Reciprocal Neutralization Research Articles

LB8. Lower SARS-CoV-2 IgG and Pseudovirus Neutralization Titers Post-mRNA Vaccination among People Living with HIV

BackgroundLimited data are available on whether there are differences in the immune response to SARS-CoV-2 vaccination by HIV status or by mRNA vaccine type.MethodsWe saved residual outpatient laboratory samples of all previously mRNA-vaccinated individuals in the adult medicine clinics of a public hospital with a large outpatient HIV clinic during May 2021, and then excluded individuals with prior SARS-CoV-2 infection. We next 1:1 matched 100 PLWH to 100 outpatient HIV-negative adult medicine patients receiving care for chronic medical conditions on days since completion of second vaccination (minimum 10), sex, age +/-5 years, and the type of mRNA vaccine received. We defined a non-response as reciprocal pseudovirus neutralizing titer< 10 and anti-RBD IgG< 10 relative fluorescent units, and compared non-response by HIV status using mixed models.ResultsIn each matched group there were 13 women; 25 received the mRNA-1273 vaccine and 75 received the BNT162b2 vaccine; the median age was 59. The median time from second vaccination was 35 days (IQR: 20–63). Among PLWH, the median CD4+ T-cell count was 511 (IQR: 351–796) and 5 individuals had HIV RNA > 200.We found 2.4-fold greater odds of pseudovirus neutralizing antibody non-response among PLWH compared to people without HIV (95% CI=1.1–5.4). Although few individuals in each group did not mount an IgG response (12 among PLWH vs. 5; p=0.08), continuous anti-RBD IgG concentrations were 43% lower among PLWH (95% CI=0.36–0.88).Among PLWH, when adjusting for age, sex, and days post-vaccination, each 100-cell increase in CD4+T-cell count was associated with 22% higher neutralizing antibody titers (GMR 1.22; 95% CI=1.09–1.37). Unsuppressed HIV RNA >200 was associated with 89% lower neutralizing antibody titers (GMR 0.11; 95% CI=0.01–0.84). Receipt of the BNT162b2 vs. mRNA-1273 vaccine was associated with 77% lower neutralizing titers (GMR 0.23; 95% CI=0.08–0.65) among PLWH.Post-mRNA Vaccination SARS-CoV-2 IgG Concentrations and Pseudovirus Neutralizing Titers by HIV Status and VaccineConclusionPLWH had lower than expected response to mRNA SARS-CoV-2 vaccines, with the highest non-response among those with low CD4+ counts, unsuppressed HIV RNA, and those who received the BNT162b2 vaccine. Immunization strategies to improve immune responses among PLWH should be studied, and may include booster vaccination or preference of the mRNA-1273 vaccine in this group. Disclosures Matthew A. Spinelli, MD, MAS, Nothing to disclose Monica Gandhi, MD, MPH, Nothing to disclose

Towards a complete Logical Phonology model of intrasegmental changes

All changes to the internal structure of phonological segments arise from combinations of rules based on two set-theoretic operations: feature deletion by set subtraction and feature insertion by unification. Apparent cases of rules targeting underspecified segments reflect two kinds of vacuous rule application, one due to unification failure and the other due to vacuous unification. Despite this reduction of all segment-internal changes to two basic mechanisms we can account for a wide variety of patterns, including the reciprocal neutralization and apparent exceptional behavior seen in Hungarian voicing assimilation.

Survey of poliovirus antibodies in Borno and Yobe States, North-Eastern Nigeria.

BackgroundNigeria remains one of only three polio-endemic countries in the world. In 2016, after an absence of 2 years, wild poliovirus serotype 1 was again detected in North-Eastern Nigeria. To better guide programmatic action, we assessed the immunity status of infants and children in Borno and Yobe states, and evaluated the impact of recently introduced inactivated poliovirus vaccine (IPV) on antibody seroprevalence.Methods and findingsWe conducted a facility-based study of seroprevalence to poliovirus serotypes 1, 2 and 3 among health-seeking patients in two sites each of Borno and Yobe States. Enrolment was conducted amongst children 6–9 and 36–47 months of age attending the paediatrics outpatient department of the selected hospitals in the two states between 11 January and 5 February 2016. Detailed demographic and immunization history of the child was taken and an assessment of the child’s health and nutritional state was conducted via physical examination. Blood was collected to test for levels of neutralizing antibody titres against the three poliovirus serotypes. The seroprevalence in the two age groups, potential determinants of seropositivity and the impact of one dose of IPV on humoral immunity were assessed. A total of 583 subjects were enrolled and provided sufficient quantities of serum for testing. Among 6-9-month-old infants, the seroprevalence was 81% (74–87%), 86% (79–91%), and 72% (65–79%) in Borno State, and 75% (67–81%), 74% (66–81%) and 69% (61–76%) in Yobe States, for serotypes-1, 2 and 3, respectively. Among children aged 36–47 months, the seroprevalence was >90% in both states for all three serotypes, with the exception of type 3 seroprevalence in Borno [87% (80–91%)]. Median reciprocal anti-polio neutralizing antibody titers were consistently >900 for serotypes 1 and 2 across age groups and states; with lower estimates for serotype 3, particularly in Borno. IPV received in routine immunization was found to be a significant determinant of seropositivity and anti-polio neutralizing antibodies among 6-9-month-old infants for serotypes 1 and 3, but demonstrated a non-significant positive association for serotype 2. Children receiving IPV through SIAs demonstrated significantly higher anti-polio neutralizing antibodies for serotypes 1 and 3.ConclusionsThe seroprevalence to poliovirus remains suboptimal in both Borno and Yobe States in Nigeria. The low seroprevalence facilitated the continued transmission of both wild serotype 1 and serotype 2 circulating vaccine-derived poliovirus detected in Borno State in 2016. Further efforts are necessary to improve the immunity status of these populations to ensure sufficient population immunity to interrupt transmission.

Amino Acid Anions in Organic Ionic Compounds. An ab Initio Study of Selected Ion Pairs

The combination of amino acids in their deprotonated and thus anionic form with a choline cation gives origin to a new and potentially important class of organic ionic compounds. A series of such neutral ion pairs has been investigated by first principle methods. The results reveal intriguing structural motives as well as regular patterns in the charge distribution and predict a number of vibrational and optical properties that could guide the experimental investigation of these compounds. The replacement of choline with its phosphocholine analogue causes the spontaneous reciprocal neutralization of cations and anions, taking place through the transfer of a proton between the two ions. Systems of this kind, therefore, provide a wide and easily accessible playground to probe the ionic/polar transition in organic systems, while the easy transfer of H(+) among neutral and ionic species points to their potential application as proton conductors. The analysis of the ab initio data highlights similarities as well as discrepancies from the rigid-ions force-field picture and suggests directions for the improvement of empirical models.

Highly efficient neutralization of human immunodeficiency viruses by plasma from antiretroviral drug treated patients is mediated by IgG fractions

Background Little is known about the neutralizing activity in patients on antiretroviral therapy (ART), as most recent studies have focused on drug naive individuals. ART may lead to a significant increase in B cell numbers and normalization of B cell subpopulations, providing a possible explanation for improved B cell responses after ART. Methods Thirty-four HIV-1 seropositive patients on ART (25 males and 9 females) within the age range of 20-55 years were recruited in this study. The patients had a median CD4 count and viral load of 283 cells and 178 RNA copies respectively, and were on treatment for a few days up to two years. Heat inactivated plasma samples were tested for neutralization against a panel of 14 subtype-A, B and C tier 1 and tier 2 viruses in TZM-bl assay. Results Of the 34 plasma samples, remarkably all the plasma samples were able to neutralize at least one virus while 32 (94%) samples were found to neutralize ≥50% viruses tested. Clustering analysis revealed that AIIMS253 (a clade-C virus) was the most sensitive while RHPA4259.7 (a clade-B isolate) was most resistant to antibody neutralization. The Immunoglobulin-G fractions from two representative samples AIIMS221 and AIIMS265 were shown to mediate neutralization exclusively. The IgG fractions retained binding to subtype-A, B and C recombinant gp120 proteins. We did not find any association of mean reciprocal ID50 neutralization titers with the plasma levels of ART drugs and clinical and immunological variables like CD4 count (p=0.35), viral load (p=0.37) and plasma total IgG (p=0.46). However we observed a positive association of neutralization with duration of ART (p=0.02) with a similar trend in two follow up patient samples. Conclusion

Neutralization of Tier-2 Viruses and Epitope Profiling of Plasma Antibodies from Human Immunodeficiency Virus Type 1 Infected Donors from India

Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19–27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.

Studies of the Antigenic relationships between Bluetongue virus serotypes 2, 9 &amp; 15 isolated in Andhra Pradesh, India

The presence of multiple serotypes of the midge-borne bluetongue virus and lack of effective vaccine are the major impediments in controlling bluetongue in sheep. Attempts are being made to develop a vaccine employing the available serotypes to control the disease in the state. Hence, it is essential to identify the antigenic relationships among the serotypes to identify the candidate strains to be incorporated in the preparation of vaccine. To understand the antigenic relationships between Bluetongue virus -2, 9 and 15 serotypes, the viruses were propagated in BHK21 cell lines, purified using PEG precipitation method and purified virus used to raise hyper immune serum in rabbits. Neutralizing antibodies for the BTV serotypes were detected by day 21 PI. Reciprocal cross neutralization test was employed to determine the R% values between BTV-2, 9 and 15 which indicated the extent of antigenic relationships among the serotypes. R% value between BTV-2 and BTV-9 was recorded as 2.8. R% value of 3.53 and 2.8 were observed between BTV-2 & 15 and BTV-9 & 15 respectively. The R% values recorded in the present study revealed a weak antigenic relationship between the BTV serotypes, indicating that the serotypes are highly divergent.

Detection of Herpes Simplex Virus Antibodies Using the Whole Virus and Recombinant gD

Background and Aims: Herpes simplex virus type 1 (HSV1) remains a potentially serious health problem world wide. All infected people, including asymptomatic ones, are potential sources for virus transmission. Virus envelope contains at least 13 glycoproteins, which glycoprotein D is the major target of immune responses. The aim of this study was development of a specific method that is a more rapid, sensitive and specific test compared to the virus neutralization test which is applied as gold standard test. Methods: In this study, the Western blot technique using crude HSV1 whole particle and baculovirus derived glycoprotein D of HSV1 as antigens was set. Human sera were analyzed by virus neutralization test and then serum samples with reciprocal virus neutralization antibody titers of 32, 64, and 128 were taken to be analyzed by Western blotting. Results: It was shown that there was a very good correlation between results obtained from virus neutralization antibody titers and those of Western blotting. Western blotting using recombinant glycoprotein D of HSV1 as an antigen showed positive results similar to the whole HSV1 antigen.

Young Infants Can Develop Protective Levels of Neutralizing Antibody after Infection with Respiratory Syncytial Virus

Humoral immunity protects against severe respiratory syncytial virus (RSV) disease, but the range and magnitude of antibody responses in RSV-naive children after RSV infection have not been completely defined. We evaluated RSV-neutralizing antibody and immunoglobulin G responses to RSV F and G glycoproteins in 65 RSV-naive Navajo and White Mountain Apache children aged 0-24 months who were hospitalized with RSV infection. In these children, antibody responses developed against RSV F and G and the central conserved region of RSV G. Twenty-seven of 41 infants <6 months old developed reciprocal log(2) RSV neutralizing antibody titers > or =8.0, which correlate with protection of the lower respiratory tract. Multivariate analysis demonstrated that the level of preexisting neutralizing antibody at infection, not age, was the most important factor influencing this response. RSV can induce substantial neutralizing antibody responses in young infants when the titer of preexisting antibodies is low.

Haemagglutination as a confirmatory test for Peste des petits ruminants diagnosis

Seven Peste des petits ruminants (PPR) viruses and a RPV (vaccine strain) and their respective antiserum raised in rabbits were used for reciprocal cross neutralization test and haemagglutination (HA) and haemagglutination inhibition (HI) test employing 0.5% chicken RBC. Although many differential diagnostic methods for PPR and RP (rinderpest) are available viz., immunocapture ELISA, ‘N’ gene radioactive cDNA probes, non radioactively labelled biotinylated cDNA probes, reverse transcription-polymerase chain reaction (RT-PCR) for ‘F’ gene along with reciprocal cross neutralization test, HA and HI tests could be used as a reliable alternative in field conditions particularly in places where RP and PPR are co-existing. The advantages of the tests have been discussed in comparison with reciprocal cross neutralization tests.

558. Anti-SARS Humoral and Cellular Immunity Evoked by an Adenovirus Vector Expressing Spike Glycoprotein from SARS Coronavirus

Top of pageAbstract Severe acute respiratory syndrome (SARS) is a newly described disease caused by SARS-CoV, a novel coronavirus. Due to the limited treatment options and concern over consequences of SARS re-emergence, we have focused on using adenovirus (Ad)-based gene transfer vectors encoding SARS-CoV specific antigens to develop an effective vaccine. We tested the hypothesis that expression of the SARS-CoV spike glycoprotein (S), or its component parts S1 and S2, in E1-E3- Ad serotype 5 vectors can elicit cellular and humoral immune responses against S in immunized mice. A synthetic spike gene was made by an overlapping PCR strategy with codon optimization for mammalian cells. An Ad vector expressing S driven by a CMV promoter was constructed and protein expression was confirmed by Western analysis. To evaluate the immune responses stimulated by this vaccine, 1010 particle units (pu) of the vector was administered by intravenous, intramuscular or subcutaneous routes to C57Bl/6 mice and serum neutralizing antibody titers were measured in an assay assessing the neutralization of SARS-CoV in vitro. Subcutaneous immunization induced anti-SARS-CoV neutralizing antibodies at 2 wk (reciprocal neutralizing titer 32 ± 15) and at 5 wk (690 ± 470) post-administration. Mice immunized by either intramuscular or intravenous routes also developed neutralizing titers (380 ± 96 and 450 ± 78 respectively) by 5 wk post-administration. Immunization by all routes resulted in statistically similar titers (p>0.3 all pairwise comparisons). At a dose of 1011 pu, anti-SARS-CoV neutralizing titers were also measurable (700 ± 160, intravenous; 130 ± 20 intravenous; 450 ± 210 subcutaneous). In contrast, naive mice and mice immunized with AdNull (an Ad with no transgene) had no detectable neutralizing antibody titers at any time point. Cellular immunity stimulated by immunization was evaluated in BALB/c mice injected intravenously with AdnS or AdNull at a dose of 1011 pu. After 2 wk, splenic CD8+ T cells were isolated and exposed to syngeneic target cells stably expressing S. Accumulation of intracellular IFNγ was observed in 8% of CD8+ cells as opposed to 2.5% of cells exposed to syngeneic cells not expressing S. Similar results were observed in C57Bl/6 mice, with 11.5% of CD8+ cells accumulating intracellular IFNγ in response to syngeneic cells expressing S, as opposed to 3.3% of CD8+ cells exposed to syngeneic cell lines not expressing S. The AdNull vaccinated controls did not stimulate antigen-specific IFNγ accumulation in the splenic CD8+ cells. To determine the location of the major cellular epitopes in the spike glycoprotein, Ad vectors and target syngeneic cell lines expressing the N-terminal domain (S1) or the C-terminal domain (S2) of spike were constructed. The specific accumulation of IFNγ in immunized BALB/c mice at 2 wk post-administration was more dependent on the S2 domain than the S1 domain (19% specific intracellular IFNγ accumulation using the S2 domain vs 6.3% with S1). We conclude that immunization with an Ad vaccine vector expressing the SARS-CoV S elicits high titers of SARS-CoV-neutralizing antibodies and that the S2 domain of spike contains the immunodominant CD8+ T-cell epitopes.

569. Increased Antigen-Specific Humoral Response by Adenoviral Vector Prime and Adeno-Associated Viral Vector Boost

Top of pageAbstract Dissemination of anthrax spores in a bioweapons event could result in inhalational anthrax, a disease that resulted in 45% mortality in the recent US anthrax attacks. The current licensed vaccine and the experimental vaccine currently being assessed by the US Army elicit protective immune responses against protective antigen (PA), a component of lethal toxin. We have recently demonstrated (Tan et al, Human Gene Therapy 2003; 14:1673-1682) that a recombinant serotype 5 adenovirus vector expressing a synthetic gene for PA (Ad5sechPA) offers more robust protection against anthrax lethal toxin challenge than that achieved with purified recombinant PA protein. In this study we have tested the hypothesis that the efficacy of Ad5sechPA vaccination can be enhanced in prime-boost vaccination regimens. BALB/c mice were given intramuscular injections of the Ad5sechPA vaccine at a dose 107, 108, 109 particle units (pu) either once at wk 0, or twice at wk 0 and 4. Anti-PA IgG titer (by ELISA) and neutralizing antibody titer (NAb; reciprocal titer required for 50% inhibition of cytotoxicity by macrophage protection assay) were assessed and lethal toxin challenge was given at wk 6. In all dose groups, boost led to a higher reciprocal anti-PA ELISA titer in pooled sera rising from 200 to 670 in the 107 pu group; from 1,730 to 32,800 in the 108 group and from 2,550 to 40,200 in the 109 group (5 mice/group). Additionally, in all dose groups, boost led to a higher reciprocal neutralizing titer rising from undetectable to 35 in the 107 pu group; from 120 to 810 in the 108 group and from 120 to 1,400 in the 109 group (pooled sera from 5 mice/group). In response to lethal toxin challenge, boost immunization significantly increased protection: 100% of mice in the 108 pu prime-boost group survived the challenge compared to only 20% survival in the 108 pu single immunization group. In the 107 group there was no survival with or without boost and in the 109 group there was 100% survival regardless of boost. We conclude that an Ad5-based anthrax vaccine can be effectively used in prime-boost immunization to achieve more robust protective immunity than single immunization, at least when given via intramuscular route. These results have implications for applying Ad5-based vaccines to human populations.

Isolation and Characterization of a Novel Antigenic Subtype of Infectious Bronchitis Virus Serotype DE072

Three infectious bronchitis virus (IBV) isolates, CU82792, CU82805, and CU82808, were recovered from sentinel chickens placed with three different layer flocks of a large commercial poultry farm in New York State. The three isolates were classified as members of the DE072 serotype on the basis of 1) their S1 genes could be amplified with only a modified primer designed for the DE072 serotype and 2) their restriction fragment length polymorphism patterns, after digestion with endonucleases HaeIII, BstyI and XcmI, were indistinguishable from that of DE072 virus. Additional characterization of one of the isolates, CU82805, revealed that its S1 gene bears approximately 96% identity at the nucleotide level and 94% identity at the amino acid level with DE072. Yet, in an in vitro reciprocal virus neutralization test, only a one-way neutralization was observed, i.e., antiserum to CU82805 neutralized DE072, whereas CU82805 was not neutralized by DE072 antiserum. Implications of these findings with regard to IBV diagnosis and immunization are discussed.

Molecular Characterization of an Infectious Bronchitis Virus Strain Isolated from an Outbreak in Vaccinated Layers

An infectious bronchitis virus (IBV) strain CT/7852/97 was isolated from a commercial layer flock experiencing decreased egg production and poor egg quality. Reciprocal virus neutralization test demonstrated that the isolate was closely related to Massachusetts, Arkansas, and JMK. With the use of both reverse transcription-polymerase chain reaction-restriction fragment length polymorphism and multiplex polymerase chain reaction methods, results consistently showed that CT/7852/97 isolate was Massachusetts serotype specific only. The nucleotide sequence of S1 polypeptide coding region of CT/7852/97 was determined and compared with the published sequences of 12 IBV strains from North America. The highest nucleotide homology with H120 (97%) and the least homology (62%) with Delaware (De/072/92) were observed. Massachusetts 41 and Beaudette shared 94% and 93%, respectively, nucleotide similarity with CT/7852/97, and Connecticut, JMK, and Arkansas 99 had 94%, 83%, and 78%, respectively, nucleotide similarity with CT/7852/97. Furthermore, no possible recombination site on S1 gene among Massachusetts 41, Arkansas 99, and JMK was detected in the isolate.

Emergence of Subtype Strains of the Arkansas Serotype of Infectious Bronchitis Virus in Delmarva Broiler Chickens

Infectious bronchitis virus (IBV) field isolates of the Arkansas (Ark) serotype were identified by reverse transcription-polymerase chain reaction (RT-PCR) as the most common serotype isolated from 1993 to 1997. These isolates were recovered from broiler flocks with respiratory disease raised on the Delmarva peninsula in spite of Ark vaccination in the region. For the purposes of investigating this apparently paradoxical finding, five RT-PCR Ark-positive field isolates recovered in 1995 and 1996 were selected for further characterization. The isolates were compared with Ark reference strains by reciprocal virus neutralization (VN) in embryonated eggs, S-1 gene sequence analysis, and challenge of immunity studies in specific-pathogen-free (SPF) chickens. Antigenic (VN) comparisons and S-1 gene analysis confirmed that the five RT-PCR Ark-positive field isolates were of the Ark serotype but also revealed that the viruses could be readily distinguished from Ark reference strains. Four of the isolates (Ark/213/96, Ark/15C/96, Ark/1529/95, Ark/1534/95) were found to have higher antigenic relatedness percentages to each other (95%-100%) than to Ark reference strains DPI (52%-72%) and Georgia variant (Georgia var) (53%-68%) by VN. Another isolate, Ark/1535/95, was found to differ antigenically from the other four RT-PCR Ark-positive field isolates (34%-61%), Ark DPI (44%), and Georgia var (43%) strains. The trends in the S-1 gene sequencing results were similar to those observed for the VN findings. Isolates Ark/213/96, Ark/15C/96, Ark/1529/95, and Ark/1534/95 demonstrated a higher degree of predicted S-1 amino acid similarity to each other (96.5%-98.7%) than to Ark DPI (92.4%-93.7%), Ark 99 (93.2%-94.7%), and Georgia var (89.3%-90.8%). Ark/1535/95 S-1 amino acid similarity values were lower compared with those of the other four RT-PCR Ark-positive field isolates (93.4%-94.8%), Ark DPI (91.9%), Ark 99 (93.0%), and Georgia var (88.7%). Furthermore, the isolates could be distinguished from the Ark reference strains by a characteristic sequence polymorphism, a six-nucleotide deletion encoding amino acids 57 (Asp) and 58 (Asp) in hypervariable region 1 of S-1. On the basis of the VN and sequencing findings, isolates Ark/213/96, Ark/15C/96, Ark/1529/95, and Ark/1534/95 were considered to be a single subtype of the Ark serotype. The fifth isolate, Ark/1535/95, may constitute another subtype of the Ark serotype. Vaccination of SPF chickens with a high-titering commercially available live vaccine containing the Ark DPI strain provided solid protection (>90%) against challenge with the RT-PCR Ark-positive field isolates. Immunization of SPF chickens with Ark/213/96 produced 100% protection against challenge with the homologous strain, as well as isolates Ark/1535/95 and Ark 99 but lower levels of protection against Ark DPI (58%) and Georgia var (55%). Primers for RT-PCR were designed to distinguish between the Ark subtypes and the Ark reference strains on the basis of the characteristic six-nucleotide deletion identified in the S-1 gene of the Ark subtypes. Retrospective analysis of RT-PCR Ark-positive isolates found that the Ark subtypes existed as early as 1992 in Delmarva broilers and became prevalent by 1995. With RT-PCR, restriction fragment length polymorphism analysis, and DNA sequencing techniques, the presence of Ark subtype viruses was demonstrated in two commercial Ark DPI strain vaccines and in our Ark DPI laboratory stocks that were the original source of the virus used for vaccine development. The demonstration of the Ark subtype and reference strains in the Ark DPI strain is evidence of the existence of IBV quasispecies. Factors possibly influencing the emergence of the Ark subtype in commercial broilers are discussed.

Relationship between serotypes and genotypes based on the hypervariable region of the S1 gene of infectious bronchitis virus.

Summary. To group infectious bronchitis virus (IBV) isolates, a genetic grouping method based on hypervariable region 1 (HVR 1, nucleotides 168 to 197) was compared with that based on the whole S1 gene. Both methods resulted in the same grouping data. So the grouping method based on HVR 1 could represent the grouping method based on the whole S1 gene. Taiwan isolates could not be placed within the existing groups. In order to test the correlation between genotype and serotype, a one-way neutralization test was used to compare 9 Taiwan isolates selected from different genotypes with Massachusetts (Mass) (H120) and Connecticut (Conn) standard strains. In addition, a two-way cross-neutralization test was performed in embryonated eggs with the β method (constant-virus, diluted-serum) and the reciprocal neutralization titers were calculated to give the relatedness (r) values. The results of two kinds of neutralizing tests showed that the serotypes of 9 isolates were different from Mass or Conn. Based on the r-values, 9 isolates were divided into two serotypes which were correlated with their genotypes. From pathogenicity tests, IBV Taiwan isolates could be divided into high, intermediate, and low pathogenicity according to their pathogenicity indexes. However, no relationship exists between pathotype and genotype. In conclusion, the genetic typing method based on HVR 1 can be used for typing IBV isolates.

Isolation and Identification of Infectious Bronchitis Virus from Chickens in Sichuan, China

A nonhemagglutinating virus was isolated from kidneys and lungs of chickens suspected of having infectious bronchitis infection. Specific-pathogen-free embryonated chicken eggs were used as the cultural system. With the use of the ciliary activity of chicken embryo tracheal organ cultures as indicator system, the physicochemical properties of one of the isolated strains (SAIB3) were shown to be similar to infectious bronchitis virus (IBV) strain M41 (standard strain); whereas electron microscopy of the isolate showed coronavirus particles. Virus-neutralization tests were performed in tracheal organ cultures to compare the serotypes of five IBV isolates and six known IBV strains on the basis of reciprocal neutralization titers and euclidean distance. The cross-neutralization pattern indicated that one isolate was of the T-strain IBV serotype, another of the M41 IBV serotype, while others had partial serotype relationship to M41 and T-strains of IBV.

Classification of rotavirus VP4 and VP7 serotypes.

Rotaviruses, members of the Reoviridae family, are major etiologic agents of acute nonbacterial gastroenteritis of the young in a wide variety of mammalian and avian species, including humans. The need for effective immunoprophylaxis against rotaviral gastroenteritis has stimulated interest in the biochemical, molecular, genetic, and clinical aspects of these agents with the aim of developing safe and effective vaccines. Because neutralizing antibodies appear to play an important role in protection against many viral diseases, rotavirus antigens that induce neutralizing antibodies have played a central role in research and development of a rotavirus vaccine. The VP7 glycoprotein and VP4 spike protein that constitute the outer capsid of a complete rotavirus particle have been shown to be independent neutralization antigens. Since type specificity of the outer capsid proteins of a rotavirus appears to play an important role in protection against disease in experimental animal models, continued efforts have been made for classification and typing of neutralization specificities on the VP7 or VP4 capsid protein. Based on a criterion of > 20-fold differences between the homologous and heterologous reciprocal neutralizing antibody titers, fourteen VP7 (G) serotypes have been established. Studies are underway to characterize and classify the VP4 (P) serotypes among the strains that exhibit the fourteen different G serotypes. Attempts to classify the VP4 serotypes based on the same criterion (i.e., > 20-fold antibody differences) that is applied to classification of VP7 serotypes are in progress. This standard of > 20-fold antibody differences can be applied with hyperimmune serum raised to a reassortant possessing the VP4 encoding gene (and an unrelated VP7 encoding gene). Genotypes can provide leads towards classification but the serotype of a strain should be based on neutralization.

Type‐specific antigenicity of avian reoviruses

The type-specificity of the neutralizing activity in chicken antiserum to avian reoviruses was affected by the method of antiserum production. The neutralizing activity produced in response to virus infection had higher type-specificity than that produced by immunization with inactivated virus emulsified in adjuvant. By using reassortant viruses the induction of type-specific neutralizing activity was shown to be associated with the sigma C (sigmaC) virion protein. Antigenic classification of virus strains based on immunoprecipitation of the sigmaC protein by chicken antiserum was attempted and the results were similar to those obtained by reciprocal serum neutralization tests. One-way immunoprecipitation of the sigmaC protein by antisera to some heterologous viruses, similar to that reported in reciprocal neutralization tests, made it difficult to assign individual viruses to serogroups and showed that the type-specificity of the sigmaC protein was not absolute. The neutralization activity of monoclonal antibodies to the sigmaC protein of the RAM1 strain of avian reovirus suggested there were separate type- and group-specific antigenic domains on the sigmaC protein, and that the group-specific domains may be associated with the induction of antibody against heterologous viruses.

Design and Construction of Rhinovirus Chimeras Incorporating Immunogens from Polio, Influenza, and Human Immunodeficiency Viruses

This paper describes the design and construction of chimeric human rhinoviruses that contain immunogenic regions from other pathogens as part of their surface coat proteins. Segments encoding the poliovirus 3 Sabin VP1 and VP2 proteins, the influenza hemagglutinin (HA) glycoprotein, and the human immunodeficiency virus gp120 surface and gp41 transmembrane glycoproteins were inserted into a full-length clone of human rhinovirus 14 (HRV14) at regions corresponding to neutralizing immunogenic sites IA (NIm-IA) and II (NIm-II). Of 12 chimeric constructs described, 3 produced viable virus. An HRV14 chimeric virus containing five amino acids of influenza HA (corresponding to 300 Å 2 of solvent-accessible surface area) had wild-type HRV14 growth characteristics and was neutralized by four of four anti-influenza HA antisera with reciprocal neutralizing titers ranging from 180 to 330. However, antisera raised in two guinea pigs against the HRV14:influenza HA chimera did not show significant neutralization of relevant strains of influenza. These results are the first to demonstrate the feasibility of making viable chimeras of human rhinoviruses displaying heterologous immunogens.