Reduced Virus Shedding Research Articles

Short- and long-term protective efficacy against clade 2.3.4.4 H5N2 highly pathogenic avian influenza virus following prime-boost vaccination in turkeys.

Highly pathogenic avian influenza virus (HPAIV) infections are frequently associated with systemic disease and high mortality in domestic poultry, particularly in chickens and turkeys. Clade 2.3.4.4 represents a genetic cluster within the Asian HPAIV H5 Goose/Guangdong lineage that has transmitted through migratory birds and spread throughout the world. In 2014, clade 2.3.4.4 strains entered the U.S. via the Pacific flyway, reassorted with local strains of the North American lineage, and produced novel HPAIV strains of the H5N1, H5N2, and H5N8 subtypes. By 2015, the H5N2 HPAIVs disseminated eastwards within the continental U.S. and Canada and infected commercial poultry, causing the largest animal health outbreak in recent history in the U.S. The outbreak was controlled by traditional mass depopulation methods, but the outbreak was of such magnitude that it led to the consideration of alternative control measures, including vaccination. In this regard, little information is available on the long-term protection of turkeys vaccinated against avian influenza. In this report, a vaccination study was carried out in turkeys using 3 prime-boost approaches with a combination of 2 different vaccines, an alphavirus-based replicon vaccine and an adjuvanted-inactivated reverse genetics vaccine. Vaccine efficacy was assessed at 6 and 16weeks of age following challenge with a prototypic novel clade 2.3.4.4 H5N2 HPAIV. All three vaccines protocols were protective with significantly reduced virus shedding and mortality after challenge at 6weeks of age. In contrast, significant variations were seen in 16-week old turkeys after challenge: priming with the alphavirus-based replicon followed by boost with the adjuvanted-inactivated vaccine conferred the best protection, whereas the alphavirus-based replicon vaccine given twice provided the least protection. Our study highlights the importance of studying not only different vaccine platforms but also vaccination strategies to maximize protection against HPAIV especially with regards to the longevity of vaccine-induced immune response.

Protection of White Leghorn chickens by U.S. emergency H5 vaccination against clade 2.3.4.4 H5N2 high pathogenicity avian influenza virus.

During December 2014-June 2015, the U.S. experienced a high pathogenicity avian influenza (HPAI) outbreak caused by clade 2.3.4.4 H5Nx Goose/Guangdong lineage viruses with devastating consequences for the poultry industry. Three vaccines, developed based on updating existing registered vaccines or currently licensed technologies, were evaluated for possible use: an inactivated reverse genetics H5N1 vaccine (rgH5N1) and an RNA particle vaccine (RP-H5), both containing the hemagglutinin gene of clade 2.3.4.4 strain, and a recombinant herpesvirus turkey vectored vaccine (rHVT-H5) containing the hemagglutinin gene of clade 2.2 strain. The efficacy of the three vaccines, alone or in combination, was assessed in White Leghorn chickens against clade 2.3.4.4 H5N2 HPAI virus challenge. In Study 1, single (rHVT-H5) and prime-boost (rHVT-H5+rgH5N1 or rHVT-H5+RP-H5) vaccination strategies protected chickens with high levels of protective immunity and significantly reduced virus shedding. In Study 2, single vaccination with either rgH5N1 or RP-H5 vaccines provided clinical protection in adult chickens and significantly reduced virus shedding. In Study 3, double rgH5N1 vaccination protected adult chickens from clinical signs and mortality when challenged 20weeks post-boost, with high levels of long-lasting protective immunity and significantly reduced virus shedding. These studies support the use of genetically related vaccines, possibly in combination with a broad protective priming vaccine, for emergency vaccination programs against clade 2.3.4.4 H5Nx HPAI virus in young and adult layer chickens.

Recombinant Newcastle disease virus expressing the infectious bronchitis virus S1 gene protects chickens against Newcastle disease virus and infectious bronchitis virus challenge

The recombinant LaSota strain expressing a chimeric IBV S1 gene (rLaSota-S1) was constructed with the S1 gene of the LX4 type IBV ck/CH/LDL/091022. The expression of the S1 protein was detected by an indirect immunofluorescence assay and Western blotting. The rLaSota-S1 strain was slightly attenuated, and its growth dynamics were similar to that of the parental LaSota strain. Vaccination of specific pathogen-free chickens with the rLaSota-S1 strain induced NDV hemagglutination inhibition antibodies, and it protected chickens from challenge with virulent NDV. In addition, vaccination with the rLaSota-S1 strain induced IBV-specific IgG antibodies and cellular immunity; however, a single vaccination provided partial protection with reduced virus shedding. Better protection efficiency was observed after a booster vaccination, which resulted in higher antibody titers, significantly fewer disease symptoms, and reduced virus replication and shedding. Our results suggest that the rLaSota-S1 strain is a bivalent vaccine candidate against both NDV and IBV.

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies.

The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

Supplementation of inactivated influenza vaccine with norovirus P particle-M2e chimeric vaccine enhances protection against heterologous virus challenge in chickens.

The current inactivated influenza vaccines provide satisfactory protection against homologous viruses but limited cross-protection against antigenically divergent strains. Consequently, there is a need to develop more broadly protective vaccines. The highly conserved extracellular domain of the matrix protein 2 (M2e) has shown promising results as one of the components of a universal influenza vaccine in different animal models. As an approach to overcome the limited, strain specific, protective efficacy of inactivated influenza vaccine (IIV), a combination of recombinant M2e expressed on the surface of norovirus P particle (M2eP) and IIV was tested in chickens. Co-immunization of birds with both vaccines did not affect the production of M2e-specific IgG antibodies compared to the group vaccinated with M2eP alone. However, the co-immunized birds developed significantly higher pre-challenge hemagglutination inhibition antibody titers against the homologous IIV antigen and heterologous challenge virus. These combined vaccine groups also had cross reactive antibody responses against different viruses (H5, H6, and H7 subtypes) compared to the IIV alone vaccinated group. Upon intranasal challenge with homologous and heterologous viruses, the combined vaccine groups showed greater reduction in viral shedding in tracheal swabs compared to those groups receiving IIV alone. Moreover, M2eP antisera from vaccinated birds were able to bind to the native M2 expressed on the surface of whole virus particles and infected cells, and inhibit virus replication in vitro. Our results support the potential benefit of supplementing IIV with M2eP, to expand the vaccine cross protective efficacy.

A Universal Influenza Vaccine Can Lead to Disease Exacerbation or Viral Control Depending on Delivery Strategies.

The development of influenza A virus (IAV) vaccines, which elicits cross-strain immunity against seasonal and pandemic viruses is a major public health goal. As pigs are susceptible to human, avian, and swine-adapted IAV, they would be key targets of so called universal IAV vaccines, for reducing both the zoonotic risk and the economic burden in the swine industry. They also are relevant preclinical models. However, vaccination with conserved IAV antigens (AGs) in pigs was reported to elicit disease exacerbation. In this study, we assessed whether delivery strategies, i.e., dendritic cell (DC) targeting by the intradermal (ID) or intramuscular (IM) routes, impact on the outcome of the vaccination with three conserved IAV AGs (M2e, NP, and HA2) in pigs. The AGs were addressed to CD11c by non-covalent binding to biotinylated anti-CD11c monoclonal antibody. The CD11c-targeted AGs given by the ID route exacerbated disease. Conversely, CD11c-targeted NP injected by the IM route promoted T cell response compared to non-targeted NP. Furthermore, the conserved IAV AGs injected by the IM route, independently of DC targeting, induced both a reduction of viral shedding and a broader IgG response as compared to the ID route. Our findings highlight in a relevant animal species that the route of vaccine delivery impacts on the protection induced by conserved IAV AGs and on vaccine adverse effects. Finally, our results indicate that HA2 stands as the most promising conserved IAV AG for universal vaccine development.

Effect of Pritelivir Compared With Valacyclovir on Genital HSV-2 Shedding in Patients With Frequent Recurrences

Current therapy of herpes infections relies on nucleoside analogues. Pritelivir is a well-tolerated novel herpes simplex virus (HSV) helicase-primase inhibitor that reduced genital shedding and lesions. To compare the efficacy of pritelivir with valacyclovir for suppression of genital HSV-2 infection. A phase 2, randomized, double-blind, crossover clinical trial at clinical research centers in 4 US cities (October 2012-July 2013) compared daily oral doses of 100 mg of pritelivir with 500 mg of valacyclovir. The planned sample size was 98 adults, allowing for detection of a 50% reduction in viral shedding between the study treatments. Healthy adults with 4 to 9 annual genital HSV-2 recurrences were eligible. 45 participants were randomized to receive pritelivir [corrected] and 46 to receive valacyclovir first when the US Food and Drug Administration placed the trial on clinical hold based on findings in a concurrent nonclinical toxicity study, and the sponsor terminated the study. Participants took the first drug for 28 days followed by 28 days of washout before taking the second drug for 28 days. Throughout treatment, the participants collected genital swabs 4 times daily for testing by HSV polymerase chain reaction assays. The primary end point was within-participant genital HSV shedding while receiving pritelivir compared with valacyclovir. Secondary end points included the quantity of HSV in positive swabs and the frequency of genital lesions and shedding episodes. Of the 91 randomized participants (median age, 48 years; 57 women [63%]), 56 had completed both treatment periods at the time of the study's termination. In intent-to-treat analyses, HSV shedding was detected in 2.4% (173 of 7276 ) of swabs during pritelivir treatment compared with 5.3% (392 of 7453) during valacyclovir treatment (relative risk [RR], 0.42 [corrected]; 95% CI, 0.21 to 0.82; P = .01). In swabs with HSV, the mean quantity of HSV was 3.2 log10 copies/mL during pritelivir treatment vs 3.7 log10 copies/mL during valacyclovir treatment (difference, -0.1; 95% CI, -0.6 to 0.5; P = .83). Genital lesions were present on 1.9% of days in the pritelivir group vs 3.9% in the valacyclovir group (RR, 0.40; 95% CI, 0.17-0.96; P = .04). The frequency of shedding episodes did not differ by group, with 1.3 per person-month for pritelivir and 1.6 per person-month for valacyclovir (RR, 0.80; 95% CI, 0.52 to 1.22; P = .29). Treatment-emergent adverse events occurred in 62.3% of participants in the pritelivir group and 69.2% of participants in the valacyclovir group. Among adults with frequently recurring genital HSV-2, the use of pritelivir compared with valacyclovir resulted in a lower percentage of swabs with HSV detection over 28 days. Further research is needed to assess longer-term efficacy and safety. clinicaltrials.gov Identifier: NCT01658826.

An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant.

BackgroundSince the end of 2011 an outbreak of pseudorabies affected Chinese pig herds that had been vaccinated with the commercial vaccine made of Bartha K61 strain. It is now clear that the outbreak was caused by an emergent PRV variant. Even though vaccines made of PRV Bartha K61 strain can confer certain cross protection against PRV variants based on experimental data, less than optimal clinical protection and virus shedding reduction were observed, making the control or eradication of this disease difficult.ResultsAn infectious clone of PRV AH02LA strain was constructed to generate a gE deletion mutant PRV(LA-AB) strain. PRV(LA-AB) strain can reach a titer of 108.43 TCID50 /mL (50% tissue culture infectious dose) on BHK-21 cells. To evaluate the efficiency of the inactivated vaccine made of PRV(LA-AB) strain, thirty 3-week-old PRV-negative piglets were divided randomly into six groups for vaccination and challenge test. All five piglets in the challenge control showed typical clinical symptoms of pseudorabies post challenge. Sneezing and nasal discharge were observed in four and three piglets in groups C(vaccinated with inactivated PRV Bartha K61 strain vaccine) and D(vaccinated with live PRV Bartha K61 strain vaccine) respectively. In contrast, piglets in both groups A(vaccinated with inactivated PRV LA-AB strain vaccine) and B(vaccinated with inactivated PRV LA-AB strain vaccine with adjuvant) presented mild or no clinical symptoms. Moreover, viral titers detected via nasal swabs were approximately 100 times lower in group B than in the challenge control, and the duration of virus shedding (3–4 days) was shorter than in either the challenge control (5–10 days) or groups C and D (5–6 days).ConclusionsThe infectious clone constructed in this study harbors the whole genome of the PRV variant AH02LA strain. The gE deletion mutant PRV(LA-AB)strain generated from PRV AH02LA strain can reach a high titer on BHK-21 cells. An inactivated vaccine of PRV LA-AB provides clinical protection and significantly reduces virus shedding post challenge, especially if accompanied by the adjuvant CVC1302. While Inactivated or live vaccines made of PRV Barth K61 strain can provide only partial protection in this test.

Pathogenicity of a TW-Like Strain of Infectious Bronchitis Virus and Evaluation of the Protection Induced against It by a QX-Like Strain.

Avian infectious bronchitis, a highly contagious disease caused by avian infectious bronchitis virus (IBV), is of considerable economic importance to the poultry industry. New IBV TW-like strains have increasingly emerged in China in recent years; hence, evaluating their pathogenicity and developing a specific vaccine to guard against their potential threat to the poultry industry is important. Here, we examined the pathogenicity of a TW-like IBV strain (GD), and evaluated the protective efficacy of the QX-like strain (JS) against GD in challenge infections in chickens. The results revealed that strain-GD-infected birds experienced severe respiratory signs, renal lesions, and 30–40% mortality. The GD virus had extensive tissue tropism, especially in the trachea, lungs, kidneys, and bursa of Fabricius, and was continuously shed via the respiratory tract and cloaca. The QX-like IBV strain JS is able to completely protect chickens from challenge with the TW-like IBV GD field strain, with no clinical signs or gross lesions, decreased tissue replication rates, lower ciliostasis score, and reduced virus shedding. These findings indicate that IBV GD is highly virulent, and that QX-like JS may serve as an effective vaccine against the threat posed by IBV TW-like viruses.

Generation and evaluation of a genetically attenuated Newcastle disease virus rGM-VIIm as a genotype-matched vaccine.

Despite intensive vaccination campaigns, outbreaks of Newcastle disease (ND) have been frequently reported in China, especially of genotype VII that first emerged in the late 1990s. Given the dire need for vaccines against the circulating genotype VII virus, we developed an alternative method to recover a highly virulent recombinant GM (rGM) virus that involves a T7 system with a hammerhead ribozyme sequence introduced downstream of the T7 promoter. By changing the F0 polybasic cleavage site RRQKR↓F to the monobasic GRQGR↓L, we generated a mutant virus (rGM-VIIm) that was found to be highly attenuated in chickens. The rGM-VIIm virus not only produced fourfold higher hemagglutination assay (HA) titers than the parental virus, but also exhibited genetic stability after 15 continuous passages in specific-pathogen-free (SPF) embryonated eggs. Whether live or inactivated, rGM-VIIm and LaSota vaccines can protect vaccinated birds from GM challenge infection. However, live and inactivated rGM-VIIm vaccines reduced virus shedding of the homologous challenge virus significantly better than the LaSota virus vaccine did. Altogether, our results suggest that rGM-VIIm vaccines could aid in the control of ND in China.

Evaluation of different chemical adjuvants on an avian influenza H6 DNA vaccine in chickens

ABSTRACTThis study assessed the ability of three adjuvants (aluminium hydroxide, Essai (microparticle) and Phema (nanoparticle)) to enhance the immune response of chickens to an H6N2 avian influenza DNA vaccine. No haemagglutination inhibition antibody was detected following two intramuscular immunizations with the adjuvanted and non-adjuvanted pCAG-HAk vaccine, which has previously been shown to induce moderate H6 haemagglutinin antibody response in SPF chickens. Following virus challenge, neither the vaccinated group without adjuvant nor the Essai-adjuvanted group showed a statistically significant reduction in virus shedding in oropharyngeal and cloacal swabs compared with the naive control group. However, the aluminium hydroxide and Phema-adjuvanted groups significantly reduced the frequency of virus shedding in oropharyngeal swabs, indicating that these adjuvants appeared to further enhance the vaccine potency. Aluminium hydroxide holds promise as an adjuvant for enhancing DNA-induced immune response in chickens owing to its low price and safety record.

Immune responses elicited by the GEN-003 candidate HSV-2 therapeutic vaccine in a randomized controlled dose-ranging phase 1/2a trial

PurposeGEN-003 is a candidate therapeutic HSV-2 vaccine containing a fragment of infected cell protein 4 (ICP4.2), a deletion mutant of glycoprotein D2 (gD2ΔTMR), and Matrix-M2 adjuvant. In a dose-ranging phase 1/2a clinical trial, immunization with GEN-003 reduced viral shedding and the percentage of reported herpetic lesion days. Here we examine the immune responses in the same trial, to characterize vaccine-related changes in antibody and cell-mediated immunity. MethodsParticipants with genital HSV-2 infection were randomized to 1 of 3 doses of GEN-003, antigens without adjuvant, or placebo. Subjects received 3 intramuscular doses, three weeks apart, and were monitored for viral shedding, lesions and immunogenicity. Antibody titers were measured by ELISA and neutralization assay in serum samples collected at baseline and 3weeks post each dose. T cell responses were assessed pre-immunization and 1week post each dose by IFN-γ ELISpot and intracellular cytokine staining. Blood was also collected at 6 and 12months to monitor durability of immune responses. ResultsAntibody and T cell responses increased with vaccination and were potentiated by adjuvant. Among the doses tested, the rank order of reduction in viral shedding follows the ranking of fold change from baseline in T cell responses. Some immune responses persisted up to 12months. ConclusionAll measures of immunity are increased by vaccination with GEN-003; however, a correlate of protection is yet to be defined.

WITHDRAWN: Protective and antiviral activities of Nigella sativa against avian influenza (H9N2) in turkeys

The main objective of this study was to determine the possible effects of Nigella sativa on immune-response and pathogenesis of H9N2 avian influenza virus in turkeys. The experiment was performed on 130 non-vaccinated mixed-sex turkey poults, divided into five experimental groups of 30 birds each. Group A was kept as non-infected and a non-treated negative control while group B was kept as infected and non-treated positive control. Turkeys in groups A and B received normal commercial feed while turkeys of groups C, D and E were fed on diets containing 2%, 4% and 4% NS seeds, respectively, from day one through the entire experiment period. All groups were challenged with H9N2 AIV at 4th week of age except group A. Infected turkeys showed clinical signs of different severity, showing the most prominent disease signs in turkeys of the group B. All infected turkeys showed positive results for virus shedding; however, the pattern of virus shedding was different, and with turkeys of the group B showing pronounced virus secretion than the turkeys in other groups receiving different levels of NS. Moreover, significantly higher antibody titer against H9N2 AIV in turkeys fed 6% NS seeds shows the immunomodulatory nature of NS. Similarly, increased cytokine gene expression suggests antiviral behavior of NS especially in dose dependent manner, leading to suppressed pathogenesis of H9N2 viruses. However, reduced virus shedding and enhanced immune responses were more pronounced in those turkeys received NS at the rate of 4% and 6%. This study showed that supplement of NS would significantly enhance immune responsiveness and suppress pathogenicity of influenza viruses in turkeys.

Immunogenicity of a recombinant parapoxvirus expressing the spike protein of Porcine epidemic diarrhea virus

The parapoxvirus Orf virus (ORFV), has long been recognized for its immunomodulatory properties in permissive and non-permissive animal species. Here, a new recombinant ORFV expressing the full-length spike (S) protein of Porcine epidemic diarrhea virus (PEDV) was generated and its immunogenicity and protective efficacy were evaluated in pigs. The PEDV S was inserted into the ORFV121 gene locus, an immunomodulatory gene that inhibits activation of the NF-κB signalling pathway and contributes to ORFV virulence in the natural host. The recombinant ORFV-PEDV-S virus efficiently and stably expressed the PEDV S protein in cell culture in vitro. Three intramuscular (IM) immunizations with the recombinant ORFV-PEDV-S in 3-week-old pigs elicited robust serum IgG, IgA and neutralizing antibody responses against PEDV. Additionally, IM immunization with the recombinant ORFV-PEDV-S virus protected pigs from clinical signs of porcine epidemic diarrhoea (PED) and reduced virus shedding in faeces upon challenge infection. These results demonstrate the suitability of ORFV121 gene locus as an insertion site for heterologous gene expression and delivery by ORFV-based viral vectors. Additionally, the results provide evidence of the potential of ORFV as a vaccine delivery vector for enteric viral diseases of swine. This study may have important implications for future development of ORFV-vectored vaccines for swine.

Delivery of an inactivated avian influenza virus vaccine adjuvanted with poly(D,L-lactic-co-glycolic acid) encapsulated CpG ODN induces protective immune responses in chickens

In poultry, systemic administration of commercial vaccines consisting of inactivated avian influenza virus (AIV) requires the simultaneous delivery of an adjuvant (water-in-oil emulsion). These vaccines are often limited in their ability to induce quantitatively better local (mucosal) antibody responses capable of curtailing virus shedding. Therefore, more efficacious adjuvants with the ability to provide enhanced immunogenicity and protective anti-AIV immunity in chickens are needed. While the Toll-like receptor (TLR) 21 agonist, CpG oligodeoxynucleotides (ODNs) has been recognized as a potential vaccine adjuvant in chickens, poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, successfully tested as vaccine delivery systems in other species, have not been extensively explored. The present study, therefore, assessed both systemic and mucosal antibody-mediated responses following intramuscular vaccination (administered at 7 and 21days post-hatch) of chickens with PLGA encapsulated H9N2 AIV plus encapsulated CpG ODN 2007 (CpG 2007), and nonencapsulated AIV plus PLGA encapsulated CpG 2007 vaccine formulations. Virus challenge was performed at 2weeks post-secondary vaccination using the oculo-nasal route. Our results showed that chickens vaccinated with the nonencapsulated AIV vaccine plus PLGA encapsulated CpG 2007 developed significantly higher systemic IgY and local (mucosal) IgY antibodies as well as haemagglutination inhibition antibody titres compared to PLGA encapsulated AIV plus encapsulated CpG 2007 vaccinated chickens. Furthermore, chickens that received CpG 2007 as an adjuvant in the vaccine formulation had antibodies exhibiting higher avidity indicating that the TLR21-mediated pathway may enhance antibody affinity maturation qualitatively. Collectively, our data indicate that vaccination of chickens with nonencapsulated AIV plus PLGA encapsulated CpG 2007 results in qualitatively and quantitatively augmented antibody responses leading to a reduction in virus shedding compared to the encapsulated AIV plus PLGA encapsulated CpG 2007 formulation.

Post-exposure antiviral treatment of norovirus infections effectively protects against diarrhea and reduces virus shedding in the stool in a mortality mouse model

Noroviruses are a leading cause of gastroenteritis across the world in all age groups and are linked to increased hospitalization and mortality in children, the elderly and immunocompromised. The development of specific antiviral treatment for norovirus gastroenteritis is urgently needed. We explored in a mouse model whether an inhibitor of norovirus replication could be used therapeutically post murine norovirus (MNV)-infection of mice.Using the MNV, we previously discovered that the viral polymerase inhibitor 2′-C-methylcytidine (2CMC) is able to protect against diarrhea and mortality in mice when used prophylactically and to block the transmission of MNV between mice. Here, we investigated whether 2CMC could be used therapeutically, starting treatment between 12 h and 3 days post-infection with 2CMC.Post-exposure treatment of MNV-infected mice with 2CMC was efficient up to 2 days after infection, preventing norovirus-induced diarrhea, delaying and reducing MNV shedding in stool of treated mice. Rehydration of 2CMC-treated animals did not result in a further improvement of the disease evolution compared to antiviral treatment only. The presence of MNV antigens and inflammation in the small intestine of infected mice inversely correlated with the effectiveness of delayed antiviral treatment. Anti-MNV IgGs were detected in re-challenged mice 10 weeks after the first contact, these protected the mice from re-infection. We here demonstrate the benefit of antiviral treatment in ongoing norovirus infections.

Human and rat epithelial cells inactivate extracellular influenza A virus via H2O2-dependent hypothiocyanite

Abstract Influenza A virus (IAV) causes ongoing epidemics characterized by high morbidity and variable mortality in several species including humans and swine. Current intervention strategies incorporate either vaccines with several strains of IAV or antiviral drugs against which the virus is developing resistance. Identifying novel anti-influenza mechanisms of the respiratory innate immune system is a potential source of influenza therapies. The first line of defense against respiratory pathogens is the respiratory epithelium. One of the innate defense mechanisms respiratory epithelial cells use to prevent infection with pathogens such as IAV is an extracellular oxidative burst at the airway surface mediated by lactoperoxidase (LPO), thiocyanate anion (SCN−), and hydrogen peroxide (H2O2) to produce antiviral hypothiocyanite ions. Cellular H2O2 is produced by two isoforms of the NADPH oxidase dual oxidase (DUOX) enzymes, DUOX1 and DUOX2. Our data has shown that differentiated, primary human or rat tracheal epithelial cells supplemented with LPO and SCN− are capable of inactivating IAV in vitro. This system has shown a 4–5 log reduction in viral titers that was eliminated by the addition of catalase (an H2O2 scavenger) demonstrating its H2O2 dependence. Additionally, we have shown that this system is capable of inactivating human-origin, swine-origin, and recombinant IAV strains. These results suggest that by modulating the DUOX system IAV infection could be prevented or at least modulated to improve viral killing thereby reducing virus shedding which could alter IAV infection dynamics within a population.

Effect of phylogenetic diversity of velogenic Newcastle disease virus challenge on virus shedding post homologous and heterologous DNA vaccination in chickens

ABSTRACTNewcastle disease (ND) is a highly devastating disease for the poultry industry as it causes high economic losses. In this present study, a DNA vaccine containing the F and HN surface antigens of a highly virulent Newcastle disease virus (NDV), NDV/1/Chicken/2005 (FJ939313), was successfully generated. Cell transfection test indicated that the vaccine expressed the F and HN genes in Hep-2 cells. The main objective of this study was to compare the extent of protection induced by DNA vaccination after homologous and heterologous NDV-challenge as determined by the amount of NDV shedding after challenge. NDV-antibody-negative chickens were vaccinated either once, twice or thrice intramuscularly at 7, 14 and 21 days old and were challenged 14 days post vaccination with either homologous virus (vaccine-matched velogenic viscerotropic Newcastle disease virus (vvNDV) strain, FJ939313), phylogenetically related to group VII, or a phylogenetically divergent heterologous virus (unmatched vvNDV strain, AY968809), which belongs to genogroup VI and shows 84.1% nucleotide similarity to the NDV-sequences of the DNA vaccine. Our data indicate that birds, which received a single dose of the DNA vaccine were poorly protected, and only 30–40% of these birds survived after challenge with high virus shedding titre. Multiple administration of the DNA vaccine induced high protection rates of 70–90% with reduced virus shedding compared to the non-vaccinated and challenged birds. Generally, homologous challenge led to reduced tracheal and cloacal shedding compared to the heterologous vvNDV strain. This study provides a promising approach for the control of ND in chickens using DNA vaccines, which are phylogenetically closely related to the circulating field strains.

Efficacy of a Recombinant Turkey Herpesvirus H5 Vaccine Against Challenge With H5N1 Clades 1.1.2 and 2.3.2.1 Highly Pathogenic Avian Influenza Viruses in Domestic Ducks (Anas platyrhynchos domesticus).

Domestic ducks are the second most abundant poultry species in many Asian countries and have played a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI).In this study, the protective efficacy of a live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAI strain (A/Swan/Hungary/4999/ 2006) (rHVT-H5/2.2), given at 3 days of age, was examined in Pekin ducks (Anas platyrhynchos domesticus). The vaccine was given alone or in combination with an inactivated H5N1 clade 2.3.2.1 reverse genetic (rgGD/2.3.2.1) vaccine given at 16 days of age, either as a single vaccination or in a prime-boost regime. At 30 days of age, ducks were challenged with one of two H5N1 HPAI viruses: A/duck/Vietnam/NCVD-2721/2013 (clade 1.1.2) or A/duck/Vietnam/NCVD-1584/2012 (clade 2.3.2.1.C). These viruses produced 100% mortality in less than 5 days in nonvaccinated control ducks. Ducks vaccinated with the rgGD/2.3.2.1 vaccine, with or without the rHVT-H5/2.2 vaccine, were 90%-100% protected against mortality after challenge with either of the two H5N1 HPAI viruses. The rHVT-H5/2.2 vaccine alone, however, conferred only 30% protection against mortality after challenge with either H5N1 HPAI virus; the surviving ducks from these groups shed higher amount of virus and for longer than the single-vaccinated rgGD/2.3.2.1 group. Despite low protection, ducks vaccinated with the rHVT-H5/2.2 vaccine and challenged with the clade 1.1.2 Vietnam virus had a longer mean death time than nonvaccinated controls (P = 0.02). A booster effect was found on reduction of virus shedding when using both vaccines, with lower oropharyngeal viral titers at 4 days after challenge with either HPAI virus (P < 0.05). Neither rHVT-H5/2.2 nor standard HVT vaccine could be detected in samples collected from multiple tissues at different time points, indicting minimal levels of viral replication. In conclusion, although a minor effect on survival was observed, this study demonstrates the suboptimal protection with the rHVT-H5/2.2 vaccine given alone in Pekin ducks against H5N1 HPAI viruses and only a minor additive effect on virus shedding reduction when used with an inactivated vaccine in a prime-boost regime.

Protective efficacy of a prime-boost protocol using H5-DNA plasmid as prime and inactivated H5N2 vaccine as the booster against the Egyptian avian influenza challenge virus.

In this study, a recombinant DNA plasmid was constructed, encoding for HA1 of a selected Egyptian H5N1 virus (isolated during the 2012 outbreaks). In the immunization and challenge experiments, SPF chickens received 1 or 2 doses of H5-DNA plasmid prime, and boosted with the inactivated H5N2 vaccine. Haemagglutination inhibition (HI) titers, protection levels, and the magnitude of virus shedding were compared to that of the chickens that received either DNA plasmid or inactivated H5N2 vaccine alone. H5N1 virus A/chicken/Egypt/128s/2012 (H5N1) highly pathogenic avian influenza (HPAI) clade 2.2.1/C was used for the challenge. Chickens immunized with 1 or 2 doses of H5-DNA vaccine failed to overcome the challenge with 0% and 10% protection, respectively. Quantitative real-time reverse transcription-PCR revealed virus shedding of 2.2 x 104 PCR copies/ml 3 days post challenge (dpc) in the only surviving bird from the group that received 2 doses of plasmid. However, chickens immunized with 1 or 2 doses of H5-DNA plasmid as prime and inactivated H5N2 vaccine as booster, showed 80% protection after challenge, with a viral shedding of 1.2 x 104 PCR copies/ml (1 dose) and 1.6 x 104 PCR copies/ml (2 doses) 3 dpc. The surviving birds in both groups did not shed the virus at 5 and 7 dpc. In H5N2-vaccinated chickens, protection levels were 70% with relatively high virus shedding (1.8 x 104 PCR copies/ml) 3 dpc. HI titers were protective to the surviving chickens. This study reports the efficacy of H5-DNA plasmid to augment reduction in viral shedding and to provide better protection when applied in a prime-boost program with the inactivated AI vaccine.