Cross Hemagglutination Inhibition Tests Research Articles

Continuous Introduction of H5 High Pathogenicity Avian Influenza Viruses in Hokkaido, Japan: Characterization of Viruses Isolated in Winter 2022–2023 and Early Winter 2023–2024

High pathogenicity avian influenza (HPAI) has impacted poultry and wild birds globally. The number of H5 HPAI virus (HPAIV) infection cases in wild birds in Hokkaido (Northern Japan) was high in the last two seasons, contributing to virus spillover to resident birds and poultry. Therefore, H5 HPAIVs in birds and mammals in Hokkaido in winter 2022–2023 and 2023–2024 were monitored and viruses were phylogenetically, antigenically, and pathogenetically characterized. Thirty HPAIV isolates were subtyped and pathotyped by sequencing the hemagglutinin (HA) gene of viruses. Phylogenetic analysis of the HA gene revealed that all isolated HPAIVs were categorized into clade 2.3.4.4b and divided into three groups (G2b, G2c, and G2d). Most isolates belonging to subgroup G2d clustered with isolates in winter 2021–2022 in Hokkaido. The other isolates were categorized into two subgroups, G2b and G2c, mainly composed of isolates in Honshu Island in winter 2021–2022 and 2022–2023, respectively. Two H5 HPAIVs isolated in Eastern Russia in spring and autumn 2022 were genetically close to most Hokkaido isolates (G2d), and a virus isolated in Hokkaido in November 2023 was also grouped in subgroup G2d. Further analysis of all eight gene segments identified six types of gene constellations. Cross-hemagglutination inhibition test indicated that the antigenicity of H5 HPAIVs isolated in the last several seasons was similar within them but slightly different from that in the 2010s. Three chicken breeds were intranasally challenged with four representative isolates to assess their pathogenicity. All chickens except one broiler chicken were dead until 5-day postchallenge with different pathogenicity of these viruses. The pathogenicity of one HPAIV strain was significantly lower in broiler chickens than in layer chickens. The mixture of multiple characteristics of HPAIVs in Hokkaido was confirmed by bird migration routes. Thus, many HPAIVs can be brought and scattered anywhere on Earth.

Efficacy of two vaccines against recent emergent antigenic variants of clade 2.3.2.1a highly pathogenic avian influenza viruses in Bangladesh

H5N1 highly pathogenic avian influenza viruses (HPAIVs) have caused outbreaks in poultry in Bangladesh since 2007. While clade 2.2.2 and 2.3.4.2 HPAIVs have not been detected since 2012, clade 2.3.2.1a viruses have caused continuous outbreaks since 2012 despite the use of vaccines. In this study, we evaluated the efficacy of two H5 vaccines licensed in Bangladesh, RE-6 inactivated vaccine, and a recombinant herpesvirus of turkeys vaccine with an H5 insert (rHVT-H5), for protection against recent field viruses in chickens. We selected three viruses for efficacy tests (A/chicken/Bangladesh/NRL-AI-3237/2017, A/crow/Bangladesh/NRL-AI-8471/2017 and A/chicken/Bangladesh/NRL-AI-8323/2017) from 36 H5 viruses isolated from Bangladesh between 2016 and 2018 by comparing the amino acid sequences at five antigenic sites (A-E) and analyzing hemagglutination inhibition (HI) titers with reference antisera. The RE-6 and rHVT-H5 vaccines both conferred 80–100% clinical protection (i.e. reduced morbidity and mortality) against the three challenge viruses with no significant differences in protection. In addition, both vaccines significantly decreased viral shedding from infected chickens as compared to challenge control chickens. Based on these metrics, the current licensed H5 vaccines protected chickens against the recent field viruses. However, the A/crow/Bangladesh/NRL-AI-8471/2017 virus exhibited antigenic divergence including: several unique amino acid changes in antigenic epitope sites A and B and was a serological outlier in cross HI tests as visualized on the antigenic map. The continuing emergence of such antigenic variants which could alter the dominant antigenicity of field viruses should be continuously monitored and vaccines should be updated if field efficacy declines.

In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutininneuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2 13 HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.

Chronological Analysis of Canine Parvovirus Type 2 Isolates in Japan

Fifty-five canine parvovirus type 2 (CPV) samples, 12 fecal specimens and 43 cell culture isolates, were examined for their genetic characteristics of VP2 gene. They were collected from the diseased dogs at various districts of Japan during 27 years from 1980 to 2006. A fragment of VP2 gene was analyzed by restriction fragment length polymorphism assay and DNA sequencing. The original antigenic type 2 of CPV (CPV-2) was no longer found in the samples since 1984, and two antigenic variants CPV-2a and CPV-2b replaced CPV-2 as predominant types for about 5 years from 1982. A new genetic variant of prototype CPV-2a with non-synonymous substitution at the VP2 amino acid residue 297 from Ser to Ala was first detected in 1987. New CPV-2b with the same amino acid substitution at position 297 as new CPV-2a was also detected from the samples collected in 1997. Since then new CPV-2b has been the predominant CPV over the field of Japan. Several additional amino acid substitutions were detected in the VP2 gene of some recent CPV strains. Neither CPV-2c(a), CPV-2c(b), nor "Glu-426" of the antigenic variants previously found outside the country was detected in any samples tested. Reactivity of new CPV-2a and 2b variants against antibodies produced by the current vaccine products was determined by a cross hemagglutination-inhibition test. The recent field CPV isolates reacted more efficiently to the antibodies produced in dogs vaccinated with the new CPV-2b vaccine strain than the conventional CPV-2 vaccine strain.

Antigenic and genetic characterization of H1 influenza viruses isolated from feral ducks and swine in Japan.

The strains of H 1 N 4 influenza A virus isolated from feral ducks in Japan in 1977-78 were compared to swine-origin H 1 N 1 viruses antigenically and genetically. Homologous characteristics were found among the H 1 N 4 isolates from feral ducks in hemagglutination-inhibition (HI) tests, viral RNA patterns on polyacrylamide gel electrophoresis and oligonucleotide mapping. Although the hemagglutinins of duck-origin viruses employed in this study were identified as H 1, the viruses were distinguishable from A/New Jersey/8/76 (H 1 N 1), A/duck/Alberta/35/76 (H 1 N 1) and the virus isolated from swine in Japan in the cross HI test. Also, the viral RNA patterns of the duck- and swine-origin H 1 viruses were found to be quite different, indicating that genetic reassortment of HA genes between them is unlikely. After H 1 N 4 virus of duck-origin was intranasally inoculated into pigs, a brief period of virus recovery with no serological response was observed; whereas swine-origin H 1 N 1 virus produced seroconversion in the pigs inoculated.

The Isolation and Properties of a Bovine Strain (T1) of Parainfluenza 3 Virus

A bovine strain of parainfluenza 3 (PI3) virus, designated T1 strain, which was isolated from the tonsillar material of each of 2 calves has been shown by cross-haemagglutination inhibition tests to be serologically indistinguishable from an American bovine strain (SF4) of PI3, virus. Cytological examination of tissue culture cells following inoculation of the T1 strain revealed the formation of syncytia, intracytoplasmic inclusions and intranuclear inclusions; electron microscope examination of the virus has shown the particle to have a structure identical with that of a Swedish bovine strain (Umea 33) of PI3 virus. The significance of the intranuclear inclusions and of the isolation of a bovine strain of PI3, virus in the United Kingdom is discussed.