Feihu Yan

Treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from Ebola virus infection

Recent successes with monoclonal antibody cocktails ZMappTM and MIL77 against Ebola virus (EBOV) infections have reignited interest in antibody-based therapeutics. Since the production process for monoclonal antibodies can be prolonged and costly, alternative treatments should be investigated. We produced purified equine antisera from horses hyperimmunized with EBOV virus-like particles, and tested the post-exposure efficacy of the antisera in a mouse model of infection. BALB/c mice were given up to 2 mg of purified equine antisera per animal, at 30 minutes, 1 or 2 days post-infection (dpi), in which all animals survived. To decrease the possibility of serum sickness, the equine antisera was digested with pepsin to generate F(ab′)2 fragments, with in vitro neutralizing activity comparable to whole immunoglobulin. Full protection was achieved with when treatment was initiated at 1 dpi, but the suboptimal protection observed with the 30 minute and 2 dpi groups demonstrate that in addition to virus neutralization, other Fc-dependent antibody mechanisms may also contribute to survival. Guinea pigs given 20 mg of antisera or F(ab′)2 at or starting at 1 or 2 dpi were also fully protected from EBOV infection. These results justify future efficacy studies for purified equine products in NHPs.

Isolation and sequence analysis of the complete NS1 and VP2 genes of canine parvovirus from domestic dogs in 2013 and 2014 in China

Canine parvovirus (CPV) can cause severe disease in animals and continuously generates new variant and recombinant strains in dogs that have a strong impact on sanitation. It is therefore necessary to investigate epidemic CPV strains to improve our understanding of CPV transmission and epidemic behavior. However, most studies have focused on the analysis of VP2, and therefore, information about recombination and relationships between strains is still lacking. Here, 14 strains of CPV were isolated from domestic dogs suspected of hosting CPV between 2013 and 2014 in China. The complete NS1 and VP2 genes were sequenced and analyzed. The results suggest that the new CPV-2a and new CPV-2b types are the prevalent strains in China. In addition to a few mutations (residues 19, 544, 545, 572 and 583 of NS1 and residues 267, 370, 377 and 440 of VP2) that were preserved during transmission, new mutations (residues 60, 630 of NS1, and residues 21, 310 of VP2) were found in the isolated strains. A phylogenetic tree based on VP2 sequences illustrated that the new CPV-2a and new CPV-2b strains from China form single clusters that are distinct from lineages from other countries. Moreover, recombination between the new CPV-2a and new CPV-2b types was also identified in the isolated strains. Due to differences in selection pressures or recombination, there were a small number of inconsistencies between the phylogenetic trees for VP2 and NS1, which indicated that phylogenetic relationships based on VP2 might not be representative of those based on NS1. The data indicated that mutations and recombination are constantly occurring along with the spread of CPV in China.

Packaging of Rift Valley fever virus pseudoviruses and establishment of a neutralization assay method

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV). RVF is mainly prevalent on the Arabian Peninsula, the African continent, and several islands in the Indian Ocean near southeast Africa. RVFV has been classified by the World Organisation for Animal Health (OIE) as a category A pathogen. To avoid biological safety concerns associated with use of the pathogen in RVFV neutralization assays, the present study investigated and established an RVFV pseudovirus-based neutralization assay. This study used the human immunodeficiency virus (HIV) lentiviral packaging system and RVFV structural proteins to successfully construct RVFV pseudoviruses. Electron microscopy observation and western blotting indicated that the size, structure, and shape of the packaged pseudoviruses were notably similar to those of HIV lentiviral vectors. Infection inhibition assay results showed that an antibody against RVFV inhibited the infective ability of the RVFV pseudoviruses, and an antibody neutralization assay for RVFV detection was then established. This study has successfully established a neutralization assay based on RVFV pseudoviruses and demonstrated that this method can be used to effectively evaluate antibody neutralization.

Equine Immunoglobulin and Equine Neutralizing F(ab′)2 Protect Mice from West Nile Virus Infection

West Nile virus (WNV) is prevalent in Africa, Europe, the Middle East, West Asia, and North America, and causes epidemic encephalitis. To date, no effective therapy for WNV infection has been developed; therefore, there is urgent need to find an efficient method to prevent WNV disease. In this study, we prepared and evaluated the protective efficacy of immune serum IgG and pepsin-digested F(ab′)2 fragments from horses immunized with the WNV virus-like particles (VLP) expressing the WNV M and E proteins. Immune equine F(ab′)2 fragments and immune horse sera efficiently neutralized WNV infection in tissue culture. The passive transfer of equine immune antibodies significantly accelerated the virus clearance in the spleens and brains of WNV infected mice, and reduced mortality. Thus, equine immunoglobulin or equine neutralizing F(ab′)2 passive immunotherapy is a potential strategy for the prophylactic or therapeutic treatment of patients infected with WNV.

Equine immunoglobulin F(ab′)2 fragments protect mice from Rift Valley fever virus infection

Background Rift Valley fever virus (RVFV) is an emerging arbovirus in Africa and the Arabian Peninsula, in which infection with RVFV poses a serious threat to humans and livestock globally. Approved treatments for RVFV infection, especially for use in humans, have not yet been developed. There is an urgent need for effective drugs to prevent RVFV disease. Methods In previous study, we developed RVFV virus like particles (VLPs) expressing the surface glycoproteins Gn and Gc. The morphology was shown to be similar to live RVFV under electron microscopy. In this study, we immunized horses with RVFV VLPs, prepared the immunoglobulin F(ab′)2 fragments, and characterized its in vitro neutralization and in vivo efficacy in mice. Results F(ab′)2 was found to potently neutralize RVFV in VeroE6 cells, and passive transfer of immunoglobulin F(ab′)2 fragments resulting in reduced mortality in RVFV infected mice. Conclusion Our results show that passive immunotherapy with equine immunoglobulin F(ab′)2 fragments is a promising strategy to treat RVFV infections. HighlightsRift Valley Fever Virus (RVFV) virus‐like particles (VLP) were used as an immunogen in horsesEquine F(ab′)2 fragments were found to potently neutralize RVFV in Vero E6 cells.Passive immunotherapy with equine immunoglobulin F(ab′)2 fragments reduced mortality in RVFV infected mice.

A Rapid and Specific Assay for the Detection of MERS-CoV

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that can cause human respiratory disease. The development of a detection method for this virus that can lead to rapid and accurate diagnosis would be significant. In this study, we established a nucleic acid visualization technique that combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualization strip (RT-LAMP-VF) to detect the N gene of MERS-CoV. The RT-LAMP-VF assay was performed in a constant temperature water bath for 30 min, and the result was visible by the naked eye within 5 min. The RT-LAMP-VF assay was capable of detecting 2 × 101 copies/μl of synthesized RNA transcript and 1 × 101 copies/μl of MERS-CoV RNA. The method exhibits no cross-reactivities with multiple CoVs including SARS-related (SARSr)-CoV, HKU4, HKU1, OC43 and 229E, and thus exhibits high specificity. Compared to the real-time RT-PCR (rRT-PCR) method recommended by the World Health Organization (WHO), the RT-LAMP-VF assay is easy to handle, does not require expensive equipment and can rapidly complete detection within 35 min.

Tangeretin, an extract from Citrus peels, blocks cellular entry of arenaviruses that cause viral hemorrhagic fever

The family Arenaviridae consists of numerous enveloped RNA viruses with ambisense coding strategies. Eight arenaviruses, including Lassa virus, are known to cause severe and fatal viral hemorrhagic fever (VHF) in humans, yet vaccines and treatments for disease caused by arenaviruses are very limited. In this study, we screened a natural product library consisting of 131 compounds and identified tangeretin, a polymethoxylated flavone widely present in citrus fruit peels, as a Lassa virus entry inhibitor that blocks viral fusion. Further analyses demonstrated the efficacy of tangeretin against seven other VHF-causing arenaviruses, suggesting that this compound, which has a history of medical usage, could be used to develop an effective therapeutic to treat infection and disease caused by Lassa virus and related viruses.

Porcine epidemic diarrhea virus virus-like particles produced in insect cells induce specific immune responses in mice

Porcine epidemic diarrhea virus (PEDV), which causes 80–100% mortality in neonatal piglets, is one of the most devastating viral diseases affecting swine worldwide. To date, the lack of effective vaccines and drugs is the main problem preventing control of the global spread of PEDV. In this study, we produced PEDV virus-like particles (VLPs) composed of S, M, and E proteins with a baculovirus expression system and tested them via indirect immunofluorescence assay (IFA)and Western blot analysis. Electron microscopy showed that the morphological structure of the PEDV VLPs was similar to that of the protovirus. Microneutralization assays and ELISpot analysis demonstrated that PEDV VLPs induced highly specific antibody responses and Th2-mediated humoral immunity. As a result, the PEDV VLPs displayed excellent immunogenicity in mice. Therefore, a VLP-based vaccine has the potential to prevent PEDV infection.