Neurovirulent Vaccine-Derived Polioviruses in Sewage from Highly Immune Populations

BACKGROUND: Vaccine-derived polioviruses (VDPVs) have caused poliomyelitis outbreaks in communities with sub-optimal vaccination. Israeli environmental surveillance of sewage from populations with high (>95%) documented vaccine coverage of confirmed efficacy identified two separate evolutionary clusters of VDPVs: Group 1 (1998–2005, one system, population 1.6×10(6)) and Group 2 (2006, 2 systems, populations 0.7×10(6) and 5×10(4)). PRINCIPAL FINDINGS: Molecular analyses support evolution of nine Group 1 VDPVs along five different lineages, starting from a common ancestral type 2 vaccine-derived Sabin-2/Sabin-1 recombinant strain, and independent evolution of three Group 2 VDPVs along one lineage starting from a different recombinant strain. The primary evidence for two independent origins was based on comparison of unique recombination fingerprints, the number and distribution of identical substitutions, and evolutionary rates. Geometric mean titers of neutralizing antibodies against Group 1 VDPVs were significantly lower than against vaccine strains in all age-group cohorts tested. All individuals had neutralizing titers >1∶8 against these VDPVs except 7% of the 20–50 year cohort. Group 1 VDPVs were highly neurovirulent in a transgenic mouse model. Intermediate levels of protective immunity against Group 2 VDPVs correlated with fewer (5.0+1.0) amino acid substitutions in neutralizing antigenic sites than in Group 1 VDPV's (12.1±1.5). SIGNIFICANCE: VDPVs that revert from live oral attenuated vaccines and reacquire characteristics of wild-type polioviruses not only threaten populations with poor immune coverage, but are also a potential source for re-introduction of poliomyelitis into highly immune populations through older individuals with waning immunity. The presence of two independently evolved groups of VDPVs in Israel and the growing number of reports of environmental VDPV elsewhere make it imperative to determine the global frequency of environmental VDPV. Our study underscores the importance of the environmental surveillance and the need to reconsider the global strategies for polio eradication and the proposed cessation of vaccination

Surveillance and Diagnosis of West Nile Virus in the Face of Flavivirus Cross-Reactivity

West Nile Virus (WNV) is an arthropod-borne flavivirus whose zoonotic cycle includes both mosquitoes and birds as amplifiers and humans and horses as dead-end hosts. In recent years WNV has been spreading globally and is currently endemic in Africa, The Middle East, India, Australia, central and southern Europe, and the Americas. Integrated surveillance schemes and environmental data aim to detect viral circulation and reduce the risk of infection for the human population emphasizing the critical role for One Health principles in public health. Approximately 20% of WNV infected patients develop West Nile Fever while in less than 1%, infection results in West Nile Neurological Disease. Currently, the diagnosis of WNV infection is primarily based on serology, since molecular identification of WNV RNA is unreliable due to the short viremia. The recent emergence of Zika virus epidemic in America and Asia has added another layer of complexity to WNV diagnosis due to significant cross-reactivity between several members of the Flaviviridae family such as Zika, dengue, Usutu, and West Nile viruses. Diagnosis is especially challenging in persons living in regions with flavivirus co-circulation as well as in travelers from WNV endemic countries traveling to Zika or dengue infected areas or vise-versa. Here, we review the recent studies implementing WNV surveillance of mosquitoes and birds within the One Health initiative. Furthermore, we discuss the utility of novel molecular methods, alongside traditional molecular and serological methods, in WNV diagnosis and epidemiological research

Antiviral Activity of 3(2H)- and 6-Chloro-3(2H)-Isoflavenes against Highly Diverged, Neurovirulent Vaccine-Derived, Type2 Poliovirus Sewage Isolates

BACKGROUND: Substituted flavanoids interfere with uncoating of Enteroviruses including Sabin-2 polio vaccine strains. However flavanoid resistant and dependent, type-2 polio vaccine strains (minimally-diverged), emerged during in vitro infections. Between 1998-2009, highly-diverged (8 to >15%) type-2, aVDPV(2)s, from two unrelated persistent infections were periodically isolated from Israeli sewage. AIM: To determine whether highly evolved aVDPV(2)s derived from persistent infections retained sensitivity to isoflavenes. METHODS: Sabin-2 and ten aVDPV(2) isolates from two independent Israeli sources were titered on HEp2C cells in the presence and absence of 3(2H)- Isoflavene and 6-chloro-3(2H)-Isoflavene. Neurovirulence of nine aVDPV(2)s was measured in PVR-Tg-21 transgenic mice. Differences were related to unique amino acid substitutions within capsid proteins. PRINCIPAL FINDINGS: The presence of either flavanoid inhibited viral titers of Sabin-2 and nine of ten aVDPV(2)s by one to two log(10). The tenth aVDPV(2), which had unique amino acid substitution distant from the isoflavene-binding pocket but clustered at the three- and five-fold axies of symmetry between capsomeres, was unaffected by both flavanoids. Genotypic neurovirulence attenuation sites in the 5'UTR and VP1 reverted in all aVDPV(2)s and all reacquired a full neurovirulent phenotype except one with amino acid substitutions flanking the VP1 site. CONCLUSION: Both isoflavenes worked equally well against Sabin 2 and most of the highly-diverged, Israeli, aVDPV(2)s isolates. Thus, functionality of the hydrophobic pocket may be unaffected by selective pressures exerted during persistent poliovirus infections. Amino acid substitutions at sites remote from the drug-binding pocket and adjacent to a neurovirulence attenuation site may influence flavanoid antiviral activity, and neurovirulence, respectively

Evolution of echovirus 11 in a chronically infected immunodeficient patient.

Deep sequencing was used to determine complete nucleotide sequences of echovirus 11 (EV11) strains isolated from a chronically infected patient with CVID as well as from cases of acute enterovirus infection. Phylogenetic analysis showed that EV11 strains that circulated in Israel in 1980-90s could be divided into four clades. EV11 strains isolated from a chronically infected individual belonged to one of the four clades and over a period of 4 years accumulated mutations at a relatively constant rate. Extrapolation of mutations accumulation curve into the past suggested that the individual was infected with circulating EV11 in the first half of 1990s. Genomic regions coding for individual viral proteins did not appear to be under strong selective pressure except for protease 3C that was remarkably conserved. This may suggest its important role in maintaining persistent infection

Genomic variation and epidemiology of SARS-CoV-2 importation and early circulation in Israel.

Severe acute respiratory disease coronavirus 2 (SARS-CoV-2) which causes corona virus disease (COVID-19) was first identified in Wuhan, China in December 2019 and has since led to a global pandemic. Importations of SARS-CoV-2 to Israel in late February from multiple countries initiated a rapid outbreak across the country. In this study, SARS-CoV-2 whole genomes were sequenced from 59 imported samples with a recorded country of importation and 101 early circulating samples in February to mid-March 2020 and analyzed to infer clades and mutational patterns with additional sequences identified Israel available in public databases. Recorded importations in February to mid-March, mostly from Europe, led to multiple transmissions in all districts in Israel. Although all SARS-CoV-2 defined clades were imported, clade 20C became the dominating clade in the circulating samples. Identification of novel, frequently altered mutated positions correlating with clade-defining positions provide data for surveillance of this evolving pandemic and spread of specific clades of this virus. SARS-CoV-2 continues to spread and mutate in Israel and across the globe. With economy and travel resuming, surveillance of clades and accumulating mutations is crucial for understanding its evolution and spread patterns and may aid in decision making concerning public health issues

Comparison of the new fully automated extraction platform eMAG to the MagNA PURE 96 and the well-established easyMAG for detection of common human respiratory viruses.

Respiratory viral infections constitute the majority of samples tested in the clinical virology laboratory during the winter season, and are mainly diagnosed using molecular assays, namely real-time PCR (qPCR). Therefore, a high-quality extraction process is critical for successful, reliable and sensitive qPCR results. Here we aimed to evaluate the performance of the newly launched eMAG compared to the fully automated MagNA PURE 96 (Roche, Germany) and to the semi-automated easyMAG (bioMerieux, France) extraction platforms. For this analysis, we assessed and compared the analytic and clinical performance of the three platforms, using 262 archived respiratory samples positive or negative to common viruses regularly examined in our laboratory (influenza A, B, H1N1pdm, Respiratory Syncytial Virus (RSV), human Metapneumovirus (hMPV), parainfluenza-3, adenovirus and negative samples). In addition, quantitated virus controls were used to determine the limit of detection of each extraction method. In all categories tested, eMAG results were comparable to those of the easyMAG and MagNa PURE 96, highly sensitive for all viruses and over 98% clinical specificity and sensitivity for all viruses tested. Together with its high level of automation, the bioMerieux eMAG is a high-quality extraction platform enabling effective molecular analysis and is mostly suitable for medium-sized laboratories

Numbers of RNA samples with different levels of inhibition of MS2 rRT-PCR by extraction protocol.

a<p>Equal amounts of stool suspensions chosen randomly from among samples sent to the laboratory from patients with acute gastroenteritis were extracted by four protocols: (A) QIAgen, (B) magNA Pure, (C) KingFisher, and (D) easyMag as described in Methods.</p>b<p>Bonferroni correction sets the significance level to <i>P</i><0.008.</p

MS2 rRT-PCR inhibition compared with enterovirus rRT-PCR inhibition for enterovirus RNA added after extraction.

<p>1. Two different concentrations of CoxB3 RNA were added to the RNA from 23 stool suspensions after extraction by protocols: (A) QIAgen, (B) MagNA Pure, (C) KingFisher, and (D) easyMag. rRT-PCR values for MS2 RNA extracted from buffer controls or the enterovirus RNA spikes were subtracted from the values for samples from stool suspensions. Results in column R¹ were obtained when enteroviral RNA (Ct 29) was added to the extracted RNA and those in column R² were obtained when 128 fold (6 CT) more enteroviral RNA was added. The values for inhibition of MS2 rRT-PCR (column M) are the mean of three replicate tests where the RNA was stored at −70°C between each of the MS2 measurements. The individual repeated MS2 measurements are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039455#pone.0039455.s002" target="_blank">Figure S2</a>. An empty white box indicates that there was no inhibition. Negative values were set to 0 and the maximum values for inhibition “C” were capped at 29 Cts. Samples with inhibition ≥10, 6 to 9, 3 to 6, and 1 to 3 CT are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. 2. The mean inhibition of MS2 and enterovirus rRT-PCR results is listed for the above by protocol. Yellow boxes indicate the pairwise comparisons where the inhibition of enteroviral rRT-PCR and MS2 differed significantly by Repeated Measurements, Analysis of Variance.</p

MS2 rRT-PCR inhibition in RNA extracted from stool samples using four different RNA extraction protocols.

<p>Equal amounts of stool suspensions prescreened by Protocol A were extracted by four protocols: (A) QIAgen, (B) MagNA Pure, (C) KingFisher, and (D) easyMag as described in methods. MS2 coliphage calculated to give 27 Ct by rRT-PCR was added to the extraction buffer. rRT-PCR values for MS2 in RNA extracted from buffer controls were subtracted from the values for MS2 in RNA extracted from stool suspensions. These differences, the number of Cts of inhibition, are shown in the boxes to the right of the sample numbers. An empty white box indicates that there was no inhibition. Negative values were set to 0 and the maximum values for inhibition “C” were capped at 29 Cts. Samples with inhibition ≥10, 6 to 9, 3 to 6, and 1 to 3 CT are indicated by the colors of the boxes: black, red, tan, and light yellow, respectively. The number of samples in each category and statistical significance are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039455#pone-0039455-t001" target="_blank">Table 1</a>, Experiment 2.</p