Reverse transcription PCR-based detection of matrix and hemagglutinin-neuraminidase genes among Avian orthoavulavirus 1 clinical isolates in the Philippines, 1991-2017

Newcastle disease (ND) is a highly infectious disease that affects devastatingly the avian population worldwide. It is caused by Avian orthoavulavirus 1 (AOAV-1), or better known as Newcastle disease virus belonging to phylum Negarnaviricota, class Monjiviricetes, order Mononegavirales and family Paramyxoviridae. This virus consists of six principal structural proteins namely: the nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and the large protein RNA dependent RNA polymerase (L).. The present study aimed to molecularly detect the M and HN gene segments of the AOAV-1 field isolates from clinical cases in the Philippines from 1991 through 2017. RT-PCR amplification and sequence analyses using primers NDV-For4359 and NDV-Rev4788 which anneal to the matrix gene and primers NDV-For6369 and NDV-Rev6598 targeting the HN genes, identified all isolates to be AOAV-1. Determining the different genes of the virus would greatly help scientists and researchers to accurately identify the viral isolates in order to improve epidemiological studies and surveillance of the disease in the country.

Genetic Characteristics of Human Parainfluenza Virus Types 1–4 From Patients With Clinical Respiratory Tract Infection in China

Human parainfluenza viruses (HPIV1–4) cause acute respiratory tract infections, thereby impacting human health worldwide. However, there are no current effective antivirals or licensed vaccines for infection prevention. Moreover, sequence information for human parainfluenza viruses (HPIVs) circulating in China is inadequate. Therefore, to shed light on viral genetic diversity and evolution, we collected samples from patients infected with HPIV1–4 in China from 2012 to 2018 to sequence the viruses. We obtained 24 consensus sequences, comprising 1 for HPIV1, 2 for HPIV2, 19 for HPIV3, and 2 for HPIV4A. Phylogenetic analyses classified the 1 HPIV1 into clade 2, and the 2 HPIV4 sequences into cluster 4A. Based on the hemagglutinin-neuraminidase (HN) gene, a new sub-cluster was identified in one of the HPIV2, namely G1c, and the 19 HPIV3 sequences were classified into the genetic lineages of C3f and C3a. The results indicated that HPIV1–4 were co-circulated in China. Further, the lineages of sub-cluster C3 of HPIV3 were co-circulated in China. A recombination analysis indicated that a putative recombination event may have occurred in the HN gene of HPIV3. In the obtained sequences of HPIV3, we found that two amino acid substitution sites (R73K in the F protein of PUMCH14028/2014 and A281V in the HN protein of PUMCH13961/2014) and a negative selection site (amino acid position 398 in the F protein) corresponded to the previously reported neutralization-related sites. Moreover, amino acid substitution site (K108E) corresponded to the negative selection site (amino acid position 108) in the 10 F proteins of HPIV3. However, no amino acid substitution site corresponded to the glycosylation site in the obtained HPIV3 sequences. These results might help in studying virus evolution, developing vaccines, and monitoring HPIV-related respiratory diseases.

Genetic analysis of human parainfluenza virus type 4 associated with severe acute respiratory infection in children in Luohe City, Henan Province, China, during 2017–2018

During 2017–2018, nasopharyngeal aspirates (NPAs) from 627 hospitalized patients with severe acute respiratory infection at Luohe Center Hospital were tested by RT-PCR for human parainfluenza virus 4 (HPIV-4). Fourteen (2.2%) of the 627 samples were positive for HPIV-4. The complete HN gene was amplified from nine positive samples and sequenced. Sequence comparisons showed that the HPIV-4 strains circulating in the city of Luohe are closely related to HPIV-4A strains. Our study indicated that there were multiple lineages of HPIV-4 circulating in Henan Province in China during the study period. This will improve our understanding of the epidemiological and clinical characteristics of HPIV-4.

Genetic Analysis of Human Parainfluenza Virus Type 4-Associated with Severe Acute Respiratory Infection Among Children in Luohe City, Henan Province, China, During 2017-2018

During 2017–2018, Nasopharyngeal aspirates (NPAs) from 627 hospitalized patients with SARI at Luohe Center Hospital were tested by RT-PCR for human parainfluenza virus 4(HPIV-4). 14 (2.2%) of 627 samples were positive for HPIV-4. The complete nucleotide sequence of the HN gene from 9 positive samples was amplified and sequenced successfully. Genetic analysis showed that the HPIV-4 strains circulating in Luohe city are more closely related to HPIV-4A. Our study indicated that there were multiple lineages of HPIV-4 circulating in Henan Province in China during the study period, which will improve our understanding of the epidemiological and clinical characteristics of HPIV-4.

Molecular and biological characterization of some circulating strains of Newcastle disease virus in broiler chickens from Eastern Saudi Arabia in 2012-2014

Background and Aim: Newcastle disease (ND) is a worldwide poultry disease that is historically known to cause severe losses in the poultry industry. In the present study, attempts were made to characterize ND virus (NDV) recovered from broiler chickens in the Eastern Region of Saudi Arabia from January 2012 to March 2014. Materials and Methods: Reverse transcription-polymerase chain reaction was used for the detection of NDV followed by partial sequencing of the fusion (F) gene. The intracerebral pathogenicity index (ICPI), mean death time (MDT), and complete sequencing of the hemagglutinin-neuraminidase (HN) gene were also used for further biological and molecular characterization. Results: NDV was detected at a rate of 9.6% (11/115) of the tested flocks, most of which were vaccinated against ND. F gene-based phylogeny and motifs of the fusion protein cleavage site (FPCS) showed segregation of Saudi isolates into two groups. The first group contained 10 isolates and was located in genotype II with the lentogenic motif 112GRQGRL117 at the FPCS. The second group contained one isolate and was located in genotype VII with velogenic motif 112RRQKRF117. Further characterization using the ICPI and MDT of two representative isolates showed virulence of both tested isolates. Phylogenetic analysis of the HN gene showed close nucleotide identity between the two isolates. A BLAST search for sequences similar to HN gene sequences showed high identity with isolates from the surrounding region. Conclusion: The present findings showed a low detection rate of NDV, possibly due to the wide application of vaccines, and the circulation of at least two NDV genotypes, II and VII, in the Eastern Region of Saudi Arabia. The present Saudi isolates may share common ancestors with isolates from the surrounding region.

The Emergence of Avian Orthoavulavirus 13 in Wild Migratory Waterfowl in China Revealed the Existence of Diversified Trailer Region Sequences and HN Gene Lengths within this Serotype

Avian orthoavulavirus 13 (AOAV-13), also named avian paramyxovirus 13 (APMV-13), has been found sporadically in wild birds around the world ever since the discovery of AOAV-13 (AOAV-13/wild goose/Shimane/67/2000) in a wild goose from Japan in 2000. However, there are no reports of AOAV-13 in China. In the present study, a novel AOAV-13 virus (AOAV-13/wild goose/China/Hubei/V93-1/2015), isolated from a wild migratory waterfowl in a wetland of Hubei province of China, during active surveillance from 2013 to 2018, was biologically and genetically characterized. Phylogenetic analyses demonstrated a very close genetic relationship among all AOAV-13 strains, as revealed by very few genetic variations. Moreover, pathogenicity tests indicated that the V93-1 strain is a low virulent virus for chickens. However, the genome of the V93-1 virus was found to be 16,158 nucleotides (nt) in length, which is 12 nt or 162 nt longer than the other AOAV-13 strains that have been reported to date. The length difference of 12 nt in strain V93-1 is due to the existence of three repeats of the conserved sequence, “AAAAAT”, in the 5′-end trailer of the genome. Moreover, the HN gene of the V93-1 virus is 2,070 nt in size, encoding 610 aa, which is the same size as the AOAV-13 strain from Japan, whereas that of two strains from Ukraine and Kazakhstan are 2,080 nt in length, encoding 579 aa. We describe a novel AOAV-13 in migratory waterfowl in China, which suggests that diversified trailer region sequences and HN gene lengths exist within serotype AOAV-13, and highlight the need for its constant surveillance in poultry from live animal markets, and especially migratory birds.

PHYLOGENETIC ANALYSIS OF NP AND HN GENE OF NEWLY ISOLATED NDV/Badung-02/AK/14

virus was isolated from suspected Newcastle disease (ND) chicken in backyard farm at Sibang Village, Badung Regency, Bali Province. The isolates were then propagated and confirmed for NDV serologically. RNA isolation was performed by standard Trizol method. Phylogenetic tree analysis of NP gene (nt1020-1561) and HN gene (nt7019-7754) were performed using sequence of Badung-02/AK/14 and selected NDV strains from GenBank. Based on the NP gene sequence, the newly isolate closely related with other NDV strains belong to genotype VII that previously isolated such as Banjarmasin-010/10, Bali-1/07, and Cockatoo/90 with genetic distance 0.2%, 12.6%, and 18.0% respectively. The genetic distance with LaSota/46 virus (genotype II) is 19.6%. Based on HN gene sequence, genetic distance of the Badung-02/AK/14 with other viruses belong to genotype VII such as Banjarmasin-010/10, Bali-1/07, and Cockatoo/90 are 0.4%, 3.7%, and 4.2% respectively. The genetic distance with LaSota/46 virus (genotype II) is 9.0%. There were no difference between the result of nucleotide and amino acid sequence analysis, both in NP and HN gene (P>0.05).