cohort 1). Another set of specimens selected on the basis of PRNT results was subsequently tested by the Euroimmun assays (cohort 2). MAC-ELISA was positive, equivocal, negative, and inconclusive in 57/223, 15/223, 147/223, and 4/223 specimens, respectively. Among the 76 specimens that were MAC-ELISA positive, equivocal, or inconclusive, 30 (39.5%) were Euroimmun IgM and/or IgG positive or equivocal. Among the 147 MAC-ELISA-negative specimens, 136 (92.5%) were Euroimmun IgM and IgG negative. The sensitivity of the combined Euroimmun IgM/IgG against the PRNT was 83% (cohort 1) and 92% (cohort 2), whereas the specificity was 81% (cohort 1) and 65% (cohort 2). The combined Euroimmun IgM/IgG showed good specificity (92.5%) but suboptimal sensitivity (39.5%) compared with that of the MAC-ELISA. However, the sensitivity of the combined Euroimmun IgM/IgG against the PRNT was significantly higher (83 to 92%). More studies are needed before commercial assays are implemented for routine ZIKV serologic diagnosis.

Recently Emerged Swine Influenza A Virus (H2N3) Causes Severe Pneumonia in Cynomolgus Macaques

The triple reassortant H2N3 virus isolated from diseased pigs in the United States in 2006 is pathogenic for certain mammals without prior adaptation and transmits among swine and ferrets. Adaptation, in the H2 hemagglutinin derived from an avian virus, includes the ability to bind to the mammalian receptor, a significant prerequisite for infection of mammals, in particular humans, which poses a big concern for public health. Here we investigated the pathogenic potential of swine H2N3 in Cynomolgus macaques, a surrogate model for human influenza infection. In contrast to human H2N2 virus, which served as a control and largely caused mild pneumonia similar to seasonal influenza A viruses, the swine H2N3 virus was more pathogenic causing severe pneumonia in nonhuman primates. Both viruses replicated in the entire respiratory tract, but only swine H2N3 could be isolated from lung tissue on day 6 post infection. All animals cleared the infection whereas swine H2N3 infected macaques still presented with pathologic changes indicative of chronic pneumonia at day 14 post infection. Swine H2N3 virus was also detected to significantly higher titers in nasal and oral swabs indicating the potential for animal-to-animal transmission. Plasma levels of IL-6, IL-8, MCP-1 and IFNγ were significantly increased in swine H2N3 compared to human H2N2 infected animals supporting the previously published notion of increased IL-6 levels being a potential marker for severe influenza infections. In conclusion, the swine H2N3 virus represents a threat to humans with the potential for causing a larger outbreak in a non-immune or partially immune population. Furthermore, surveillance efforts in farmed pig populations need to become an integral part of any epidemic and pandemic influenza preparedness.

Evaluation of Altona Diagnostics RealStar Zika Virus Reverse Transcription-PCR Test Kit for Zika Virus PCR Testing

ABSTRACT With the emerging Zika virus (ZIKV) epidemic, accessible real-time reverse transcription-PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR test kit (Altona PCR) has been approved for emergency use authorization by the U.S. FDA. Our aim was to verify the Altona PCR by comparing it to the CDC-designed dual-target ZIKV rRT-PCR reference assay (reference PCR) and describe the demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada. A large set of clinical specimens was tested for ZIKV by the Altona PCR and the reference PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting the ZIKV NS5 gene. A total of 671 serum specimens were tested by the reference PCR: 58 (8.6%) were positive, 193 (28.8%) were equivocal, and 420 (62.6%) were negative. Ninety percent of the reference PCR-positive patients were tested in the first 5 days after symptom onset. The Altona PCR was performed on 284/671 specimens tested by the reference PCR. The Altona PCR was positive for 53/58 (91%) reference PCR-positive specimens and 16/193 (8%) reference PCR-equivocal specimens; the ZIKV NS5 PCR was positive for all 68 Altona PCR-positive specimens and negative for all 181 Altona PCR-negative specimens that underwent the NS5 PCR. The Altona PCR has very good sensitivity (91%) and specificity (97%) compared to the reference PCR. The Altona PCR can be used for ZIKV diagnostic testing and has less extensive verification requirements than a laboratory-developed test.

Neonatal pigs are susceptible to experimental Zika virus infection

Emerging Microbes & Infections (2017) 6, e6; doi:10.1038/emi.2016.133; published online 15 February 2017

Risk of travel-related cases of Zika virus infection is predicted by transmission intensity in outbreak-affected countries

BackgroundZika virus (ZIKV) infection is emerging globally, currently causing outbreaks in the Caribbean, and Central and South America, and putting travellers to affected countries at risk. Model-based estimates for the basic reproduction number (R0) of ZIKV in affected Caribbean and Central and South American countries, obtained from 2015 to 2016 human case surveillance data, were compared by logistic regression and Receiver-Operating Characteristic (ROC), with the prevalence of ZIKV-positive test results in Canadians who travelled to them.ResultsEstimates of R0 for each country were a good predictor of the ZIKV test result (ROC area under the curve = 0.83) and the odds of testing positive was 11-fold greater for travellers visiting countries with estimated R0 ≥ 2.76, compared to those visiting countries with R0 < 2.76.ConclusionsRisk to travellers varies widely amongst countries affected by ZIKV outbreaks. Estimates of R0 from surveillance data can assist in assessing levels of risk for travellers and may help improve travel advice. They may also allow better prediction of spread of ZIKV from affected countries by travellers.

Intramuscular Adeno-Associated Virus–Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice

The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

Human polyclonal antibodies produced in transchromosomal cattle prevent lethal Zika virus infection and testicular atrophy in mice

ABSTRACT Zika virus (ZIKV) is rapidly spreading throughout the Americas and is associated with significant fetal complications, most notably microcephaly. Treatment with polyclonal antibodies for pregnant women at risk of ZIKV‐related complications could be a safe alternative to vaccination. We found that large quantities of human polyclonal antibodies could be rapidly produced in transchromosomal bovines (TcB) and successfully used to protect mice from lethal infection. Additionally, antibody treatment eliminated ZIKV induced tissue damage in immunologically privileged sites such as the brain and testes and protected against testicular atrophy. These data indicate that rapid development and deployment of human polyclonal antibodies could be a viable countermeasure against ZIKV. Graphical abstract Figure. No Caption available. HighlightsTranschromosomal Bovine (TcB) were developed to produce human polyclonal antibody.Vaccinated TcB produce high‐titer neutralizing antibody to Zika virus.Human ZIKV‐TcB antibodies prevent lethal disease in two mouse models.ZIKV‐TcB antibodies prevent tissue damage and testicular atrophy in mice.Delayed treatment results in significant protection from lethal ZIKV infection.

Zika Virus Causes Persistent Infection in Porcine Conceptuses and may Impair Health in Offspring

Outcomes of Zika virus (ZIKV) infection in pregnant women vary from the birth of asymptomatic offspring to abnormal development and severe brain lesions in fetuses and infants. There are concerns that offspring affected in utero and born without apparent symptoms may develop mental illnesses. Therefore, animal models are important to test interventions against in utero infection and health sequelae in symptomatic and likely more widespread asymptomatic offspring. To partially reproduce in utero infection in humans, we directly inoculated selected porcine conceptuses with ZIKV. Inoculation resulted in rapid trans-fetal infections, persistent infection in conceptuses, molecular pathology in fetal brains, fetal antibody and type I interferon responses. Offspring infected in utero showed ZIKV in their fetal membranes collected after birth. Some in utero affected piglets were small, depressed, had undersized brains, and showed seizures. Some piglets showed potentially increased activity. Our data suggest that porcine model of persistent in utero ZIKV infection has a strong potential for translational research and can be used to test therapeutic interventions in vivo.

Evaluation of the Diasorin Liaison® XL Zika Capture IgM CMIA for Zika virus serological testing

Due to the increase of Zika virus (ZIKV) transmission throughout the world, many commercial kits have recently become available to aid in laboratory diagnosis of ZIKV infections in clinical samples. Here, we analyze the fully automated Liaison® XL Zika Capture immunoglobulin M (IgM) assay against the recommended IgM-capture enzyme-linked immunosorbent assay.