Exchange of C-Terminal Variable Sequences within Morbillivirus Nucleocapsid Protein Are Tolerated: Development and Evaluation of Two Marker (DIVA) Vaccines (Sungri/96 DIVA, Nigeria/75/1 DIVA) against PPR

Across Africa, the Middle East, and Asia, peste des petits ruminants virus (PPRV) places a huge disease burden on agriculture, affecting, in particular, small ruminant production. The recent PPR outbreaks in Northern Africa, the European part of Turkey, and Bulgaria represent a significant threat to mainland Europe, as a source of disease. Although two safe and efficacious live attenuated vaccines (Sungri/96 and Nigeria/75/1) are available for the control of PPR, current serological tests do not enable the differentiation between naturally infected and vaccinated animals (DIVA). The vaccinated animals develop a full range of immune responses to viral proteins and, therefore, cannot be distinguished serologically from those that have recovered from a natural infection. This poses a serious problem for the post-vaccinal sero-surveillance during the ongoing PPR eradication program. Furthermore, during the latter stages of any eradication program, vaccination is only possible if the vaccine used is fully DIVA compliant. Using reverse genetics, we have developed two live attenuated PPR DIVA vaccines (Sungri/96 DIVA and Nigeria/75/1 DIVA), in which the C-terminal variable region of the PPRV N-protein has been replaced with dolphin morbillivirus (DMV). As a proof of principle, both the DIVA vaccines were evaluated in goats in pilot studies for safety and efficacy, and all the animals were clinically protected against the intranasal virulent virus challenge, similar to the parent vaccines. Furthermore, it is possible to differentiate between infected animals and vaccinated animals using two newly developed ELISAs. Therefore, these DIVA vaccines and associated tests can facilitate the sero-monitoring process and speed up the implementation of global PPR eradication through vaccination.

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Rescue of a chimeric rinderpest virus with the nucleocapsid protein derived from peste-des-petits-ruminants virus: use as a marker vaccine

Journal of General Virology ◽

10.1099/vir.0.82913-0 ◽

2007 ◽

Vol 88 (7) ◽

pp. 2019-2027 ◽

Cited By ~ 23

Author(s):

Satya Parida ◽

Madhuchhanda Mahapatra ◽

Sai Kumar ◽

Subash C. Das ◽

Michael D. Baron ◽

...

Keyword(s):

Matrix Protein ◽

Reverse Genetics ◽

Variable Region ◽

N Gene ◽

Peste Des Petits Ruminants ◽

Rinderpest Virus ◽

Eradication Programme ◽

Reading Frame ◽

N Protein ◽

Marker Vaccine

The nucleocapsid (N) protein of all morbilliviruses has a highly conserved central region that is thought to interact with and encapsidate the viral RNA. The C-terminal third of the N protein is highly variable among morbilliviruses and is thought to be located on the outer surface and to be available to interact with other viral proteins such as the phosphoprotein, the polymerase protein and the matrix protein. Using reverse genetics, a chimeric rinderpest virus (RPV)/peste-des-petits-ruminants virus (PPRV) was rescued in which the RPV N gene open reading frame had been replaced with that of PPRV (RPV–PPRN). The chimeric virus maintained efficient replication in cell culture. Cattle vaccinated with this chimeric vaccine showed no adverse reaction and were protected from subsequent challenge with wild-type RPV, indicating it to be a safe and efficacious vaccine. The carboxyl-terminal variable region of the rinderpest N protein was cloned and expressed in Escherichia coli. The expressed protein was used to develop an indirect ELISA that could clearly differentiate between RPV- and PPRV-infected animals. The possibility of using this virus as a marker vaccine in association with a new diagnostic ELISA in the rinderpest eradication programme is discussed.

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Probing Morbillivirus Antisera Neutralization Using Functional Chimerism between Measles Virus and Canine Distemper Virus Envelope Glycoproteins

Viruses ◽

10.3390/v11080688 ◽

2019 ◽

Vol 11 (8) ◽

pp. 688 ◽

Cited By ~ 7

Author(s):

Miguel Angel Muñoz-Alía ◽

Stephen J. Russell

Keyword(s):

Neutralizing Antibodies ◽

Measles Virus ◽

Canine Distemper Virus ◽

Reverse Genetics ◽

Canine Distemper ◽

Virus Envelope ◽

Live Virus ◽

Virus Challenge ◽

Virus Attachment ◽

Globular Head

Measles virus (MeV) is monotypic. Live virus challenge provokes a broadly protective humoral immune response that neutralizes all known measles genotypes. The two surface glycoproteins, H and F, mediate virus attachment and entry, respectively, and neutralizing antibodies to H are considered the main correlate of protection. Herein, we made improvements to the MeV reverse genetics system and generated a panel of recombinant MeVs in which the globular head domain or stalk region of the H glycoprotein or the entire F protein, or both, were substituted with the corresponding protein domains from canine distemper virus (CDV), a closely related morbillivirus that resists neutralization by measles-immune sera. The viruses were tested for sensitivity to human or guinea pig neutralizing anti-MeV antisera and to ferret anti-CDV antisera. Virus neutralization was mediated by antibodies to both H and F proteins, with H being immunodominant in the case of MeV and F being so in the case of CDV. Additionally, the globular head domains of both MeV and CDV H proteins were immunodominant over their stalk regions. These data shed further light on the factors constraining the evolution of new morbillivirus serotypes.

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Expression of the Nucleoprotein of the Puumala Virus from the Recombinant Semliki Forest Virus Replicon: Characterization and Use as a Potential Diagnostic Tool

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.4.658-663.2003 ◽

2003 ◽

Vol 10 (4) ◽

pp. 658-663 ◽

Cited By ~ 9

Author(s):

A. Billecocq ◽

D. Coudrier ◽

F. Boué ◽

B. Combes ◽

H. Zeller ◽

...

Keyword(s):

Mammalian Cells ◽

Semliki Forest Virus ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Puumala Virus ◽

Wild Rodents ◽

Serological Tests ◽

N Protein ◽

Antigenic Properties ◽

Native Antigen

ABSTRACT Puumala virus (Bunyaviridae family, Hantavirus genus) causes a mild form of hemorrhagic fever with renal syndrome (HFRS) called nephropathia epidemica in northern and central Europe. Serological tests are used for diagnosis, but antigen production is difficult because the virus grows poorly in tissue culture. We expressed the N protein (nucleoprotein) of Puumala virus via the Semliki Forest virus (SFV) replicon in mammalian cells and compared its antigenic properties with those of the native antigen derived from Puumala virus-infected cells. Detection of immunoglobulin G or immunoglobulin M by enzyme-linked immunosorbent assay (ELISA), μ-capture ELISA, and indirect immunofluorescence assay was (at least) as effective with the recombinant antigen as with the native antigen when HFRS patient sera or organ washes from wild rodents were tested. No nonspecific reaction was observed. Thus, the SFV-expressed N protein of Puumala virus appears as a valid antigen, specific and sensitive for serological investigations.

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The Hemagglutinin-Neuraminidase Protein of Newcastle Disease Virus Determines Tropism and Virulence

Journal of Virology ◽

10.1128/jvi.78.8.4176-4184.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 4176-4184 ◽

Cited By ~ 125

Author(s):

Zhuhui Huang ◽

Aruna Panda ◽

Subbiah Elankumaran ◽

Dhanasekaran Govindarajan ◽

Daniel D. Rockemann ◽

...

Keyword(s):

Newcastle Disease Virus ◽

Disease Virus ◽

Newcastle Disease ◽

Reverse Genetics ◽

Index Test ◽

Death Time ◽

Virulent Virus ◽

Hn Gene ◽

Chimeric Viruses

ABSTRACT The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.

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NATURAL OCCURRENCE OF TOBACCO RINGSPOT VIRUS IN PELARGONIUM IN ONTARIO

Canadian Journal of Plant Science ◽

10.4141/cjps67-052 ◽

1967 ◽

Vol 47 (3) ◽

pp. 295-300 ◽

Cited By ~ 4

Author(s):

W. G. Kemp

Keyword(s):

Natural Infection ◽

First Record ◽

Ringspot Virus ◽

Natural Occurrence ◽

Serological Tests ◽

Tobacco Ringspot Virus ◽

Simultaneous Infection

Simultaneous-infection and serological tests demonstrate that a ringspot virus associated with a disease of Pelargonium hortorum cv. Mme. Salleron is related to tobacco ringspot virus. This report is believed to constitute the first record of natural infection of Pelargonium by this virus.

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Kinetics, Longevity, and Cross-Reactivity of Antineuraminidase Antibody after Natural Infection with Influenza A Viruses

Clinical and Vaccine Immunology ◽

10.1128/cvi.00248-17 ◽

2017 ◽

Vol 24 (12) ◽

Author(s):

Don Changsom ◽

Li Jiang ◽

Hatairat Lerdsamran ◽

Sopon Iamsirithaworn ◽

Rungrueng Kitphati ◽

...

Keyword(s):

Influenza A ◽

Reverse Genetics ◽

H1n1 Virus ◽

Natural Infection ◽

Seroconversion Rate ◽

Disease Onset ◽

Cross Reactivity ◽

Plasma Samples ◽

Influenza A Viruses ◽

Serum Samples

ABSTRACT The kinetics, longevity, and breadth of antibodies to influenza virus neuraminidase (NA) in archival, sequential serum/plasma samples from influenza A virus (IAV) H5N1 infection survivors and from patients infected with the 2009 pandemic IAV (H1N1) virus were determined using an enzyme-linked lectin-based assay. The reverse-genetics-derived H4N1 viruses harboring a hemagglutinin (HA) segment from A/duck/Shan Tou/461/2000 (H4N9) and an NA segment derived from either IAV H5N1 clade 1, IAV H5N1 clade 2.3.4, the 2009 pandemic IAV (H1N1) (H1N1pdm), or A/Puerto Rico/8/1934 (H1N1) virus were used as the test antigens. These serum/plasma samples were also investigated by microneutralization (MN) and/or hemagglutination inhibition (HI) assays. Neuraminidase-inhibiting (NI) antibodies against N1 NA of both homologous and heterologous viruses were observed in H5N1 survivors and H1N1pdm patients. H5N1 survivors who were never exposed to H1N1pdm virus developed NI antibodies to H1N1pdm NA. Seroconversion of NI antibodies was observed in 65% of the H1N1pdm patients at day 7 after disease onset, but an increase in titer was not observed in serum samples obtained late in infection. On the other hand, an increase in seroconversion rate with the HI assay was observed in the follow-up series of sera obtained on days 7, 14, 28, and 90 after infection. The study also showed that NI antibodies are broadly reactive, while MN and HI antibodies are more strain specific.

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Expression and novel alternative purification of the recombinant nucleocapsid (N) protein of SARS-CoV-2 in Escherichia coli for the serodiagnosis of COVID-19

10.1101/2021.11.10.467990 ◽

2021 ◽

Author(s):

Jose David Rosales ◽

William Quintero ◽

Jhon Cruz ◽

Balbino Perdomo ◽

Militza Quintero ◽

...

Keyword(s):

Escherichia Coli ◽

Protein S ◽

Exchange Chromatography ◽

Diagnostic Methods ◽

Enzyme Linked Immunosorbent Assay ◽

Metal Exchange ◽

Serological Tests ◽

N Protein ◽

Global Pandemic ◽

Novel Strategy

The SARS-CoV-2 coronavirus causes severe acute respiratory syndrome and has caused a global pandemic by causing the COVID-19 disease. To monitor and control it, diagnostic methods such as molecular and serological tests are necessary. The serological approach use SARS-CoV-2 antigens to detect the antibodies present in patients using quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or qualitative rapid tests such as lateral flow chromatography (RDT's). The main antigens used are the spike protein (S) and the nucleocapsid protein (N). Both proteins are obtained in different expression systems, in eukaryotic cells, their production is expensive, so in this work we chose a simpler and cheaper system such as prokaryotic to express and purify the N protein. Thereore, the nucleotide sequence had to being optimized to be expressed in Escherichia coli. The protein N is sensitive to E.coli proteases and also has the ability to self-proteolyze under native conditions, degrading into different fragments. However, under denaturing conditions, using urea and at pH 5.3 it is stable and efficiently purified using metal exchange chromatography (IMAC). In our purification strategy, we surprisingly found that by not using a sonicator, a homogeneous and time-stable preparation of the recombinant antigen is obtained. An approximate yield of 200 mg / L was obtained. It was then tested with healthy sera and sera from COVID-19 convalescent patients in Wester-blot tests that were able to recognize it. Our work provides a novel strategy to produce the SARS-CoV-2 protein N so that it can be used as an input in the development and innovation of serological tests in the diagnosis of COVID-19.

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Retrospective study of COVID-19 seroprevalence among tissue donors at the onset of the outbreak before implementation of strict lockdown measures in France

10.1101/2020.09.11.20192518 ◽

2020 ◽

Author(s):

Nicolas Germain ◽

Stephanie Herwegh ◽

Anne Sophie Hatzfeld ◽

Laurence Bocket ◽

Brigitte Prevost ◽

...

Keyword(s):

Tissue Bank ◽

Transmission Probability ◽

Risk Level ◽

Intermediate Risk ◽

Serum Samples ◽

Tissue Donors ◽

Serological Tests ◽

Systematic Screening ◽

N Protein ◽

Selection Of

Background: The COVID-19 pandemic has altered organ and tissue donations as well as transplantation practices. SARS-CoV-2 serological tests could help in the selection of donors. We assessed COVID-19 seroprevalence in a population of tissue donors, at the onset of the outbreak in France, before systematic screening of donors for SARS-CoV-2 RNA. Methods: 235 tissue donors at the Lille Tissue bank between November 1, 2019 and March 16, 2020 were included. Archived serum samples were tested for SARS-CoV-2 antibodies using two FDA-approved kits. Results: Most donors were at higher risks for severe COVID-19 illness including age over 65 years (142/235) and/or presence of co-morbidities (141/235). According to the COVID-19 risk assessment of transmission, 183 out of 235 tissue donors presented with a low risk level and 52 donors with an intermediate risk level of donor derived infection. Four out of the 235 (1.7%) tested specimens were positive for anti-SARS-CoV-2 antibodies: 2 donors with anti-N protein IgG and 2 other donors with anti-S protein total Ig. None of them had both type of antibodies. Conclusion: Regarding the seroprevalence among tissue donors, we concluded that the transmission probability to recipient via tissue products was very low at the beginning of the outbreak.

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Determination of an optimal dilution of virulent feline infectious enteritis (panleucopaenia) virus for challenge purposes

Epidemiology and Infection ◽

10.1017/s0022172400042479 ◽

1970 ◽

Vol 68 (4) ◽

pp. 549-556 ◽

Cited By ~ 8

Author(s):

K. J. O'Reilly

Keyword(s):

Influenza Virus ◽

Blood Cells ◽

White Blood Cells ◽

Geometric Means ◽

Virus Challenge ◽

Cell Counts ◽

Blood Cell Counts ◽

Virulent Virus ◽

White Blood Cell Counts

SUMMARYWhen ten cats were infected orally with undiluted or a 10−1 dilution of virulent feline infectious enteritis (panleucopaenia) virus, all developed severe leucopaenia followed by the development of demonstrable antibody, but none died. Eighteen of 29 cats given a 10−2 dilution of virus died of the disease. Three of the survivors had white blood cell counts of less than 4000 and three had counts between 4000 and 6000 cells. Although the remaining five animals never had individual counts of less than 6000 cells, the geometric means of these counts showed that a marked depression in the leucocyte counts had occurred. All surviving cats developed antibody.Among the ten cats dosed with either 10−3 or 10−4 dilution of virus, four died of feline infectious enteritis and three developed antibody after falls in the leucocyte counts. It is suspected that low dilutions of feline infectious enteritis virulent virus in cats produce a phenomenon similar to that reported by von Magnus (1954) with influenza virus in eggs.Leucopaenia is commonly defined as less than 4000 white blood cells/mm.3 of blood. Counts lower than this are usual in cats which either die of the disease or have received large doses of virus; they are less common in cats surviving after administration of diluted virus. Challenge of cats with pre-existing antibody did not provoke a depression in the leucocyte counts.

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