A hepatitis B virus causes chronic infections in equids worldwide.

Preclinical testing of novel therapeutics for chronic hepatitis B (CHB) requires suitable animal models. Equids host homologs of hepatitis C virus (HCV). Because coinfections of hepatitis B virus (HBV) and HCV occur in humans, we screened 2,917 specimens from equids from five continents for HBV. We discovered a distinct HBV species (Equid HBV, EqHBV) in 3.2% of donkeys and zebras by PCR and antibodies against EqHBV in 5.4% of donkeys and zebras. Molecular, histopathological, and biochemical analyses revealed that infection patterns of EqHBV resembled those of HBV in humans, including hepatotropism, moderate liver damage, evolutionary stasis, and potential horizontal virus transmission. Naturally infected donkeys showed chronic infections resembling CHB with high viral loads of up to 2.6 × 109 mean copies per milliliter serum for >6 mo and weak antibody responses. Antibodies against Equid HCV were codetected in 26.5% of donkeys seropositive for EqHBV, corroborating susceptibility to both hepatitis viruses. Deltavirus pseudotypes carrying EqHBV surface proteins were unable to infect human cells via the HBV receptor NTCP (Na+/taurocholate cotransporting polypeptide), suggesting alternative viral entry mechanisms. Both HBV and EqHBV deltavirus pseudotypes infected primary horse hepatocytes in vitro, supporting a broad host range for EqHBV among equids and suggesting that horses might be suitable for EqHBV and HBV infections in vivo. Evolutionary analyses suggested that EqHBV originated in Africa several thousand years ago, commensurate with the domestication of donkeys. In sum, EqHBV naturally infects diverse equids and mimics HBV infection patterns. Equids provide a unique opportunity for preclinical testing of novel therapeutics for CHB and to investigate HBV/HCV interplay upon coinfection.

Viral Hepatitis and Human Immunodeficiency Virus Testing and Linkage to Care for Individuals Enrolled in an Opioid Treatment Program.

BACKGROUND: In the United States, many opioid treatment programs (OTPs) do not offer viral hepatitis (VH) or human immunodeficiency virus (HIV) testing despite high prevalence among OTP clients. We initiated an opt-out VH and HIV testing and linkage-to-care program within our OTP. METHODS: All OTP intakes are screened for VH and HIV and evaluated for rescreening annually. A patient navigator reviews laboratory results and provides counseling in the OTP clinic. The medical record is queried to identify individuals with previously diagnosed, untreated VH or HIV. Navigation support is provided for linkage or relinkage to VH or HIV care. RESULTS: Between March 2018 and Februrary 2019, 532 individuals were screened for hepatitis C virus (HCV), 180 tested HCV antibody positive (34%), and 108 were HCV-ribonucleic acid (RNA) positive (20%). Sixty individuals were identified with previously diagnosed, untreated HCV. Of all HCV RNA+, 49% reported current injection drug use (82 of 168). Ninety-five individuals were seen by an HCV specialist (57% of HCV RNA+), 72 started treatment (43%), and 69 (41%) completed treatment. Individuals with primary care providers were most likely to start treatment. Four individuals were diagnosed with hepatitis B; 0 were diagnosed with HIV. CONCLUSIONS: The implementation of an OTP-based screening and navigation protocol has enabled significant gains in the identification and treatment of VH in this high prevalence setting.

Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies.

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

Development of the suppository form of human immunoglobulin preparation with high titers of antibodies to herpes simplex virus types 1 and 2 for the treatment of chronic forms of herpetic disease.

In spite of the vast arsenal of therapeutic agents, therapy of herpes virus infection (HVI) is very difficult, particularly in pregnant women, newborns and children in the first years of life, as well as in patients with immune deficiency. In this regard, possibility of using immunoglobulins for the treatment of HVI is currently attracting the attention of doctors. The aim of this work was to develop a suppository form of the drug containing donor immunoglobulins with high levels of neutralizing antibodies to herpes simplex virus types 1 and 2 for the treatment of chronic forms of herpetic disease. The study included the following steps: 1) selection of gamma-globulins with high antibody titer for HSV-1 and HSV-2 ELISA test; 2) determination of the level of neutralizing antibodies in the selected series of gamma-globulins in tests in tissue cultures and animals; 3) lyophilization of immunoglobulins; 4) development of the suppository form of the preparation containing gamma-globulin donors with high levels of neutralizing antibodies to HSV-1 and HSV-2; 5) study of the safety of the activity of neutralizing antibodies to HSV-1 and HSV-2 in the suppository form of the drug with hyaluronic acid used as immunomodulator. As the result of this work, immunoglobulin preparation in the suppository form was developed. The developed preparation meets the requirements for safety and efficacy. It is not toxic or pyrogenic. The problems of clinical use of this drug as a method of HVI therapy are discussed.

[Reducing viral load in life-threatening viral diseases using snowdrops]. / Schneeglöckchen zur Senkung der Viruslast bei lebensbedrohlichen Viruserkrankungen.

In the case of life-threatening viral diseases, viral load is associated with mortality. A new and innovative therapeutic approach is the reduction of viral load by extracorporeal elimination without simultaneously weakening the immune system by removing specific antibodies. Basis of this therapy is a modified plasma filter coated with a lectin derived from the snowdrop.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein.

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014-2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies.

A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV.

BACKGROUND: Evidence points to the emergence of a novel human coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), which causes a severe acute respiratory syndrome (SARS)-like disease. In response, the development of effective vaccines and therapeutics remains a clinical priority. To accomplish this, it is necessary to evaluate neutralizing antibodies and screen for MERS-CoV entry inhibitors. METHODS: In this study, we produced a pseudovirus bearing the full-length spike (S) protein of MERS-CoV in the Env-defective, luciferase-expressing HIV-1 backbone. We then established a pseudovirus-based inhibition assay to detect neutralizing antibodies and anti-MERS-CoV entry inhibitors. RESULTS: Our results demonstrated that the generated MERS-CoV pseudovirus allows for single-cycle infection of a variety of cells expressing dipeptidyl peptidase-4 (DPP4), the confirmed receptor for MERS-CoV. Consistent with the results from a live MERS-CoV-based inhibition assay, the antisera of mice vaccinated with a recombinant protein containing receptor-binding domain (RBD, residues 377-662) of MERS-CoV S fused with Fc of human IgG exhibited neutralizing antibody response against infection of MERS-CoV pseudovirus. Furthermore, one small molecule HIV entry inhibitor targeting gp41 (ADS-J1) and the 3-hydroxyphthalic anhydride-modified human serum albumin (HP-HSA) could significantly inhibit MERS-CoV pseudovirus infection. CONCLUSION: Taken together, the established MERS-CoV inhibition assay is a safe and convenient pseudovirus-based alternative to BSL-3 live-virus restrictions and can be used to rapidly screen MERS-CoV entry inhibitors, as well as evaluate vaccine-induced neutralizing antibodies against the highly pathogenic MERS-CoV.

Recombinant expression of Garlic virus C (GARV-C) capsid protein in insect cells and its potential for the production of specific antibodies.

Garlic cultivars in Brazil are infected by a complex of viruses and for some virus species, such as the allexivirus, purification of the virions is sometimes cumbersume. To overcome this problem, recombinant expression of viral proteins in heterologous systems is an alternative method for producing antibodies. The capsid gene from Garlic virus C (GarV-C), an Allexivirus, was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) generating the recombinant virus vSynGarV-C. The recombinant protein expression was confirmed by SDS-PAGE and western-blot of extracts from recombinant virus infected insect cells, where a protein band of approximately 32KDa was observed only in extracts from recombinant infected cells. This protein corresponded to the predicted size of the capsid protein of the GarV-C. A rabbit polyclonal antibody was raised against this protein, shown to be specific for the GarV-C protein in western-blot and dot-Elisa, however with a low titer.

West Nile virus in farmed alligators.

Seven alligators were submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory for necropsy during two epizootics in the fall of 2001 and 2002. The alligators were raised in temperature-controlled buildings and fed a diet of horsemeat supplemented with vitamins and minerals. Histologic findings in the juvenile alligators were multiorgan necrosis, heterophilic granulomas, and heterophilic perivasculitis and were most indicative of septicemia or bacteremia. Histologic findings in a hatchling alligator were random foci of necrosis in multiple organs and mononuclear perivascular encephalitis, indicative of a viral cause. West Nile virus was isolated from submissions in 2002. Reverse transcription-polymerase chain reaction (RT-PCR) results on all submitted case samples were positive for West Nile virus for one of four cases associated with the 2001 epizootic and three of three cases associated with the 2002 epizootic. RT-PCR analysis was positive for West Nile virus in the horsemeat collected during the 2002 outbreak but negative in the horsemeat collected after the outbreak.

Origin of maternally transferred antibodies against rabies in foxes (Vulpes vulpes).

During previous experiments, maternal antibodies against rabies were detected in the sera of fox cubs whelped by orally immunised vixens. These antibodies appear to be transferred exclusively via the colostrum. No evidence of maternally transferred immunity in the form of immunoglobulin G was found in 80 fox embryos collected from 19 rabies-immune vixens originating from areas where oral rabies vaccine baits had been distributed.

Ascorbic acid supplementation improved antibody response to infectious bursal disease vaccination in chickens.

The purpose of the present study was to determine if supplementation of ascorbic acid (AA) to the diet would have a beneficial effect on infectious bursal disease (IBD) vaccination of chickens for protection against infectious bursal disease virus (IBDV) infection. Two hundred forty specific pathogen-free (SPF) chickens were divided into eight experimental groups. A 2 x 2 x 2 factorial arrangement in a completely randomized design was used; AA supplementation at 1,000 ppm in the diet, vaccination, and challenge were the main effects. Prior to challenge and 10 d after challenge, serum AA concentration, serum corticosterone concentration, ELISA antibody titer to IBDV, body weight, bursa-to-body weight (B:B) ratio, and bursal histological score (BHS) were determined. Nonvaccinated chickens fed a diet supplemented with AA did not exhibit clinical signs or mortality following challenge, whereas AA-unsupplemented counterparts had 100% cumulative morbidity and 30% cumulative mortality. Serum AA levels of AA-supplemented and vaccinated chickens were significantly (P < 0.05) higher than AA-unsupplemented and vaccinated chickens. Fourteen days following vaccination, significantly (P < 0.05) higher ELISA titers to IBDV were observed in vaccinated chickens supplemented with AA as compared to AA-unsupplemented counterparts. Ascorbic acid-supplemented chickens, especially those also vaccinated, had higher body weight gains as compared to the AA-unsupplemented chickens. Ascorbic acid-supplemented chickens challenged with IBDV did not show any clinical signs or mortality. The results suggest that supplementation of AA at 1,000 ppm in the diet has beneficial effects on antibody response to IBD vaccination and body weight gain.

An antibody which binds to the membrane-proximal end of influenza virus haemagglutinin (H3 subtype) inhibits the low-pH-induced conformational change and cell-cell fusion but does not neutralize virus.

A monoclonal antibody, LMBH6, was derived from mice which had been sequentially immunized with bromelain-cleaved haemagglutinin (BHA) from influenza virus A/Aichi/2/68, A/Victoria/3/75 and A/Philippines/2/82 (all H3N2). LMBH6 recognizes the haemagglutinin (HA) of all H3N2 influenza A strains tested, which were isolated between 1968 and 1989. HA in the low-pH-induced conformation is not recognized, and cleavage of the HA0 precursor to HA1 and HA2 is needed to obtain efficient binding. Compared to other monoclonal antibodies, binding of LMBH6 to virus and to virus-infected cells is weak, while binding to BHA is comparable. Electron microscopy demonstrates binding to the membrane proximal end of the stem structure. The antibody shows no haemagglutination-inhibition activity, but inhibits polykaryon formation and the low-pH-induced conformational change of BHA. However, LMBH6 cannot prevent infection of MDCK cells but slows the growth of virus when included in a plaque assay overlay.

Neonatal immune response and growth performance of chicks hatched from single comb White Leghorn breeders fed diets supplemented with beta-carotene, canthaxanthin, or lutein.

Forty Single Comb White Leghorn (SCWL) hens and 8 SCWL cocks were randomly divided into four treatment groups. Each group was fed a diet containing .02% beta-carotene, canthaxanthin, lutein, or basal control. After 20 d of feeding, eggs were collected daily from each experimental group for incubation. Two different hatches were set and chicks from each hatch were used for one of two different experiments. In both experiments, 24 chicks per treatment were vaccinated against Newcastle disease virus at 1 d of age and raised for 5 wk on a basal diet. In the second experiment, birds were revaccinated at 3 wk of age. In both experiments, at the end of 5 wk birds were killed and bursa of Fabricius, liver, and spleen were collected. For both experiments, there were no differences in antibody titers, weight gain, feed conversion ratio, and relative bursa weights of chicks. However in the second experiment, birds hatched from breeders fed lutein had significantly lower relative liver weights than chicks of the other treatments, whereas birds hatched from the breeders fed beta-carotene and canthaxanthin had significantly lower spleen weights than the control. These experiments suggest that carotenoids may not be effective in increasing neonatal immune response when they supplement practical breeder diets.

[The protective effect of hemagglutinin-specific monoclonal antibodies in experimental influenza infection]. / Protektivnyi éffekt gemaggliutininspetsifichnykh monoklonal'nykh antitel pri éksperimental'noi grippoznoi infektsii.

Experiments in mice showed a high protective effect of monoclonal antibodies (MCA) to influenza A/Krasnodar/101/59 (H2N2) virus hemagglutinin, possessing neutralizing activity in ovo. A 100% protective effect was observed upon intranasal administration of MCA Kp/101-3 48 hours before infection, and 90% effect upon administration of MCA 96 hours before infection. A 100% therapeutic effect was observed upon intranasal administration of MCA Kp/101-3 less than 24 hours postinfection and 70% therapeutic effect was achieved by administration of these MCA 48-72 hours postinfection.

[The presence of antibodies against glycoprotein I (gI) of the virus of Aujeszky's disease in colostrum and milk of sows]. / De aanwezigheid van antistoffen tegen glycoproteïne I (gI) van het virus van de ziekte van Aujeszky in biest en melk van zeugen.

The possibility of using sow's milk to detect antibodies to the gI protein of Aujeszky's virus was studied by the present authors. Antibody titres in serum (collected less than 30 days ante partum) and colostrum from sixty-three sows were determined using a commercially available ELISA; twenty-four of thirty-seven animals vaccinated with gI-positive vaccines showed an antibody response in both colostrum and serum. The titre in colostrum was significantly higher than it was in serum. Antibodies could not be detected in serum or in colostrum of the twenty-six animals which were only vaccinated with gI-negative vaccines. Subsequently, anti-gI titres were determined in milk collected during lactation. Antibodies were also detectable in milk of animals showing high colostrum titres (greater than 128) for at least seventeen days. This period decreased as the initial colostrum titre became lower. Antibodies could still be detected in milk of animals vaccinated with gI-positive vaccine, which were sero-negative at the time of the experiment. Milk therefore seems to be a useful alternative to serum in detecting anti-gI antibodies in sows.

Rabies Vaccine Adsorbed: neutralizing antibody titers after three-dose pre-exposure vaccination.

Field trials at several schools of veterinary medicine showed that three-dose pre-exposure rabies vaccination with Rabies Vaccine Adsorbed developed by the Michigan Department of Health elicited neutralization antibody in practically all recipients two to three weeks after immunization. Titers declined during the first six months after vaccination. However, by 18 to 24 months, 98 percent of recipients still had titers equal or greater than a 1:5 dilution of serum.

Antiviral activity of colostrum and serum immunoglobulins A and G.

Enteric virus-specific IgA and IgG present in paired human sera and colostrums were measured by the enzyme-linked immunosorbent assay (ELISA). Virus-specific IgA was present in all colostrums, but virus-specific IgG could not be detected. The reverse was true when sera were assayed. Most of these colostrums also neutralized either polio virus or reovirus, as did IgA, which was separated from a pool of colostrums by exclusion chromatography. No correlation could be made between levels of neutralizing and ELISA antibody titers in colostrums.

[Rational method of obtaining sera with a high titre of virus-neutralizing antibodies. Report 2]. / Ratsional'nyi metod polucheniia syvorotok s vysokim titrom virusneitralizuiushchikh antitel. Soobshchenie 2.

In addition to report I (ZHMEI, 1977, No. 1) a study was made of 9 more schemes of rabbit immunization with the poliomyelitis virus, type I, for the purpose of obtaining the neutralizing sera of high titre. Vitamins A and C were used in the experiments in the capacity of the activators of the organism reaction; Freund's adjuvant of different make was tested; different reimmunization periods and different amounts of the adjuvant were administered. Titration of rabbit sera in the process of immunization and reimmunization showed immunization into the lymph nodes with the subsequent single reimmunization in one month to be the most effective and economical method of obtaining high effective sera.