Neurovirulence of yellow fever 17DD vaccine virus to rhesus monkeys

The yellow fever 17D virus is attenuated and used for human vaccination. Two of its substrains, 17D-204 and 17DD, are used for vaccine production. One of the remarkable properties of this vaccine is limited viral replication in the host but with significant dissemination of the viral mass, yielding a robust and long-lived neutralizing antibody response. The vaccine has excellent records of efficacy and safety and is cheap, used as a single dose, and there are well-established production methodology and quality control procedures which include the monkey neurovirulence test (MNTV). The present study aims at a better understanding of YF 17DD virus attenuation and immunogenicity in the MNVT with special emphasis on viremia, seroconversion, clinical and histological lesions scores, and their intrinsic variability across the tests. Several MNVTs were performed using the secondary seed lot virus 17DD 102/84 totaling 49 rhesus monkeys. Viremia was never higher than the accepted limits established in international requirements, and high levels of neutralizing antibodies were observed in all animals. None of the animals showed visceral lesions. We found that the clinical scores for the same virus varied widely across the tests. There was a direct correlation between the clinical scores in animals with clinical signs of encephalitis and a higher degree of central nervous system (CNS) histological lesions, with an increase of lesions in areas of the CNS such as the substantia nigra, nucleus caudatus, intumescentia cervicalis, and intumescentia ventralis. The histological scores were shown to be less prone to individual variations and had a more homogeneous value distribution among the tests. Since 17DD 102/84 seed virus has been used for human vaccine production and immunization for 16 years with the vaccine being safe and efficacious, it demonstrates that the observed variations across the MNVTs do not influence its effect on humans.     

Junin virus access to CNS by extraneural rat inoculation

This study was carried out to determine the pathways along which two strains of Junin virus (JV), the pathogenic XJV and the attenuated XJC13V, reach the CNS following IP inoculation of 2-day-old rats. A sequential study of infectivity and antigen distribution in peritoneal macrophages, spleen, and brain was performed. Mortality was 85% with the former strain, but only 15% with the latter. At 4-7 days PI, XJV-infected animals had viral antigen in 10% of peritoneal macrophages. Viremia and spleen virus lasted for 10-15 days. Low brain titers were detected at day 7, with a peak at day 15. Brain antigen correlated with virus titers. In contrast, XJC13V-infected rats, macrophage antigen appeared later and to a lesser degree (1% of cells). Viremia and spleen virus were transient, while both the titer of brain virus and the viral antigen proved lower. Antibody titers were over twofold higher for XJ-infected animals. It is suggested that the different replication rate at the inoculation site could account for the greater ability of the XJV strain to reach the CNS. A greater antigen mass and/or more numerous antigenic determinants presented by the macrophage could explain the higher antibody titers found in XJ-injected rats, which were unable, however, to prevent viral spread.     

Calomys callidus as a potential Junin virus reservoir

The present study investigated whether C. callidus, a species belonging to the Calomys genus, is capable of developing experimentally a persistent Junin virus (JV) infection. Newborn and adult cricetids were inoculated with the attenuated XJ-Clone 3 strain of JV by intracerebral or mucosal route. The present results indicate that the species is susceptible to JV infection, capable of shedding virus chronically through saliva and developing a persistent infection as shown by the detection of virus in brain tissue at 60 days post infection. These findings, and the fact that this cricetid shares its distribution areas with Calomys musculinus and Akodon azarae, support C. callidus as a potential JV reservoir.     

Alteration of blood coagulation and complement system in neotropical primates infected with Junin virus

The neotropical primate Callithrix jacchus infected with Junin virus presented an acute disease with hematological and neurological manifestations and died 17 to 24 days after infection. This picture is similar to that of human Argentine hemorrhagic fever (AHF). Blood coagulation and complement studies were performed in ten C jacchus animals inoculated with 10³ TCID₅₀ of Junin virus, the prototype pathogenic XJ strain. Four monkeys were used as normal controls. Infected monkeys and normal controls were bled to death on days 7, 14, 17, and 21. A progressive decrease in the number of platelets was found after day 7 of infection. On day 21, the last monkey had a value of 24,000/μl. The levels of blood clotting factors did not change until day 17, when a shortened partial thromboplastin time activated with Kaolin (PTTK) (36 sec) and increased factors VIII (192.2%) and VII-X (266.6%) were found. On day 21, the PTTK was prolonged (50.7 sec) and factors II, V, and VIII, were decreased. Thrombin time was found prolonged from day 14 onward. Fibrinogen and fibrin degradation products (FDPs) were increased on days 17 (754 mg/dl and 9.2 μg/ml) and 21 (457 mg/dl and 29.4 μg/ml). No changes in the levels of α₂ macroglobulin were observed. Complement hemolytic levels were found to be low on day 7 (58.3 UCH₅₀, increased on day 14 (165.1), and within normal range at the end of infection (107.2). C3 levels showed a similar pattern. The bone marrow was active and hypercellular, and the number and morphology of megakaryocytes were normal in all but one of infected animals. The results of blood clotting suggest a limited activation. The complement system presented a profile of activation followed by a rebound phenomenon. The activation of complement appeared ten days before the alteration of the clotting system was evident.     

Pathogenesis of attenuated Junin virus in the guinea pig model

The purpose of this work was to elucidate the pathogenesis of attenuated Junin virus (JV) strains in the guinea pig model. Three groups of guinea pigs were infected by the IM route with 10(3) PFU of the XJC13 and XJO-attenuated strains or with the XJ pathogenic strain of JV, respectively. Viremia was studied at 3, 5, 7, 9, 12, and 14 days postinfection (pi) (a) in serum samples of all animals and in washed cells from XJC13-infected guinea pigs by conventional techniques and (b) in whole blood samples from XJC13 and XJO animals by coculture with Vero cells. Virus spread was studied at 14 days pi in brain, spleen, lymph nodes, and bone marrow by parallel suckling mouse inoculation or organ homogenates and coculture of cell suspensions with Vero cells. By coculture techniques of whole blood, an otherwise undetectable viremia was demonstrated for both attenuated strains throughout the observation period. In contrast, XJ viremia was easily detected by direct techniques, as has already been shown. Attenuated virus was also shown to reach brain and bone marrow when coculture methods were employed. But titers were always markedly lower than those of the pathogenic strain. The sustained viremia demonstrated in guinea pigs infected with either attenuated strain explains the mode of viral dissemination and accounts for viral rescue and antigen detection from some organs. These results suggest that attenuated strains do not differ greatly in their invasive capacity in guinea pigs, but later on viral replication is impaired. Therefore, these findings reveal potential risks and should be noted when developing human vaccines.     

Role of Calomys musculinus peritoneal macrophages in age-related resistance to Junin virus infection

In nature, the cricetid Calomys musculinus is the principal host of Junin virus, the etiological agent of Argentine hemorrhagic fever. In the experimental infection, adult C. musculinus survived whereas newborns died after intraperitoneal inoculation with the XJ.Cl3 strain of Junin virus. The role of peritoneal macrophages in this age-related resistance was studied. Junin virus multiplied in cultivated macrophages from either neonatal or adult animals and, therefore, it was not possible to correlate the susceptibility of peritoneal macrophages to Junin virus infection with the age-dependent resistance. When adult and neonatal animals were treated with silica prior to Junin virus infection, deaths occurred in the adults, while a delay and decrease in the mortality rate were observed in neonatals. These results suggest that in neonatal C. musculinus macrophages could be permissive cells for Junin virus multiplication, whereas in adult cricetids, these cells would act as a barrier against viral infection by means of an extrinsic antiviral activity.     

Attenuated Junin virus infection in Callithrix jacchus

Twenty marmosets, male Callithrix jacchus, were used during this study. Fifteen of the marmosets were inoculated with 5,000 TCID50 of the attenuated XJC13 strain of Junin virus by intramuscular route and five were left as uninoculated controls. Animals were observed for a 420-day period. In order to carry out virologic, hematologic, serologic, and histologic studies the animals were bled and/or killed at different days post infection(pi). Results obtained showed that the attenuated strain produced an infection with no mortality or signs of illness. There was only a slight loss of weight at 18-40 days pi, which was soon recovered. Viremia was present from day 6 to 22, titers peaking at 4.0 log. Viral spread was limited to the lungs, spleen, lymph nodes, and bone marrow in the animal killed on day 14. No virus was found in the organs of the animal killed on day 23, and neither hematologic alterations nor pathologic lesions were seen in these monkeys except for ganglionar hypertrophy with immunoblast proliferation. Antigen was detected by immunofluorescence (IF) in lymph nodes, spleen, adrenals, lungs and brain. Neutralizing antibodies were detected from the third week onward. Protection conferred by the XJC13 strain proved effective when XJC13-inoculated monkeys were challenged with 1,000 TCID50 of the pathogenic XJ strain at days 60 or 380 pi, while normal controls died. When viral persistence was searched for on days 370, 390, and 420 pi, no infectious virus was detected, but viral antigen was seen in certain organs, which, however, lacked tissue damage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Junin virus activity in two rural populations of the Argentine hemorrhagic fever (AHF) endemic area

To determine the prevalence of inapparent infection with Junin virus among the rural population and its relation to the clinical disease, a serological study was carried out in two zones of the endemic area of Argentine hemorrhagic fever (AHF). From the first appearance of AHF in the zones (1963) and the moment of the survey (1977), 14 years had passed. A total of 695 serum samples were obtained, 540 from Córdoba and 155 from Buenos Aires. Of the 695 serum samples, 83 were positive for neutralizing antibodies against Junin virus. Total infection (clinical and inapparent cases) reached 11.6% and 12.03% in the Buenos Aires and Córdoba zones, respectively, showing that the total prevalence of infection in two zones separated by 320 miles, are very much alike. In Córdoba province, the prevalence of clinical infection was 7.59%, while that for inapparent infection was 4.44%. Values for the Province of Buenos Aires were 9.67% and 1.93%, respectively. In addition to a low prevalence of inapparent infections, the results of this survey show that roughly 90% of the population is susceptible to contract the disease; this stresses the need to immunize susceptible individuals in the endemic area.     

Recent developments on Junin virus,a causative agent for Argentine haemorrhagic fever

Junin virus consists of ribonucleic acid as the genome and is responsible for a rapidly changing tendency of the virus. The virus is accountable for ailments in the human body and causes Argentine Haemorrhagic Fever (AHF). The infection is may be transmitted through contact between an infected animal/host and a person, and later between person to person. Prevention of outbreaks of AHF in humans can be a tough practice, as their occurrence is infrequent and unpredictable. In this review, recent information from the past 5 years available on the Junin virus including the risk of its emergence, infectious agents, its pathogenesis in humans, available diagnostic and therapeutic approaches, and disease management has been summarised. Altogether, this article would be highly significant in understanding the mechanistic basis behind virus interaction and other processes during the life cycle. Currently, no specific therapeutic options are available to treat the Junin virus infection. The information covered in this review could be important for finding possible treatment options for Junin virus infections.     

Comparative studies of five strains of mumps virus in vitro and in neonatal hamsters: evaluation of growth,cytopathogenicity, and neurovirulence

The growth and Cytopathogenicity of five strains of mumps virus were examined in six types of cell cultures and in neonatal hamsters. These strains included the MJ and RW strains, both recent cerebrospinal fluid isolates; the neuroadapted Kilham strain; the Enders strain adapted to chick embryo; and the Jeryl Lynn vaccine strain adapted to chick cell culture. The MJ, RW, and Kilham strains all produced infectious virus without restriction in vitro, but the RW strain did not cause obvious cytopathic effect; the MJ and Kilham strains were cytopathic. The Enders and Jeryl Lynn strains adapted to chick embryo cells were cytopathic and productive in chick cell culture but were restricted in ability to grow productively in vitro on mammalian cell types, even failing to produce noninfectious particles in some cases. In vitro Cytopathogenicity was a host-independent property of a specific virus strain, but the type of cytopathic effect manifest in culture (eg, fusion, cytoplasmic vacuoles) depended on both the strain and the host cell. The ability of a virus strain to invade the brain parenchyma and infect neurons in vivo appeared to correlate with the strain's Cytopathogenicity and not with passage history or adaptive status.     

Monkeypox virus diagnosis and laboratory testing

The multi-country outbreak of monkeypox virus (MPXV) infection, while the coronavirus disease 2019 pandemic is still an ongoing issue, has caused a new challenge. The re-emergence of MPXV and the rising incidence in non-endemic countries is turning into an upcoming threat to global health. Hence, rapid identification of the virus with appropriate methodology with the lowest false results plays a critical role in estimating the global extent of the crisis and providing preventive measures. This review summarised the main applicable strategies for primary detection and confirmation of MPXV and highlighted available data in biosafety, requirements, standard operating procedures, specimen collection, transportation and storage of clinical samples, and waste disposal of the viral agent. Also, various assays including molecular techniques, immunoassays, histopathological methods, electron microscopy, genomic sequencing, and cell culture have been illustrated. Moreover, we reflected on current knowledge of the advantages and disadvantages of each approach.     

Antigenic relationships between attenuated and pathogenic strains of junin virus

Antigenic relationships between attenuated and pathogenic strains of Junin virus (JV) were investigated. Five strains of either human or rodent origin were tested by cross-neutralization assay with hyperimmune antisera, raised in rabbits, against each strain. Polyclonal antisera could be used to distinguish among these JV strains, as the titer values differed significantly with ratios of homologous to heterologous titers, which ranged from 1.3 to 22.3. This demonstrates, independent of their virulence, a heterogeneity among the JV strains tested. The relatedness among JV strains was expressed quantitatively through a dendrogram based on taxonomic distance coefficients. The field strains of JV were grouped into two clusters, according to their geographic origin.     

Neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates

Although vesicular stomatitis virus (VSV) neurovirulence and pathogenicity in rodents have been well studied, little is known about VSV pathogenicity in non-human primates. To address this question, we measured VSV viremia, shedding, and neurovirulence in macaques. Following intranasal inoculation, macaques shed minimal recombinant VSV (rVSV) in nasal washes for 1 day post-inoculation; viremia was not detected. Following intranasal inoculation of macaques, wild type (wt) VSV, rVSV, and two rVSV-HIV vectors showed no evidence of spread to CNS tissues. However, macaques inoculated intrathalamically with wt VSV developed severe neurological disease. One of four macaques receiving rVSV developed clinical and histological signs similar to the wt group, while the remaining three macaques in this group and all of the macaques in the rVSV-HIV vector groups showed no clinical signs of disease and reduced severity of histopathology compared to the wt group. The implications of these findings for rVSV vaccine development are discussed.     

Neurovirulence of influenza virus in mice I. Neurovirulence of recombinants between virulent and avirulent virus strains

The neurovirulence of influenza A/WSN (HONI) virus in mice was studied using recombinants between neurovirulent WSN and nonneurovirulent A/Hong Kong (HK, H3N2) viruses. Parental derivation of genes in recombinants were analyzed by the electrophoresis of viral RNA in urea-polyacrylamide gel. The neurovirulence was tested by intracerebral inoculation of recombinants into mice. It was found that five large genes, P₃, P₁, P₂, HA, and NP, were not essential for the virulence, because recombinants having these five genes derived from HK parent were virulent. Recombinants in which any of three remaining WSN genes, NA, M, and NS, was replaced with the one from HK parent, failed to kill mice. Therefore, these three genes were responsible for the difference of neurovirulence between the two virus strains. However, when tested in mice immunosuppressed by the administration of cyclophosphamide, recombinants containing either M or NS protein from HK parent were virulent, but viruses containing HK neuraminidase were still avirulent. Viruses containing HK neuraminidase appeared incapable of multiplying in the mouse brain, while those containing either M or NS protein derived from HK virus multiplied to a limited extent. It was suggested that WSN neuraminidase was the principal determining factor of the neurovirulence of WSN virus, without which no virus multiplication occurred, while M and probably NS proteins of WSN virus played a role of helper or accessory virulence factor(s), enabling the efficient virus replication.     

Cytotoxicity and cytoadherence of human and murine polymorphonuclear leukocytes against Junin virus infected targets in the absence of anti-viral antibody and complement.

Human and murine polymorphonuclear leukocytes (PMN) lysed L cells infected with Junin Virus (JV) in an in vitro system free of antiviral antibody and complement. Infected VERO cells proved to be resistant to the cytolytic effect. Murine PMN showed an increased adherence on JV-infected L cells but did not attach to VERO cells. The opposite was observed with human PMN, which did adhere to infected VERO cells but not to infected L cells. These results indicate that PMN activity may be an early mechanism of defense in JV infection by lysing virus-infected cells when no immune response has been established, and that cytoadherence is not a necessary step for target lysis.     

Changes in the neuraminidase of neurovirulent influenza virus strains

The influenza virus A/WS/33 has been adapted to mouse brain to produce two neurovirulent derivatives, A/NWS/33 (NWS) and A/WSN/33 (WSN), with the viral neuraminidase gene shown to be the major determinant of neurovirulence. The complete nucleotide sequence of the NA genes from each strain has been determined, which has allowed the identification of changes that have occurred during adaptation to mouse brain. Five changes are shared by the neurovirulent strains. Comparison to the known neuraminidase structure has identified four of these that may affect the active site of the enzyme. In addition, significant differences in the properties of the neuraminidase from the neurovirulent strains were observed relative to the parent strain. While no correlation was observed between neurovirulence and overall neuraminidase activity or preference for a particularN-substitution, the enzymes from both neurovirulent strains showed an increased preference for small substrates and those with 23 linkages, and their activity was potentiated by Ca²⁺ ions.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers L25815-7.     

Formalin inactivated Junin virus: immunogenicity and protection assays

The aim of this study was to determine if Junin virus inactivated with formalin (FA) was immunogenic and able to elicit a protective response in the guinea pig. The XJ-Clone 3 strain of Junin virus grown in Vero cells was exposed to FA at 0 degrees C. The following inactivated antigens were prepared: A1, 0.1% FA for 50 hr; A2, 0.1% FA for 50 hr followed by concentration with polyethylene glycol (PEG); B1, 0.05% FA for 70 hr; B2, 0.05% FA for 70 hr plus PEG concentration; C, 0.1% FA for 50 hr followed by ultracentrifugation and purification by sucrose gradient. No residual infectivity was detected in any inactivated antigen either after two passages in newborn mice or by coculture with Vero cells. After immunization, high-neutralizing and low-immunofluorescent antibody titers wer obtained in adult mice and guinea pigs, thus showing that antigenicity was preserved. However, in spite of the presence of neutralizing antibodies, guinea pigs gained no protection against challenge with the highly pathogenic XJ strain. These results suggest that antibody amount and/or quality may be inadequate; alternatively, mechanisms other than humoral immunity, such as cellular response, not elicited by inactivated antigens may be essential for protection against Junin virus.     

Immunogenicity of wild and attenuated varicella-zoster virus strains in rhesus monkeys

Thirty susceptible rhesus monkeys were inoculated with cell-free varicella-zoster virus strain OKA or strain KMcC. Both wild and attenuated strains were used. No clinical signs characteristic of human varicella were seen in any of the animals. Virus was not isolated from throat swabs, blood, or cerebrospinal fluid. Antibodies were measured by an enhanced plaque neutralization test. The wild and attenuated OKA strains produced comparable levels of antibodies for 3 months after inoculation. Attenuated KMcC strains produced lower titers than the wild strain. On rechallenge 3 months after primary inoculation animals boostered with the attenuated OKA strain developed significantly higher antibody titers than animals receiving the wild strain. Animals primed and challenged with the attenuated KMcC strains showed significantly lower antibody titers than animals which received the wild strain. The results indicate that the immunogenicity of attenuated OKA and KMcC strains in rhesus monkeys parallels the experience obtained with these strains in humans.     

Pathogenicity of mumps virus in the marmoset.

The neurovirulence of two mumps virus strains was compared using marmosets. Marmosets were inoculated intravenously with the wild-type mumps virus Odate strain, resulting in evident meningitis in 1 of 3 marmosets at each of the weeks 3, 4, and 5 postinoculation, representing a total of 3 out of 9 marmosets. Nephritis, parotitis, pancreatitis, and tonsillitis were manifest in addition to central nervous system (CNS) sequelae. On the other hand, the Jeryl Lynn vaccine strain did not induce histopathological changes in the CNS and multiplication of the Jeryl Lynn strain was distinctly lower compared to that of the Odate strain in the marmoset. This is the first report to describe the induction of meningitis in non-human primates after peripheral inoculation of a wild-type mumps virus, presenting findings useful for the elucidation of the mechanism of infection and pathology of mumps virus in the CNS. The distinction observed between the Odate and Jeryl Lynn strains suggests the applicability of the marmoset model for the evaluation of any neurovirulence potential of vaccine strains.     

Neurotropism of a high-passage XJ strain of Junín virus

Guinea pig infection with a highly passaged XJ prototype strain of Junín virus by the intramuscular route (IM) was carried out in order to study viral tropism modification in this host. The neurotropism of this strain was demonstrated by viral isolation, meningitis, and by the presence of Junín antigens as shown by immunofluorescence. These events, not previously observed with the same lower-passaged strain, revealed the appearance of neurotropism after multiple passages in guinea pigs.