Multimodal Surveillance Model for Enterovirus D68 Respiratory Disease and Acute Flaccid Myelitis among Children in Colorado, USA, 2022.

Surveillance for emerging pathogens is critical for developing early warning systems to guide preparedness efforts for future outbreaks of associated disease. To better define the epidemiology and burden of associated respiratory disease and acute flaccid myelitis (AFM), as well as to provide actionable data for public health interventions, we developed a multimodal surveillance program in Colorado, USA, for enterovirus D68 (EV-D68). Timely local, state, and national public health outreach was possible because prospective syndromic surveillance for AFM and asthma-like respiratory illness, prospective clinical laboratory surveillance for EV-D68 among children hospitalized with respiratory illness, and retrospective wastewater surveillance led to early detection of the 2022 outbreak of EV-D68 among Colorado children. The lessons learned from developing the individual layers of this multimodal surveillance program and how they complemented and informed the other layers of surveillance for EV-D68 and AFM could be applied to other emerging pathogens and their associated diseases.

Multistate Outbreak of Salmonella Thompson Infections Linked to Seafood Exposure - United States, 2021.

In July 2021, the Colorado Department of Public Health and Environment (CDPHE) laboratory identified a cluster of five Salmonella enterica serotype Thompson isolates related to one another within one allele difference, using whole genome multilocus sequence typing (wgMLST). These five isolates, submitted to the public health laboratory as is routine process for confirmatory testing of Salmonella, were highly related to those identified in a 2020 multistate investigation, during which traceback was conducted for sushi-grade tuna and salmon; a common supplier was not identified. The 2021 investigation commenced on August 5, 2021, with five patients living in Colorado, and one each in Missouri, Washington, and Wisconsin. During August-December 2021, CDC, CDPHE, public health and regulatory officials in several states, and the Food and Drug Administration (FDA) conducted epidemiologic, environmental, and laboratory investigations of this multistate outbreak of Salmonella Thompson. Isolates were genetically related to one another and to 2020 isolates within zero to one allele difference. Implicated seafood products were traced to a single seafood distributor, in which the outbreak strain was identified through environmental sampling, and in which inspection identified inadequate sanitization and opportunities for cross-contamination of raw fish. The distributor issued a voluntary recall of 16 seafood items with high potential for contamination and completed remediation actions. This outbreak illustrated the importance of effective cleaning and sanitizing procedures and implementation of controls. When multiple products are recalled during an outbreak investigation, collaboration between public health agencies and implicated facilities can help provide food safety information to restaurants, retailers, and consumers, and to ensure disposal of all recalled products.

Evaluation of Correctional Facility COVID-19 Outbreaks With Layered Mitigation Strategies Including Vaccination: Colorado, 2020-2021.

In 2020-2021, a Colorado corrections facility experienced four COVID-19 outbreaks. Case counts, attack rates (ARs) in people who are detained or incarcerated (PDI), and mitigation measures used in each outbreak were compared to evaluate effects of combined strategies. Serial PCR testing, isolation/quarantine, and masking were implemented in outbreak 1. Daily staff antigen testing began in outbreak 2. Facility-wide COVID-19 vaccination started in outbreak 3 and coverage increased by the end of outbreak 4 (PDI: <1% to 59%, staff: 27% to 40%). Despite detection of variants of concern, outbreaks 3 and 4 had 97% lower PDI ARs (both 1%) than outbreak 2 (29%). Daily staff testing and increasing vaccination coverage, with other outbreak mitigation strategies, are important to reduce COVID-19 transmission in congregate settings.

Investigation of COVID-19 Outbreak among Wildland Firefighters during Wildfire Response, Colorado, USA, 2020.

A COVID-19 outbreak occurred among Cameron Peak Fire responders in Colorado, USA, during August 2020-January 2021. The Cameron Peak Fire was the largest recorded wildfire in Colorado history, lasting August-December 2020. At least 6,123 responders were involved, including 1,260 firefighters in 63 crews who mobilized to the fire camps. A total of 79 COVID-19 cases were identified among responders, and 273 close contacts were quarantined. State and local public health investigated the outbreak and coordinated with wildfire management teams to prevent disease spread. We performed whole-genome sequencing and applied social network analysis to visualize clusters and transmission dynamics. Phylogenetic analysis identified 8 lineages among sequenced specimens, implying multiple introductions. Social network analysis identified spread between and within crews. Strategies such as implementing symptom screening and testing of arriving responders, educating responders about overlapping symptoms of smoke inhalation and COVID-19, improving physical distancing of crews, and encouraging vaccinations are recommended.

Household Transmission and Symptomology of Severe Acute Respiratory Syndrome Coronavirus 2 Alpha Variant among Children-California and Colorado, 2021.

OBJECTIVE: To assess the household secondary infection risk (SIR) of B.1.1.7 (Alpha) and non-Alpha lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among children. STUDY DESIGN: During January to April 2021, we prospectively followed households with a SARS-CoV-2 infection. We collected questionnaires, serial nasopharyngeal swabs for reverse transcription polymerase chain reaction testing and whole genome sequencing, and serial blood samples for serology testing. We calculated SIRs by primary case age (pediatric vs adult), household contact age, and viral lineage. We evaluated risk factors associated with transmission and described symptom profiles among children. RESULTS: Among 36 households with pediatric primary cases, 21 (58%) had secondary infections. Among 91 households with adult primary cases, 51 (56%) had secondary infections. SIRs among pediatric and adult primary cases were 45% and 54%, respectively (OR, 0.79; 95% CI, 0.41-1.54). SIRs among pediatric primary cases with Alpha and non-Alpha lineage were 55% and 46%, respectively (OR, 1.52; 95% CI, 0.51-4.53). SIRs among pediatric and adult household contacts were 55% and 49%, respectively (OR, 1.01; 95% CI, 0.68-1.50). Among pediatric contacts, no significant differences in the odds of acquiring infection by demographic or household characteristics were observed. CONCLUSIONS: Household transmission of SARS-CoV-2 from children and adult primary cases to household members was frequent. The risk of secondary infection was similar among child and adult household contacts. Among children, household transmission of SARS-CoV-2 and the risk of secondary infection was not influenced by lineage. Continued mitigation strategies (eg, masking, physical distancing, vaccination) are needed to protect at-risk groups regardless of virus lineage circulating in communities.

Comparison of Home Antigen Testing With RT-PCR and Viral Culture During the Course of SARS-CoV-2 Infection.

Importance: As self-collected home antigen tests become widely available, a better understanding of their performance during the course of SARS-CoV-2 infection is needed. Objective: To evaluate the diagnostic performance of home antigen tests compared with reverse transcription-polymerase chain reaction (RT-PCR) and viral culture by days from illness onset, as well as user acceptability. Design, Setting, and Participants: This prospective cohort study was conducted from January to May 2021 in San Diego County, California, and metropolitan Denver, Colorado. The convenience sample included adults and children with RT-PCR-confirmed infection who used self-collected home antigen tests for 15 days and underwent at least 1 nasopharyngeal swab for RT-PCR, viral culture, and sequencing. Exposures: SARS-CoV-2 infection. Main Outcomes and Measures: The primary outcome was the daily sensitivity of home antigen tests to detect RT-PCR-confirmed cases. Secondary outcomes included the daily percentage of antigen test, RT-PCR, and viral culture results that were positive, and antigen test sensitivity compared with same-day RT-PCR and cultures. Antigen test use errors and acceptability were assessed for a subset of participants. Results: This study enrolled 225 persons with RT-PCR-confirmed infection (median [range] age, 29 [1-83] years; 117 female participants [52%]; 10 [4%] Asian, 6 [3%] Black or African American, 50 [22%] Hispanic or Latino, 3 [1%] Native Hawaiian or Other Pacific Islander, 145 [64%] White, and 11 [5%] multiracial individuals) who completed 3044 antigen tests and 642 nasopharyngeal swabs. Antigen test sensitivity was 50% (95% CI, 45%-55%) during the infectious period, 64% (95% CI, 56%-70%) compared with same-day RT-PCR, and 84% (95% CI, 75%-90%) compared with same-day cultures. Antigen test sensitivity peaked 4 days after illness onset at 77% (95% CI, 69%-83%). Antigen test sensitivity improved with a second antigen test 1 to 2 days later, particularly early in the infection. Six days after illness onset, antigen test result positivity was 61% (95% CI, 53%-68%). Almost all (216 [96%]) surveyed individuals reported that they would be more likely to get tested for SARS-CoV-2 infection if home antigen tests were available over the counter. Conclusions and Relevance: The results of this cohort study of home antigen tests suggest that sensitivity for SARS-CoV-2 was moderate compared with RT-PCR and high compared with viral culture. The results also suggest that symptomatic individuals with an initial negative home antigen test result for SARS-CoV-2 infection should test again 1 to 2 days later because test sensitivity peaked several days after illness onset and improved with repeated testing.

Rapid Increase in Circulation of the SARS-CoV-2 B.1.617.2 (Delta) Variant - Mesa County, Colorado, April-June 2021.

On May 5, 2021, the Colorado Department of Public Health and Environment (CDPHE) identified the first five COVID-19 cases caused by the SARS-CoV-2 B.1.617.2 (Delta) variant in Mesa County in western Colorado (population 154,933, <3% of the state population). All five initial cases were associated with school settings. Through early June, Mesa County experienced a marked increase in the proportion of Delta variant cases identified through sequencing: the 7-day proportion of sequenced specimens identified as B.1.617.2 in Mesa County more than doubled, from 43% for the week ending May 1 to 88% for the week ending June 5. As of June 6, more than one half (51%) of sequenced B.1.617.2 specimens in Colorado were from Mesa County. CDPHE assessed data from surveillance, vaccination, laboratory, and hospital sources to describe the preliminary epidemiology of the Delta variant and calculate crude vaccine effectiveness (VE). Vaccination coverage in early May in Mesa County was lower (36% of eligible residents fully vaccinated) than that in the rest of the state (44%). Compared with that in all other Colorado counties, incidence, intensive care unit (ICU) admissions, and COVID-19 case fatality ratios were significantly higher in Mesa County during the analysis period, April 27-June 6, 2021. In addition, during the same time period, the proportion of COVID-19 cases in persons who were fully vaccinated (vaccine breakthrough cases) was significantly higher in Mesa County compared with that in all other Colorado counties. Estimated crude VE against reported symptomatic infection for a 2-week period ending June 5 was 78% (95% confidence interval [CI] = 71%-84%) for Mesa County and 89% (95% CI = 88%-91%) for other Colorado counties. Vaccination is a critical strategy for preventing infection, serious illness, and death from COVID-19. Enhanced mitigation strategies, including masking in indoor settings irrespective of vaccination status, should be considered in areas with substantial or high case rates.

A National Antimicrobial Resistance Monitoring System Survey of Antimicrobial-Resistant Foodborne Bacteria Isolated from Retail Veal in the United States.

ABSTRACT: Little is known about the prevalence of antimicrobial-resistant (AMR) bacteria in veal meat in the United States. We estimated the prevalence of bacterial contamination and AMR in various veal meats collected during the 2018 U.S. National Antimicrobial Resistance Monitoring System (NARMS) survey of retail outlets in nine states and compared the prevalence with the frequency of AMR bacteria from other cattle sources sampled for NARMS. In addition, we identified genes associated with resistance to medically important antimicrobials and gleaned other genetic details about the resistant organisms. The prevalence of Campylobacter, Salmonella, Escherichia coli, and Enterococcus in veal meats collected from grocery stores in nine states was 0% (0 of 358), 0.6% (2 of 358), 21.1% (49 of 232), and 53.5% (121 of 226), respectively, with ground veal posing the highest risk for contamination. Both Salmonella isolates were resistant to at least one antimicrobial agent as were 65.3% (32 of 49) of E. coli and 73.6% (89 of 121) of Enterococcus isolates. Individual drug and multiple drug resistance levels were significantly higher (P < 0.05) in E. coli and Enterococcus from retail veal than in dairy cattle ceca and retail ground beef samples from 2018 NARMS data. Whole genome sequencing was conducted on select E. coli and Salmonella from veal. Cephalosporin resistance (blaCMY and blaCTX-M), macrolide resistance (mph), and plasmid-mediated quinolone resistance (qnr) genes and gyrA mutations were found. We also identified heavy metal resistance genes ter, ars, mer, fieF, and gol and disinfectant resistance genes qac and emrE. An stx1a-containing E. coli was also found. Sequence types were highly varied among the nine E. coli isolates that were sequenced. Several plasmid types were identified in E. coli and Salmonella, with the majority (9 of 11) of isolates containing IncF. This study illustrates that veal meat is a carrier of AMR bacteria.

Identification of and Surveillance for the SARS-CoV-2 Variants B.1.427 and B.1.429 - Colorado, January-March 2021.

The B.1.427 and B.1.429 variants of SARS-CoV-2, the virus that causes COVID-19, were first described in Southern California on January 20, 2021 (1); on March 16 they were designated variants of concern* (2). Data on these variants are limited, but initial reports suggest that, compared with other lineages, they might be more infectious (1,2), cause more severe illness (2), and be less susceptible to neutralizing monoclonal antibody products such as bamlanivimab, an investigational treatment for mild-to-moderate COVID-19 (1-3). On January 24, the Colorado Department of Public Health and Environment (CDPHE) identified the first Colorado case of COVID-19 attributed to these variants. B.1.427 and B.1.429 were considered a single variant described as CAL.20C or B.1.427/B.1.429 in the 20C clade (1,3); in this report "B.1.427/B.1.429" refers to B.1.427 or B.1.429 lineage, including those reported as B.1.427/B.1.429 without further differentiation.

Porcine circovirus type 2a or 2b based experimental vaccines provide protection against PCV2d/porcine parvovirus 2 co-challenge.

With the discovery of Porcine circovirus type 2d (PCV2d) in the USA in 2012 and subsequent genotype shift from the previously predominant PCV2b to PCV2d in the face of widespread PCV2a vaccination, concerns over PCV2 vaccine efficacy were raised. The objective of this study was to evaluate the efficacy of two similarly produced PCV2 vaccines, one containing the PCV2a capsid and the other one containing the PCV2b capsid, in the conventional pig model against PCV2d/porcine parvovirus 2 (PPV2) co-challenge. A co-challenge was added since there is evidence that PPV2 may exacerbate PCV2 infection and since PCV2 only rarely causes disease in experimentally infected pigs, hence vaccine efficacy can be difficult to assess. In brief, sixty 3-week-old-pigs from a PCV2 seropositive farm without evidence of active virus replication (no PCV2 viremia, low antibody titers with no evidence of increase after two consecutive bleedings) were blocked by PCV2 antibody titer and then randomly divided into three groups with 20 pigs each, a non-vaccinated group (challenge control), a PCV2a vaccinated group (VAC2a) and a PCV2b vaccinated group (VAC2b). Vaccinations were done at 4 and again at 6 weeks of age. At 8 weeks of age, all pigs were challenged with a PCV2d strain via intranasal and intramuscular routes of inoculation followed by intramuscular administration of PPV2 one day later. PCV2 vaccination, regardless of PCV2 genotype, resulted in significantly higher humoral and cellular immunity compared to non-vaccinated challenge control pigs as evidenced by increased numbers of interferon (IFN) γ secreting cells after PCV2d stimulation of peripheral blood mononuclear cells collected prior to challenge. Furthermore, PCV2a and PCV2b vaccinations both reduced PCV2d viremia and PCV2-associated pathological lesions. Under the study conditions, the PCV2a and PCV2b vaccine preparations each induced immune responses and clinical protection against a heterologous PCV2d/PPV2 co-challenge.

A Porcine circovirus type 2b (PCV2b)-based experimental vaccine is effective in the PCV2b-Mycoplasma hyopneumoniae coinfection pig model.

Porcine circovirus type 2 (PCV2) is one of the major swine pathogens causing high economic losses due to PCV2-associated disease (PCVAD). PCV2 infection is not only immunosuppressive by damaging lymphoid tissues but is also exacerbated by co-infections with other pathogens including Mycoplasma hyopneumoniae. While PCV2 can be divided into several genotypes, currently only PCV2a, PCV2b and PCV2d are globally prevalent and considered of major importance. Most commercial PCV2 vaccines are based on PCV2a isolates; however, the high prevalence of PCV2b and PCV2d in the global pig population is raising concerns among pig veterinarians. The objective of this study was to evaluate the efficacy of an experimental PCV2b-based subunit vaccine in a combined PCV2b and M. hyopneumoniae coinfection model. Briefly, a total of 49 PCV2- and M. hyopneumoniae-free 3-week-old pigs were randomly divided into four groups: A non-vaccinated, non-infected NEG-CONTROL group, a non-vaccinated, PCV2b-infected, POS-CONTROL group, and two vaccinated and PCV2b-infected groups (SINGLE-VAC, DUAL-VAC). SINGLE-VAC and DUAL-VAC pigs were vaccinated at 3 weeks of age and DUAL-VAC pigs received a booster dose at 5 weeks of age. All pigs, except NEG-CONTROLs, were experimentally infected with M. hyopneumoniae 28 days after initial vaccination and challenged with PCV2b one week later. The pigs were necropsied 21 days after PCV2b challenge. Prior to PCV2b challenge, both vaccinated groups had detectable humoral and cell-medicated immune responses to PCV2. Vaccination significantly reduced PCV2b viremia and also reduced or eliminated PCV2-associated lymphoid lesions compared to the POS-CONTROL pigs. Under the study conditions, an experimental PCV2b vaccine protected conventional growing pigs against PCV2b viremia and associated lesions in a coinfection model with some advantages of the two-dose regimen versus the one dose regimen. Both protocols induced neutralizing antibodies against PCV2a and PCV2d prior to challenge.

CD8+ lymphocytes but not B lymphocytes are required for protection against chronic hepatitis E virus infection in chickens.

Hepatitis E is an important global disease, causing outbreaks of acute hepatitis in many developing countries and sporadic cases in industrialized countries. Hepatitis E virus (HEV) infection typically causes self-limiting acute hepatitis but can also progress to chronic disease in immunocompromised individuals. The immune response necessary for the prevention of chronic infection is T cell-dependent; however, the arm of cellular immunity responsible for this protection is not currently known. To investigate the contribution of humoral immunity in control of HEV infection and prevention of chronicity, we experimentally infected 20 wild-type (WT) and 18 immunoglobulin knockout (JH-KO) chickens with a chicken strain of HEV (avian HEV). Four weeks postinfection (wpi) with avian HEV, JH-KO chickens were unable to elicit anti-HEV antibody but had statistically significantly lower liver lesion scores than the WT chickens. At 16 wpi, viral RNA in fecal material and liver, and severe liver lesions were undetectable in both groups. To determine the role of cytotoxic lymphocytes in the prevention of chronicity, we infected 20 WT and 20 cyclosporine and CD8+ antibody-treated chickens with the same strain of avian HEV. The CD8 + lymphocyte-depleted, HEV-infected chickens had higher incidences of prolonged fecal viral shedding and statistically significantly higher liver lesion scores than the untreated, HEV-infected birds at 16 wpi. The results indicate that CD8 + lymphocytes are required for viral clearance and reduction of liver lesions in HEV infection while antibodies are not necessary for viral clearance but may contribute to the development of liver lesions in acute HEV infection.

Evaluation of the pathogenicity of mammalian orthoreovirus type 3 (MRV3) in germ-free gnotobiotic pigs and of the efficacy of an inactivated vaccine against MRV3 infection in neonatal conventional piglets.

A novel U.S. strain of mammalian orthoreovirus type 3 (MRV3) isolated from diarrheic pigs in 2015 was reportedly highly pathogenic in pigs. In this study, we first developed an inactivated MRV3 vaccine and determined its protective efficacy against MRV3 infection in conventional neonatal piglets. A pathogenicity study was also conducted in gnotobiotic pigs to further assess the pathogenicity of MRV3. To evaluate if piglets could be protected against MRV3 infection after immunization of pregnant sows with an inactivated MRV3 vaccine, pregnant sows were vaccinated with 2 or 3 doses of the vaccine or with PBS buffer. Four-day-old piglets born to vaccinated and unvaccinated sows were subsequently challenged with MRV3. The results showed that piglets born from vaccinated sows had lower levels of fecal viral RNA shedding at 1, 3, and 4 days post-challenge, suggesting that the inactivated MRV3 vaccine can reduce MRV3 replication. Surprisingly, although the conventional piglets were infected, they did not develop severe enteric disease as reported previously. Therefore, in an effort to further definitively assess the pathogenicity of MRV3, we experimentally infected gnotobiotic pigs, a more sensitive model for pathogenicity study, with the wild-type MRV3 virus. The infected gnotobiotic piglets all survived and exhibited only very mild diarrhea in some pigs. Taken together, the results indicate that the novel strain of MRV3 recently isolated in the United States infected but caused only very mild diarrhea in pigs, and that maternal immunity acquired from sows vaccinated with an inactivated vaccine can reduce MRV3 replication in neonatal pigs.

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout JH-/- Gnotobiotic Piglets.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH-/- knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH-/- knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH-/- knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH-/- knockout pigs, suggesting that the Ig heavy chain JH-/- knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH-/- knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH-/- knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH-/- knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH-/- knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

A Lipid/DNA Adjuvant-Inactivated Influenza Virus Vaccine Protects Rhesus Macaques From Uncontrolled Virus Replication After Heterosubtypic Influenza A Virus Challenge.

Background: Influenza A virus (IAV) vaccines offer little protection from mismatched viruses with antigenically distant hemagglutinin (HA) glycoproteins. We sought to determine if a cationic lipid/DNA complex (CLDC) adjuvant could induce heterosubtypic protection if added to a whole inactivated IAV vaccine (WIV). Methods: Adult rhesus macaques (RMs) were vaccinated and at 2 weeks boosted with either an H1N1-WIV or an H3N2-WIV, with and without CLDC adjuvant. Four weeks postboost, animals were challenged with an H1N1 IAV matched to the H1N1-WIV vaccine. Results: After challenge, viral RNA (vRNA) levels in the trachea of control RMs and RMs vaccinated with the unadjuvanted H1 or H3 WIV vaccines were similar. However, vRNA levels in the trachea of both the H1-WIV/CLDC- and the H3-WIV/CLDC-vaccinated RMs (P < 0.01 and P < 0.05, respectively) were significantly lower than in unvaccinated control RMs. Heterosubtypic protection in H3-WIV/CLDC RMs was associated with significantly higher levels of nucleoprotein (NP) and matrix-1-specific immunoglobulin G antibodies (P < 0.05) and NP-specific nonneutralizing antibody-dependent natural killer cell activation (P < 0.01) compared with unprotected H3-WIV RMs. Conclusions: Addition of the CLDC adjuvant to a simple WIV elicited immunity to conserved virus structural proteins in RMs that correlate with protection from uncontrolled virus replication after heterosubtypic influenza virus challenge.

Isolation of Peripheral Blood CD8 T Cells Specific to Porcine Reproductive and Respiratory Syndrome Virus Utilizing Porcine CD137 Activation Marker.

CD137 is a costimulatory molecule transiently expressed on activated T cells after mitogen or antigen stimulation that can be exploited for isolating antigen-specific T cells as reported in mouse models. By utilizing an antiporcine CD137 monoclonal antibody (mAb, clone 3B9) developed in our laboratory, we isolated virus-specific CD8ß T cells from peripheral blood of pigs experimentally infected with different porcine reproductive and respiratory syndrome virus (PRRSV) strains. Similar to mouse, porcine CD8ß T cells also express CD137 transiently upon Concavalin A stimulation while the unstimulated cells did not. Most frequently, virus-specific CD8ß T cells were isolated at low levels from peripheral blood of pigs experimentally infected with PRRSV strains VR2385, NADC20, and MN184B at 49 and 63 days postinfection. The results suggest that porcine CD137-specific mAb is a useful tool for isolating virus-specific CD8 T cells from peripheral blood and tissues of pigs after in vitro stimulation with viral antigen.

Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are three of the most economically important swine pathogens, causing immense economic losses to the global swine industry. Monovalent commercial vaccines against each of the three viruses are routinely used in pig farms worldwide. A trivalent vaccine against all three pathogens would greatly simplify the vaccination programme and reduce the financial burden to the swine industry. In this study, by using an attenuated strain of PRRSV (strain DS722) as a live virus vector, we generated a multi-component vaccine virus, DS722-SIV-PCV2, which expresses the protective antigens from SIV and PCV2. The DS722-SIV-PCV2 trivalent vaccine virus replicates well, and expresses PCV2 capsid and SIV HA proteins in vitro. A subsequent vaccination and challenge study in 48 pigs revealed that the DS722-SIV-PCV2-vaccinated pigs had significantly reduced lung lesions and viral RNA loads when challenged with PRRSV. Upon challenge with PCV2, the vaccinated pigs had partially reduced lymphoid lesions and viral DNA loads, and when challenged with SIV the vaccinated pigs had significantly reduced acute respiratory sign scores. The results from this study demonstrate the potential of DS722-SIV-PCV2 as a candidate trivalent vaccine, and also shed light on exploring PRRSV as a potential live virus vaccine vector.

Markedly different immune responses and virus kinetics in littermates infected with porcine circovirus type 2 or porcine parvovirus type 1.

Porcine parvovirus type 1 (PPV1) and porcine circovirus type 2 (PCV2) are small single-stranded DNA viruses with high prevalence in the global pig population. The aim of this study was to compare and contrast PCV2 and PPV1 infections in high-health status pigs and to describe PCV2 long-term infection dynamics. Six caesarian-derived colostrum-deprived pigs were randomly divided into two groups and were experimentally infected with PCV2 or PPV1 at 5 weeks of age. All pigs had detectable viremia by day (D) 3 post-infection. Pigs infected with PPV1 had a detectable INF-α response by D3 followed by a high IFN-γ response by D6. The PPV1 pigs developed antibodies against PPV1 by D6 resulting in decreasing virus titers until PPV1 DNA became undetectable from D28 until D42. In contrast, PCV2-infected pigs had no detectable INF-α or IFN-γ response after PCV2 infection. PCV2-infected pigs had no detectable anti-PCV2 humoral response until D49 and had a sustained high level of PCV2 DNA for the duration of the study. While PPV1-infected pigs were clinically normal, PCV2-infected pigs developed severe clinical illness including fatal systemic porcine circovirus associated disease (PCVAD) by D28, fatal enteric PCVAD by D56 and chronic PCVAD manifested as decreased weight gain and periods of diarrhea. Microscopically, all three PCV2-infected pigs had lymphoid lesions consistent with PCVAD and associated with low (chronic disease) to high (acute disease) levels of PCV2 antigen. Under the study conditions, there was a lack of early IFN-γ and INF-α activation followed by a delayed and low humoral immune response and persisting viremia with PCV2 infection. In contrast, PPV1-infected pigs had IFN-γ and INF-α activation and an effective immune response to the PPV1 infection.