How Can Vaccines Against Influenza and Other Viral Diseases Be Made More Effective?

A large fraction of the world’s most widespread and problematic pathogens, such as the influenza virus, seem to persist in nature by evading host immune responses by inducing immunity to genetically and phenotypically plastic epitopes (aka antigenic variation). The more recent re-emergence of pandemic influenza A/ H1N1 and avian H5N1 viruses has called attention to the urgent need for more effective influenza vaccines. Developing such vaccines will require more than just moving from an egg-based to a tissueculture–based manufacturing process. It will also require a new conceptual understanding of pathogen–host interactions, as well as new approaches and technologies to circumvent immune evasion by pathogens capable of more genetic variation. Here, we discuss these challenges, focusing on some potentially fruitful directions for future research

Experimental infection of calves by two genetically-distinct strains of rift valley fever virus

Citation: Wilson, W. C., Davis, A. S., Gaudreault, N. N., Faburay, B., Trujillo, J. D., Shivanna, V., . . . Richt, J. A. (2016). Experimental infection of calves by two genetically-distinct strains of rift valley fever virus. Viruses, 8(5). doi:10.3390/v8050145Additional Authors: McVey, D. S.Recent outbreaks of Rift Valley fever in ruminant livestock, characterized by mass abortion and high mortality rates in neonates, have raised international interest in improving vaccine control strategies. Previously, we developed a reliable challenge model for sheep that improves the evaluation of existing and novel vaccines in sheep. This sheep model demonstrated differences in the pathogenesis of Rift Valley fever virus (RVFV) infection between two genetically-distinct wild-type strains of the virus, Saudi Arabia 2001 (SA01) and Kenya 2006 (Ken06). Here, we evaluated the pathogenicity of these two RVFV strains in mixed breed beef calves. There was a transient increase in rectal temperatures with both virus strains, but this clinical sign was less consistent than previously reported with sheep. Three of the five Ken06-infected animals had an early-onset viremia, one day post-infection (dpi), with viremia lasting at least three days. The same number of SA01-infected animals developed viremia at 2 dpi, but it only persisted through 3 dpi in one animal. The average virus titer for the SA01-infected calves was 1.6 logs less than for the Ken06-infected calves. Calves, inoculated with either strain, seroconverted by 5 dpi and showed time-dependent increases in their virus-neutralizing antibody titers. Consistent with the results obtained in the previous sheep study, elevated liver enzyme levels, more severe liver pathology and higher virus titers occurred with the Ken06 strain as compared to the SA01 strain. These results demonstrate the establishment of a virulent challenge model for vaccine evaluation in calves. © 2016 by the authors; licensee MDPI, Basel, Switzerland

Rift Valley Fever Phlebovirus Reassortment Study in Sheep

Rift Valley fever (RVF) in ungulates and humans is caused by a mosquito-borne RVF phlebovirus (RVFV). Live attenuated vaccines are used in livestock (sheep and cattle) to control RVF in endemic regions during outbreaks. The ability of two or more different RVFV strains to reassort when co-infecting a host cell is a significant veterinary and public health concern due to the potential emergence of newly reassorted viruses, since reassortment of RVFVs has been documented in nature and in experimental infection studies. Due to the very limited information regarding the frequency and dynamics of RVFV reassortment, we evaluated the efficiency of RVFV reassortment in sheep, a natural host for this zoonotic pathogen. Co-infection experiments were performed, first in vitro in sheep-derived cells, and subsequently in vivo in sheep. Two RVFV co-infection groups were evaluated: group I consisted of co-infection with two wild-type (WT) RVFV strains, Kenya 128B-15 (Ken06) and Saudi Arabia SA01-1322 (SA01), while group II consisted of co-infection with the live attenuated virus (LAV) vaccine strain MP-12 and a WT strain, Ken06. In the in vitro experiments, the virus supernatants were collected 24 h post-infection. In the in vivo experiments, clinical signs were monitored, and blood and tissues were collected at various time points up to nine days post-challenge for analyses. Cell culture supernatants and samples from sheep were processed, and plaque-isolated viruses were genotyped to determine reassortment frequency. Our results show that RVFV reassortment is more efficient in co-infected sheep-derived cells compared to co-infected sheep. In vitro, the reassortment frequencies reached 37.9% for the group I co-infected cells and 25.4% for the group II co-infected cells. In contrast, we detected just 1.7% reassortant viruses from group I sheep co-infected with the two WT strains, while no reassortants were detected from group II sheep co-infected with the WT and LAV strains. The results indicate that RVFV reassortment occurs at a lower frequency in vivo in sheep when compared to in vitro conditions in sheep-derived cells. Further studies are needed to better understand the implications of RVFV reassortment in relation to virulence and transmission dynamics in the host and the vector. The knowledge learned from these studies on reassortment is important for understanding the dynamics of RVFV evolution

Rift Valley fever viral RNA detection by in situ hybridization in formalin-fixed, paraffin-embedded tissues

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.This work was done in partial completion of Izabela Ragan’s PhD dissertation.The USDA Agricultural Research Service and the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University’s startup funds for Dr. Davis. This work was also supported through the Department of Homeland Security (grant no. 2010-ST061-AG0001), Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD).http://online.liebertpub.com/VBZ2020-06-26hj2019Paraclinical Science

Rift Valley Fever virus M and L genome segment detection : a comparison of field-deployable reverse transcription insulated isothermal PCR (RT-iiPCR) and laboratory-based multiplex reverse transcription real-time PCR

SUPPLEMENTARY MATERIAL : TABLE S1 (JCM00430-23-S0001.pdf). RVFV reference panel performance and reproducibility.Rift Valley Fever phlebovirus (RVFV) is a mosquito-borne zoonotic pathogen that causes major agricultural and public health problems in Africa and the Arabian Peninsula. It is considered a potential agro-bioterrorism agent for which limited countermeasures are available. To address diagnostic needs, here we describe a rapid and sensitive molecular method immediately employable at sites of suspected outbreaks in animals that commonly precede outbreaks in humans. The strategy involves the concurrent detection of two of the three RVFV genome segments (large and medium) using reverse transcription insulated isothermal PCR (RT-iiPCR) performed on a portable, touch screen nucleic acid analyzer, POCKIT. The analytical sensitivity for both the RT-iiPCR and a laboratory-based L and M multiplex reverse transcription real-time PCR assay was estimated at approximately 0.1–3 copies/reaction using synthetic RNA or viral RNA. The diagnostic sensitivity and specificity of detection of RVFV on the POCKIT, determined using sera from sheep and cattle (n = 181) experimentally infected with two strains of RVFV (SA01 and Ken06), were 93.8% and 100% (kappa = 0.93), respectively. Testing of ruminant field sera (n = 193) in two locations in Africa demonstrated 100% diagnostic sensitivity and specificity. We conclude that the POCKIT dual-gene RVFV detection strategy can provide reliable, sensitive, and specific point-of-need viral RNA detection. Moreover, the field detection of RVFV in vectors or susceptible animal species can aid in the surveillance and epidemiological studies to better understand and control RVFV outbreaks.The US Department of Homeland Security, Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD); the National Bio and Agro-Defense Facility (NBAF) Transition Fund from the State of Kansas; the MCB and AMP Core of the Center on Emerging and Zoonotic Infectious Diseases (CEZID) of the National Institutes of General Medical Sciences; the College of Veterinary Medicine at Iowa State University; and the Center of Advanced Host Defenses, Immunobiotics and Comparative Translational Medicine at Iowa State University.https://journals.asm.org/journal/jcmhj2024Production Animal StudiesVeterinary Tropical DiseasesSDG-03:Good heatlh and well-bein

Immunization of pigs with replication-incompetent adenovirus-vectored African swine fever virus multi-antigens induced humoral immune responses but no protection following contact challenge

IntroductionAfrican swine fever virus (ASFV) is a pathogen of great economic importance given that continues to threaten the pork industry worldwide, but there is no safe vaccine or treatment available. Development of a vaccine is feasible as immunization of pigs with some live attenuated ASFV vaccine candidates can confer protection, but safety concerns and virus scalability are challenges that must to be addressed. Identification of protective ASFV antigens is needed to inform the development of efficacious subunit vaccines.MethodsIn this study, replication-incompetent adenovirus-vectored multicistronic ASFV antigen expression constructs that covered nearly 100% of the ASFV proteome were generated and validated using ASFV convalescent serum. Swine were immunized with a cocktail of the expression constructs, designated Ad5-ASFV, alone or formulated with either Montanide ISA-201™ (ASFV-ISA-201) or BioMize® adjuvant (ASFV-BioMize).ResultsThese constructs primed strong B cell responses as judged by anti-pp62-specific IgG responses. Notably, the Ad5-ASFV and the Ad5-ASFV ISA-201, but not the Ad5-ASFV BioMize®, immunogens primed significantly (p < 0.0001) higher anti-pp62-specific IgG responses compared with Ad5-Luciferase formulated with Montanide ISA-201™ adjuvant (Luc-ISA-201). The anti-pp62-specific IgG responses underwent significant (p < 0.0001) recall in all the vaccinees after boosting and the induced antibodies strongly recognized ASFV (Georgia 2007/1)-infected primary swine cells. However, following challenge by contact spreaders, only one pig nearly immunized with the Ad5-ASFV cocktail survived. The survivor had no typical clinical symptoms, but had viral loads and lesions consistent with chronic ASF.DiscussionBesides the limited sample size used, the outcome suggests that in vivo antigen expression, but not the antigen content, might be the limitation of this immunization approach as the replication-incompetent adenovirus does not amplify in vivo to effectively prime and expand protective immunity or directly mimic the gene transcription mechanisms of attenuated ASFV. Addressing the in vivo antigen delivery limitations may yield promising outcomes

Persistence of African Swine Fever Virus in Feed and Feed Mill Environment over Time after Manufacture of Experimentally Inoculated Feed

To reduce the risk of disease from harmful feed-based pathogens, some feed manufacturers quarantine high-risk ingredients prior to their inclusion in feed. Data exist that confirms this practice is effective, but to our knowledge there is no information about porcine pathogen survival in mill environments. The objective of this study was to determine survival of African swine fever virus (ASFV) in swine feed and on mill surfaces after manufacture of experimentally inoculated swine feed. A pilot-scale feed mill was placed within a biosecurity level (BSL) 3 facility to manufacture batches of feed. The priming batch, Batch 1, was ASFV-free feed and was followed with Batch 2 which was experimentally inoculated with ASFV (5.6 × 104 TCID50/gram). Four subsequent ASFV-free batches were then manufactured (Batch 3-6). After each batch of feed, 10 feed samples were aseptically collected in a double ‘X’ pattern. During feed manufacturing, 24 steel coupons were placed on the floor of the manufacturing area and feed dust was allowed to settle onto them overnight. Once feed manufacturing was completed, feed samples and steel coupons were stored at room temperature. On the day of (day 0) and d 3, 7, 14, 28, 60, 90, and 180 after feed manufacturing, feed samples and 3 steel coupons were randomly selected, taken out of storage, and analyzed for ASFV DNA. For feed samples there was a statistically significant (P = 0.023) batch × day interaction for log10 genomic copies per gram of feed, and a marginal statistical significance (P = 0.072) for batch × day interaction for cycle threshold (Ct) values. This indicates that the batch of feed and days held at room temperature impacted the amount of the detectable ASFV DNA in feed samples. There was no evidence (P = 0.433) of ASFV degradation on environmental coupons over the 180-d storage period. This study found that quarantine time can help reduce, but not eliminate ASFV DNA in feed over time. Surprisingly, ASFV DNA is detectable on feed manufacturing surfaces for at least 180 days

Effect of mixing and feed batch sequencing on the prevalence and distribution of African swine fever virus in swine feed

It is critical to have methods that can detect and mitigate the risk of African swine fever virus (ASFV) in potentially contaminated feed or ingredients bound for the United States. The purpose of this work was to evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and to determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University's Biosafety Research Institute in Manhattan, Kansas. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for final concentration of 5.6 × 104 TCID50/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a collection container, 10 samples were collected in a double ‘X’ pattern. Samples were analysed using a qPCR assay for ASFV p72 gene then the cycle threshold (Ct) and Log10 genomic copy number (CN)/g of feed were determined. The qPCR Ct values (p < .0001) and the Log10 genomic CN/g (p < .0001) content of feed samples were impacted based on the batch of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest amounts of ASFV p72 DNA across all criteria (p < .05). Quantity of ASFV p72 DNA decreased sequentially as additional batches of feed were manufactured, but was still detectable after batch sequence 4. This subsampling method was able to identify ASFV genetic material in feed samples using p72 qPCR. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients can be accurately evaluated for ASFV contamination by collecting 10 subsamples using the sampling method described herein. Future research is needed to evaluate if different mitigation techniques can reduce ASFV feed contamination

Evaluating the distribution of African swine fever virus within a feed mill environment following manufacture of inoculated feed

11 Pág. Centro de Investigación en Sanidad Animal (CISA)It is critical to understand the role feed manufacturing may have regarding potential African swine fever virus (ASFV) transmission, especially given the evidence that feed and/or ingredients may be potential vectors. The objective of the study was to evaluate the distribution of ASFV in a feed mill following manufacture of contaminated feed. To accomplish this, a pilot-scale feed mill consisting of a mixer, bucket elevator, and spouting was constructed in a BSL-3Ag facility. First, a batch of ASFV-free feed was manufactured, followed by a batch of feed that had an ASFV-contaminated ingredient added to feed, which was then mixed and discharged from the equipment. Subsequently, four additional ASFV-free batches of feed were manufactured using the same equipment. Environmental swabs from 18 locations within the BSL-3Ag room were collected after each batch of feed was discharged. The locations of the swabs were categorized into four zones: 1) feed contact surface, 2) non-feed contact surface 1 meter from feed, and 4) transient surfaces. Environmental swabs were analyzed using a qPCR specific for the ASFV p72 gene and reported as genomic copy number (CN)/mL of environmental swab processing buffer. Genomic copies were transformed with a log10 function for statistical analysis. There was no evidence of a zone × batch interaction for log10 genomic CN/mL (P = 0.625) or cycle threshold (Ct) value (P = 0.608). Sampling zone impacted the log10 p72 genomic CN/mL (P < 0.0001) and Ct values (P < 0.0001), with a greater amount of viral genome detected on transient surfaces compared to other surfaces (P < 0.05). This study illustrates that once ASFV enters the feed mill environment it becomes widespread and movement of people can significantly contribute to the spread of ASFV in a feed mill environment.Funding for this work was obtained from the NBAF Transition Funds from the state of Kansas (JAR), the National Pork Board under award number 20-018 (CKJ), the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006 (JAR), and the AMP Core of the NIGMS COBRE Center on Emerging and Zoonotic Infectious Diseases (CEZID) under award number P20GM13044 (JAR)Peer reviewe

Prevalence and Distribution of African Swine Fever Virus in Swine Feed After Mixing and Feed Batch Sequencing

As the United States maintains trade with countries where African swine fever virus (ASFV) is endemic, it is critical to have methods that can detect and mitigate the risk of ASFV in potentially contaminated feed or ingredients. Therefore, the objectives of this study were to 1) evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and 2) determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University’s Biosafety Research Institute in Manhattan, KS. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for a final concentration of 5.6 × 104 TCID50/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a biohazard tote, 10 samples were collected in a double ‘X’ pattern. Samples were analyzed using a qPCR assay specific for the ASFV p72 gene to determine the cycle threshold (Ct) and log10 genomic copy number (CN)/g of feed. Batch of feed affected the qPCR Ct values (P \u3c 0.0001) and the log10 genomic CN/g (P \u3c 0.0001) content of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest (P \u3c 0.05) amounts of ASFV p72 DNA across all criteria. Quantity of ASFV p72 DNA decreased sequentially as additional batches of initially ASFV-free feed were manufactured, but it was still detectable after batch sequence 4, suggesting cross contamination between batches. This subsampling method was able to identify ASFV genetic material in feed samples using the PCR assay specific for the ASFV p72 gene. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients or feed can be accurately evaluated for ASFV contamination by collecting 10 evenly distributed subsamples, representing 0.05% of the volume of the container, using the sampling method described herein