Circulating vaccine-derived polioviruses in the Extreme North region of Cameroon

BackgroundThe World Health Organization (WHO) poliovirus eradication program includes careful surveillance of acute-flaccid paralysis (AFP) and mass and routine immunization with oral polio vaccine (OPV). In populations with low vaccine coverage, the live-attenuated Sabin strains, OPV types 1, 2 and 3, can evolve into virulent vaccine-derived polioviruses (VDPVs) and circulate in the community. Until recently, circulating VDPVs (cVDPVs) had not been reported in Cameroon despite the fact that VDPV2 outbreaks have occurred in nearby countries.ObjectivesThis study aimed to characterize virus isolates from four AFP patients infected with cVDPV2 in the Extreme North region of Cameroon in 2013.Study designThe complete VP1 region of the four VDPV strains was sequenced and the relationships with cVDPVs from neighboring countries were investigated.ResultsAll four patients were infected by cVDPV2 strains showing 1.2–2.0% nucleotide difference compared to the reference Sabin 2 VP1 sequence. Phylogenetic analysis indicated that the VDPV strains were genetically linked to cVDPV2 lineages of the recent Chad cVDPV2 outbreak.ConclusionsThe circulation of pathogenic VDPVs suggests that there are localized immunization gaps in some districts like Makary, Mada and Kolofata in Cameroon. To avoid poliomyelitis outbreaks in Cameroon, especially in the districts close to neighboring countries with ongoing cVDPV outbreaks, high polio vaccine coverage is essential.     

Isolation of recombinant type 2 vaccine-derived poliovirus (VDPV) from a Nigerian child

A type 2 vaccine-derived poliovirus (VDPV), differing from Sabin 2 at 2.5% (22/903) of VP1 nucleotide (nt) positions, was isolated from an incompletely immunized 21-month-old Nigerian child who developed acute flaccid paralysis in 2002. Sequences upstream of nt position 620 (within the 5'-untranslated region [5'-UTR]) and downstream of nt position 5840 (in the 3C(pro) region) were derived from species C enteroviruses unrelated to the oral poliovirus vaccine (OPV) strains. The two substitutions associated with the attenuated phenotype had either recombined out (A(481)-->G in the 5'-UTR) or reverted (Ile(143)-->Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype of Sabin 2 and it was antigenically distinct from the parental OPV strain, having amino acid substitutions in or near neutralizing antigenic sites 1 and 3. The date of the initiating OPV dose, calculated from the number of synonymous substitutions in the capsid region, was estimated to be approximately 16 to 18 months before onset of paralysis, a finding inconsistent with the most recent mass OPV campaign (conducted 12 days before onset of paralysis) as being the source of infection. Although no related type 2 VDPVs were detected in Nigeria or elsewhere, the VDPV was found in an area where conditions favor VDPV emergence and spread.     

Circulation of a type 1 recombinant vaccine-derived poliovirus strain in a limited area in Romania

Summary After intensive immunisation campaigns with the oral polio vaccine (OPV) as part of the Global Polio Eradication Initiative, poliomyelitis due to wild viruses has disappeared from most parts of the world, including Europe. Here, we report the characterization of a serotype 1 vaccine-derived poliovirus (VDPV) isolated from one acute flaccid paralysis (AFP) case with tetraplegia and eight healthy contacts belonging to the same small socio-cultural group having a low vaccine coverage living in a small town in Romania. The genomes of the isolated strains appeared to be tripartite type 1/type 2/type 1 vaccine intertypic recombinant genomes derived from a common ancestor strain. The presence of 1.2% nucleotide substitutions in the VP1 capsid protein coding region of most of the strains indicated a circulation time of about 14 months. These VDPVs were thermoresistant and, in transgenic mice expressing the human poliovirus receptor, appeared to have lost the attenuated phenotype. These results suggest that small populations with low vaccine coverage living in globally well-vaccinated countries can be the origin of VDPV emergence and circulation. These results reaffirm the importance of active surveillance for acute flaccid paralysis and poliovirus in both polio-free and polio-endemic countries.     

Characterization of persistent poliovirus mutants selected in human neuroblastoma cells

Six Sabin-derived persistent poliovirus mutants were selected in human neuroblastoma IMR-32 cells. The mutants had a titer 30 to 10⁵ times lower in nonneural HEp-2c cells than in IMR-32 cells. When the growth cycles of persistent viruses in the two cell lines were compared, the most striking feature was a delay of 2 to 4 hr in virus release from HEp-2c cells. In HEp-2c cells, type 1 mutants could spontaneously establish a persistent infection in the absence of any exogenous viral inhibitor. Mutations at a rate of 1 every 210 nucleotides had accumulated in the genome of the type 1 mutants selected in neuroblastoma cells, modifying cell specificity and conferring the ability to persist in some non-neural cells. These results indicate that mutants of poliovirus with highly modified biological properties can be selected in vitro in cells of neural origin.     

Clearing Vaccine-Derived Poliovirus Infection Following Hematopoietic Stem Cell Transplantation: a Case Report and Review of Literature

The use of oral poliovirus vaccine in a worldwide scale has led to a 99.9% decrease in annual incidence of wild-type poliomyelitis and the eradication of serotype 2 poliovirus. However, the emergence of vaccine-derived polioviruses (VDPVs) is endangering the eradication program. Patients with combined immunodeficiencies are at increased risk of both vaccine-associated poliomyelitis and prolonged asymptomatic infection with immunodeficiency-associated VDPVs (iVDPVs). Herein, we present a severe combined immunodeficiency patient with prolonged and asymptomatic iVDPV infection. He continued to shed poliovirus during immunoglobulin replacement therapy and cleared the infection following successful hematopoietic stem cell transplantation (HSCT). To explain the efficiency of HSCT in clearing the infection, we reviewed the literature for all reports of HSCT in iVDPV-excreting patients and discussed novel ideas about the role of different immune mechanisms, including cell-mediated interactions, in mounting immune responses against poliovirus infections. This study could provide further insights into the immune mechanisms contributing to the clearance of enteroviral infections.     

Influence of cellular functions on the evolution of persistent infections with Junin virus

Summary Vero cell cultures persistently infected with Junin virus and subjected to different cultural conditions were established. The production of infectious plaque-forming virus,ts mutants and interfering viral particles was determined at different times during 110 days after infection. Carrier cultures maintained in stationary conditions continuously released PFU while proliferating persistent cultures exhibited a cyclical pattern which tends to a rapid PFU disappearance. Concomitantly, in stationary cultures the production of interfering particles was delayed and was lower than in actively growing persistent cells. The metabolic state of the infected cells did not affect the release ofts mutants. The results suggest that a cellular function is involved on the regulation of Junin virus persistent infections.With 2 FiguresMember of Research Career from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).     

Potential of bovine herpesvirus 4 as a gene delivery vector

A cloning system was developed for construction of BHV-4 recombinants and recombinant virus BHV-4EGFPDeltaTK containing an enhanced green fluorescent protein (EGFP) gene was constructed. The host range of BHV-4EGFPDeltaTK was characterized in vitro. When cell lines from various species and tissues were infected, most of the non-bovine cell lines exhibited neither cytopathic effect (CPE) nor supported viral replication, but EGFP expression was clearly observed. Next, embryonic stem cells were infected and induced to either non-specific or neural differentiation to determine whether they could survive and differentiate after BHV-4EGFPDeltaTK infection. Embryonic stem cells were infected successfully, as indicated by EGFP expression prior to differentiation, and EGFP expression could be detected in many differentiated cells. No CPE was noted. Therefore, BHV-4EGFPDeltaTK infection caused neither cell death nor interfered with non-specific or neural differentiation of embryonic stem cells. Finally, to assess the capability of BHV-4EGFPDeltaTK to infect post-mitotic neurons, cultures from brains of 2-weeks old mice were infected. No death of neuronal cells due to infection was observed and EGFP expression persisted for at least 15 days. Several biological characteristics of BHV-4 demonstrated previously make it a good candidate for a gene delivery vector. These include: little or no pathogenicity, unlikely oncogenicity, ability to establish persistent infection, and capability of herpesviruses to accommodate large amounts of foreign genetic material. These findings add the ability to infect several cell types coming from different animal species, usually without CPE, lack of interference with differentiation, and ability to maintain transgene expression in both undifferentiated and differentiated cells.     

Mosquito cells accommodate balanced, persistent co-infections with a densovirus and Dengue virus

To study persistent viral co-infections in arthropods, we first produced stable, persistently infected C6/36 mosquito cell cultures by serial passage of exponentially growing whole cells infected with either a densovirus (AalDNV) or Dengue virus (DEN-2). We then obtained stable, persistent co-infections by reciprocal super-challenge and similar passaging. Persistently infected cultures did not differ from naïve-cell cultures in growth rate and cell morphology. Nor did they differ in high production of both viruses with high infection rates for naïve C6/36 cells. Immunocytochemistry revealed that 99–100% of the cells were coinfected but that super-infection order had some effect on antigen distribution for the two viruses. Our results combined with existing field information and previously published experimental work suggest that the capacity to support stable, viral co-infections may be a general phenomenon for arthropod cells, and that they may be achieved easily and rapidly by serial passaging of whole cultured cells. Such persistent infections would facilitate studies on interactions between co-infecting viruses.     

Inactivated polio vaccine: Time to introduce it in India's national immunization schedule

Polio is a communicable disease caused by poliovirus that may attack nerve cells of the brain and spinal cord. The victims develop neurological complications, likes stiffness of the neck, muscular weakness, or paralysis of one or more limbs. In severe cases, it may be fatal due to respiratory paralysis. The world has seen tremendous gains in polio eradication over the past year. India and Nigeria saw a reduction in cases of almost 95% from 2009 to 2010, and cases of wild poliovirus type 3 (WPV3) fell by 92% globally over the same period. In fact, no case has been reported in India since February 2011, such that India may be on the verge of eradicating polio. Nevertheless, polio control experts are particularly worried about Vaccine-Derived Poliovirus (VDPV). Global surveillance efforts picked up 430 cases of VDPV from several countries between July 2009 and March 2011. In India, 7 cases of VDPV were reported during the year 2011. As long as OPV is used, virologists say that the world is at risk of VDPV causing polio in unprotected children. Achieving a polio-free world will require the "cessation of all OPV" and with it the elimination of the risk of vaccine-associated paralytic polio (VAPP) or VDPV infections. To this effect, in 2011 the Global Polio Eradication Initiative (GPEI) will produce and develop a new roadmap for VDPV Elimination. Several countries have shifted from all OPV to sequential OPV-IPV schedules and all-IPV schedules with elimination of live poliovirus. IPV will be indispensable in the post-eradication era when use of OPV has to stop but "vaccination against polio" cannot stop. IPV offers complete individual protection and has been considered as an additional tool at present for those who can afford the vaccine, and since we are nearing the eradication of polio, it is time to shift from OPV to sequential OPV-IPV schedule in India. Such a strategy will avoid inevitable problems with VAPP.     

Infectivity of a strain of Cryptosporidium found in the guinea-pig (Cavia porcellus) for guinea-pigs, mice and lambs

Cryptosporidiosis was diagnosed in guinea-pigs bred by a commercial laboratory supplier on histological examination of the intestine. Oral transmission to laboratory guinea-pigs aged up to 16 weeks and to infant mice, with gut contents containing oocysts, was successful, but the organism failed to infect adult mice. From day 5 post-inoculation (pi) in guinea-pigs, infection of the ileum was associated with villous stunting and fusion, and with infiltrates of macrophages and other mononuclear cells, and eosinophils. Some guinea-pigs died; others were depressed and anorectic, with diarrhoea or watery caecal contents. Mouse infections were subclinical and caused no significant pathological changes. By contrast, a bovine Cryptosporidium isolate infected infant mice but failed to infect young guinea-pigs. Guinea-pigs and infant mice excreted oocysts in faeces after a prepatent period of 3 to 4 days. Some guinea-pigs excreted oocysts for up to 2 weeks, but excretion in mice lasted only about 4 days. Infection of guinea-pigs by contact with a contaminated environment occurred, with excretion of oocysts between 17 and 27 days after exposure. An indirect fluorescent antibody test (IFA) showed that antibody was present by day 17 pi with infected bowel contents, but none was detected in the guinea-pigs exposed to the contaminated environment. The IFA test demonstrated a serological relationship between the guinea-pig isolate and a bovine strain used to infect gnotobiotic lambs. Transmission electron microscopy of intestine from infected guinea-pigs and mice showed that more than one schizont generation occurred. The first consisted of 8 merozoite packets attached to enterocytes, but many packets of 2 or 4 merozoites of the second or subsequent generations were apparently released into the gut lumen. Fixed microgametocytes contained lipid vacuoles and had microneme-like structures in their cytoplasm. Oocysts and sporocysts were also identified, with sporulation occurring within the parasitiphorous vacuole. A sparse infection was established in 1 of 2 12-day-old specific pathogen-free lambs by day 3 pi, but no oocysts were detected in its caecal contents or those of a second lamb killed 4 days later.     

Ultrastructural phenotype of "intestinal-type" cells in columnar-lined esophagus

An intestinal-type epithelium is often present at columnar-lined esophagus, gastroesophageal junction or within the so-called short segment Barrett's esophagus, but ultrastructural study failed to detect enterocytes in columnar-lined esophagus. The authors have analyzed the intestinal aspects present in areas of columnar-lined esophagus in a population of patients with reflux esophagitis to better understand the morphology and histogenesis of the proliferating elements. Columnar-lined mucosa was studied in 35 patients. Columnar surface cells displayed a wide spectrum of ultrastructural features. Well-differentiated columnar secretory cells, secretory-absorptive cells, poorly differentiated columnar cells, atypical columnar cells, and goblet cells were detected. Well-differentiated absorptive cells were never found. These results demonstrate that the areas of intestinal metaplasia show a wide spectrum of ultrastructural phenotypes, ranging from poorly to well-differentiated cells. However, true enterocytes were not found and the most represented phenotype is that of secretory-absorptive cells, whose principal characteristic is the presence of secretory and absorptive aspects together. They can be described as secretory enterocytes or cells with double specialization. To the authors' knowledge, similar cells were not previously described in normal intestinal mucosa, and ultrastructural studies are consistent in describing a broad spectrum of ultrastructural features, suggesting that Barrett's specialized metaplasia is derived from cells with the capacity for a wide range of differentiation. Therefore, despite the wide use of term intestinal metaplasia in the medical literature, experimental data clearly failed to detect enterocytes in the columnar-lined esophagus, and ultrastructural data do not support the concept of intestinal metaplasia. The cellular heterogeneity seems to be the result of a "phenotypic shift" of undifferentiated elements, which show a different pattern of evolution. The result of this process is the formation of new cell types dissimilar from those normally present in esophageal, gastric, or duodenal mucosa.     

Ultrastructural Phenotype of "Intestinal-Type" Cells in Columnar-Lined Esophagus

An intestinal-type epithelium is often present at columnar-lined esophagus, gastroesophageal junction or within the so-called short segment Barrett's esophagus, but ultrastructural study failed to detect enterocytes in columnar-lined esophagus. The authors have analyzed the intestinal aspects present in areas of columnar-lined esophagus in a population of patients with reflux esophagitis to better understand the morphology and histogenesis of the proliferating elements. Columnar-lined mucosa was studied in 35 patients. Columnar surface cells displayed a wide spectrum of ultrastructural features. Well-differentiated columnar secretory cells, secretory-absorptive cells, poorly differentiated columnar cells, atypical columnar cells, and goblet cells were detected. Well-differentiated absorptive cells were never found. These results demonstrate that the areas of intestinal metaplasia show a wide spectrum of ultrastructural phenotypes, ranging from poorly to well-differentiated cells. However, true enterocytes were not found and the most represented phenotype is that of secretory-absorptive cells, whose principal characteristic is the presence of secretory and absorptive aspects together. They can be described as secretory enterocytes or cells with double specialization. To the authors' knowledge, similar cells were not previously described in normal intestinal mucosa, and ultrastructural studies are consistent in describing a broad spectrum of ultrastructural features, suggesting that Barrett's specialized metaplasia is derived from cells with the capacity for a wide range of differentiation. Therefore, despite the wide use of term intestinal metaplasia in the medical literature, experimental data clearly failed to detect enterocytes in the columnar-lined esophagus, and ultrastructural data do not support the concept of intestinal metaplasia. The cellular heterogeneity seems to be the result of a "phenotypic shift" of undifferentiated elements, which show a different pattern of evolution. The result of this process is the formation of new cell types dissimilar from those normally present in esophageal, gastric, or duodenal mucosa.     

Ultrastructural study of viral replication in human fetal intestinal organ culture

Summary Human fetal intestinal organ cultures were employed to study the growth patterns of various viruses and resultant cytoarchitectural changes using electron microscopic techniques. Representative strains of adenovirus, adenovirusassociated virus, herpes simplex virus, poliovirus, and echovirus were chosen for the study. The intracellular localization of these viruses was demonstrated, and the occurrence and rate of cellular destruction were observed. The difficulty of visualizing smaller viruses represented was emphasized. The use of these techniques to detect certain fastidious viruses of the gastrointestinal tract was suggested, since human fetal intestinal organ culture provides differentiated cell types which may be important for the initiation and support of viral replication.     

Enterocytes: active cells in tolerance to food and microbial antigens in the gut

Enterocytes used to be studied particularly in terms of digestion protagonists. However, as the immune functions of the intestinal tract were better understood, it became clear that enterocytes are not mere bystanders concerning the induction of immune tolerance to dietary peptides and gut microbiota. In fact, enterocytes are involved actively in shaping the intestinal immune environment, designed for maintaining a non-belligerent state. This tolerant milieu of the gut immune system is achieved by keeping a balance between suppression and stimulation of the inflammatory responses. Our review presents the current state of knowledge concerning the relationship between enterocytes and immune cells (dendritic cells, lymphocytes), with emphasis on the enterocytes' impact on the mechanisms leading to the induction of oral tolerance.     

Gamma-Interferon-Mediated Down-Regulation of Electrolyte Secretion by Intestinal Epithelial Cells: A Local Immune Mechanism?

Active chloride (Cl-) secretion by intestinal crypt enterocytes is the central pathophysiological disturbance in most cases of acute diarrhoea. We examined monolayers of the human intestinal cell line T84 mounted in Ussing chambers to see whether the T-cell lymphokine gamma interferon (IFN-gamma) might affect the Cl- secretory properties of these cells, which morphologically and functionally resemble native crypt enterocytes. Pretreatment of T84 cell layers with IFN-gamma for 24 h (but not for 3 h) markedly decreased the Cl- secretory response to vaso-active intestinal polypeptide (VIP) and to cholera toxin and carbachol without appreciably affecting the overall morphology, electrical resistance, or cyclic AMP response of the T84 cell monolayer. The IFN-gamma treatment, however, did induce subtle changes in the T84 cell membrane protein composition which might have affected ion channels regulating Cl- secretion. Our results may indicate a possible novel 'cell-mediated' immune mechanism through which activated gut T cells could modulate the extent of intestinal electrolyte and fluid secretion in, for example, enteric infections.     

Type 1 fimbriae of Salmonella enterica serovar Typhimurium bind to enterocytes and contribute to colonization of swine in vivo

Salmonella enterica serovar Typhimurium strain 798 is a clinical isolate from a pig and is known to be able to cause persistent, asymptomatic infections. This strain also is known to exist in two phenotypes (adhesive and nonadhesive to enterocytes) and can switch between the two phenotypes at a rate consistent with phase variation. Cells in the adhesive phenotype are more readily phagocytosed by leukocytes than nonadhesive cells. Once in a leukocyte, adhesive-phase cells survive while nonadhesive-phase cells die. In the present study, nonadhesive mutants were obtained with the transposon TnphoA. A nonadhesive mutant was selected for study and was shown by electron microscopy not to produce fimbriae. The gene encoding the adhesin was cloned and sequenced. Based on its sequence, the adhesin was shown to be FimA, the major subunit of type 1 fimbriae. The nonadhesive mutant was attenuated in its ability to colonize both mouse and pig intestines, but remained capable of systemic spread in mice. The nonadhesive mutant was phagocytosed to the same extent as parental cells in the adhesive phase and then survived intracellularly. These results demonstrated that type 1 fimbriae were important for attachment to enterocytes and promoted intestinal colonization. However, they were not important in promoting phagocytosis or intracellular survival.     

Heparin-binding EGF-like growth factor protects intestinal stem cells from injury in a rat model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is an often catastrophic disease that typically affects premature newborns. Although the exact etiology of NEC is uncertain, the disease is associated with formula feeding, bacterial colonization of the gut, hypoxia and hypoperfusion. In light of the pathogenesis of NEC, the integrity and function of the intestinal mucosa has a major defensive role against the initiation of NEC. Various forms of intestinal injury, including NEC, injure the intestinal epithelial cell (IEC) lineages, including the intestinal stem cells (ISCs), thereby disrupting the normal homeostasis needed to maintain gut barrier function. In the current study, we examined the effects of heparin-binding EGF-like growth factor (HB-EGF) administration on enterocytes, goblet cells, neuroendocrine cells and ISCs in a newborn rat model of experimental NEC. We also examined the cytoprotective effects of HB-EGF on ISCs in in vitro cell cultures and in ex vivo crypt-villous organoid cultures. We found that HB-EGF protects all IEC lineages, including ISCs, from injury. We further found that HB-EGF protects isolated ISCs from hypoxic injury in vitro, and promotes ISC activation and survival, and the expansion of crypt transit-amplifying cells, in ex vivo crypt-villous organoid cultures. The protective effects of HB-EGF were dependent on EGF receptor activation, and were mediated via the MEK1/2 and PI3K signaling pathways. These results show that the intestinal cytoprotective effects of HB-EGF are mediated, at least in part, through its ability to protect ISCs from injury.     

Rapid appearance of M cells after microbial challenge is restricted at the periphery of the follicle-associated epithelium of Peyer's patch

M cells within the follicle-associated epithelium (FAE) of the gut play a central role in the initiation of mucosal immune responses by transporting antigens to the intestinal lymphoid tissue. We have previously demonstrated that the instillation into the gut of a nonenteric microorganism, Streptococcus pneumoniae R36a, is an excellent experimental model to investigate the highly dynamic nature of the FAE in response to microbial challenge. In the present study, S. pneumoniae was introduced into rabbit ileal loops, each one containing a Peyer's patch (PP), and the number of M cells was assessed by morphological and functional characteristics in different areas of the FAE after a short time (1-3 hours). We report that a marked increase in the number of M cells was detected in the periphery, but not in the apical area, of the FAE as early as 1 hour after exposure to S. pneumoniae. Furthermore, a variant of this experiment enabled us to establish that the increased numbers of M cells led to an improved capability of the FAE to transport latex fluorescent microspheres (0.5 microm), highly specific to rabbit M cells, from the gut lumen to the intestinal lymphatic system. In these animals the cisterna chyli was cannulated, and the microparticles were introduced into the intestinal loops after stimulation with pneumococci. The microparticles reaching the lymph were then counted by flow cytometer. We interpreted these results as showing that only enterocytes located within the periphery of the FAE are converted to fully operational M cells by certain microbial interaction and the ability of enterocytes to undergo this conversion may depend on their stage of differentiation.     

Intestinal immunity to poliovirus develops only after repeated infections in monkeys

To establish gut immunity in monkeys to polio-virus type 1, we fed four juvenile bonnet monkeys (Macaca radiata) with 100 median infectious doses of the Mahoney strain of virus. The duration of viral shedding in the throat and faeces and the titre of virus in faeces were measured. Eighteen days after the last monkey stopped shedding virus, they were inoculated once again with the same amount of virus. All monkeys got re-infected; the duration and titres of viral shedding were very similar to those after the first inoculation. This was interpreted to mean a lack of effective gut immunity. After a third inoculation the re-infection was of very short duration, and viral titres were one log10 less, indicating a high degree of gut immunity. These results suggest that one infection by poliovirus does not induce gut immunity; at least two infections are necessary to induce it. Even after two consecutive infections, immunity was not sufficient to prevent re-infection.     

Characterization of west nile virus persistent infections in genetically resistant and susceptible mouse cells. II. Generation of temperature-sensitive mutants

Long-term persistent infections were established with the flavivirus, West Nile virus (WNV), strain E101, in embryofibroblast cultures derived from susceptible C3H/HE and congenic-resistant C3H/RV mice. Cultures were initially maintained by weekly subculture at 37 degrees, but at passage 6 sister cultures were shifted to 32 degrees. Virus progeny titers were observed to increase after the shift to 32 degrees indicating the possible presence of temperature-sensitive mutants. Temperature-sensitive mutants were found to arise in cultures of both susceptible and resistant cells. However, only in the resistant cultures did temperature-sensitive virus become the majority population. Temperature-sensitive mutants did not appear to be essential for either initiation or maintenance of WNV-persistant infections. The resistant cells appear to provide an environment which is advantageous for the amplification of temperature-sensitive mutants.