Correction: Detection of novel coronaviruses in bats in Myanmar.

[This corrects the article DOI: 10.1371/journal.pone.0230802.].

Sylvatic Transmission of Chikungunya Virus among Nonhuman Primates in Myanmar.

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

First Report of Lumpy Skin Disease in Myanmar and Molecular Analysis of the Field Virus Isolates.

Lumpy skin disease virus (LSDV) causes lumpy skin disease in cattle and buffaloes, which is associated with significant animal production and economic losses. Since the 2000s, LSDV has spread from Africa to several countries in the Middle East; Europe; and Asia; including, more recently, several south-east Asian countries. In November 2020, Myanmar reported its first LSD outbreak. This study reports on the first incursion of LSD in Myanmar and the molecular analysis of the LSDV detected. Staff from the Livestock Breeding and Veterinary Department (LBVD) of the Ministry of Agriculture, Livestock, and Irrigation collected samples from cattle with suspected LSD infection. The Food and Agriculture Organization (FAO) of the United Nations' Emergency Centre for Transboundary Animal Diseases (ECTAD) and the Joint International Atomic Energy Agency (IAEA)/FAO program's Animal Health and Production laboratory provided LSDV diagnostic support to two regional veterinary diagnostic laboratories in Myanmar. Samples from 13 cattle tested positive by real-time PCR. Selected samples underwent sequence analysis in IAEA laboratories. The results show that the Myanmar LSDV sequences clustered with LSDV isolates from Bangladesh and India, LSDV Kenya, and LSDV NI-2490. Further characterization showed that the Myanmar LSDV is 100% identical to isolates from Bangladesh and India, implying a common source of introduction. These findings inform diagnosis and development of control strategies.

Longitudinal Analysis of Influenza A(H5) Sero-Surveillance in Myanmar Ducks, 2006-2019.

Between 2006 and 2019, serological surveys in unvaccinated domestic ducks reared outdoors in Myanmar were performed, using a haemagglutination inhibition (HI) test, to confirm H5 avian influenza virus circulation and assess temporal and spatial distribution. Positive test results occurred every year that samples were collected. The annual proportion of positive farms ranged from 7.1% to 77.2%. The results revealed silent/sub-clinical influenza A (H5) virus circulation, even in years and States/Regions with no highly pathogenic avian influenza (HPAI) outbreaks reported. Further analysis of the 2018/19 results revealed considerable differences in seroconversion rates between four targeted States/Regions and between years, and showed seroconversion before and during the sampling period. By the end of the trial, a high proportion of farms were seronegative, leaving birds vulnerable to infection when sold. Positive results likely indicate infection with Gs/GD/96-lineage H5Nx HPAI viruses rather than other H5 subtype low-pathogenicity avian influenza viruses. The findings suggested persistent, but intermittent, circulation of Gs/GD/96-lineage H5Nx HPAI viruses in domestic ducks, despite the veterinary services' outbreak detection and control efforts. The role of wild birds in transmission remains unclear but there is potential for spill-over in both directions. The findings of this study assist the national authorities in the design of appropriate, holistic avian influenza control programs.

Movements of Indian Flying Fox in Myanmar as a Guide to Human-Bat Interface Sites.

Frugivorous bats play a vital role in tropical ecosystems as pollinators and seed dispersers but are also important vectors of zoonotic diseases. Myanmar sits at the intersection of numerous bioregions and contains habitats that are important for many endangered and endemic species. This rapidly developing country also forms a connection between hotspots of emerging human diseases. We deployed Global Positioning System collars to track the movements of 10 Indian flying fox (Pteropus giganteus) in the agricultural landscapes of central Myanmar. We used clustering analysis to identify foraging sites and high-utilization areas. As part of a larger viral surveillance study in bats of Myanmar, we also collected oral and rectal swab samples from 29 bats to test for key emerging viral diseases in this colony. There were no positive results detected for our chosen viruses. We analyzed their foraging movement behavior and evaluated selected foraging sites for their potential as human-wildlife interface sites.

Seroepidemiologic Survey of Crimean-Congo Hemorrhagic Fever Virus in Logging Communities, Myanmar.

Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in Asia, infecting many animal hosts, but CCHFV has not been reported in Myanmar. We conducted a seroepidemiologic survey of logging communities in Myanmar and found CCHFV exposure was common (9.8%) and exposure to wild animal blood and body fluids was associated with seropositivity.

Detection of novel coronaviruses in bats in Myanmar.

The recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. Of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and most recently, Coronavirus Disease 2019 (COVID-19), an epidemic of acute respiratory illness originating from Wuhan, China in December 2019. Viral detection, discovery, and surveillance activities were undertaken in Myanmar to identify viruses in animals at high risk contact interfaces with people. Free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. Sequences from positives were compared to known coronaviruses. Three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast Asian countries were detected for the first time in bats in Myanmar. Ongoing land use change remains a prominent driver of zoonotic disease emergence in Myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales.

Emerging Zoonotic Influenza A Virus Detection in Myanmar: Surveillance Practices and Findings.

We describe 2-season, risk-based, virological surveillance for zoonotic avian influenza in Myanmar and report the first detection of influenza A subtypes H5N6 and H9N2 in Myanmar. The study focused mainly on the live bird markets in border townships, where illegal poultry importation from China usually takes place. The objective was to enhance early warning for low pathogenic avian influenza A(H7N9) incursion. The study followed the guidelines of the Food and Agriculture Organization (FAO) of the United Nations for influenza A(H7N9) surveillance in uninfected countries. The sampling strategy was risk-based at all sampling levels. Sample collection and laboratory analysis were carried out with the government of the Union of the Republic of Myanmar. Laboratory testing was according to a previously published FAO laboratory protocol and algorithm designed to detect a range of influenza A subtypes. Challenges to implementation are outlined. The study provided evidence that the H7N9 subtype had not entered Myanmar but detected other subtypes, including H5N6 and H9N2. Although there were logistical difficulties associated with nation-related issues, the results highlight the importance and feasibility of this risk-based active surveillance, which should be urgently established in other countries, especially those located at the east-southeast influenza epicenter.

Highly pathogenic avian influenza (H5N1) in Myanmar, 2006-2010.

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first reported in Myanmar in 2006. In this study, we have characterized 6 HPAI (H5N1) viruses recovered from 2007-2010 as well as three additional available nucleotide sequences representing Myanmar AI outbreaks. Phylogenetic analysis showed that the Myanmar viruses belong to HPAI (H5N1) clades 7, 2.3.2 and 2.3.4. The result suggested that the HPAI (H5N1) viruses recovered from Myanmar had been introduced into the country by multiple introductions. Genetic analysis of the viruses confirmed the HPAI characteristics of the viruses.

Detection of vaccine-like infectious bursal disease (IBD) virus in IBD vaccine-free chickens in Japan.

The prevalence of infectious bursal disease virus (IBDV) was studied in chickens, which had not been vaccinated against IBD. Fifty sera and forty-six bursae of Fabricius from chickens showing impaired growth, collected from 7 IBD vaccination-free farms in Japan were used for virus neutralization (VN) tests and RT-PCR for detection of IBDV genome corresponding to the VP2 hypervariable region. Of the fifty sera, 39 sera (78%) from 6 farms were VN antibodies positive. Of the forty-six bursae, 37 bursae (80.4%) from 6 farms were positive in the RT-PCR assay. The sequences of all the RT-PCR products detected in this study were closely related or identical to those of the vaccine strains. These results show that vaccine-like IBDV is prevalent even in IBD vaccine-free chicken farms in Japan.

Chicken B lymphoma DT40 cells as a useful tool for in vitro analysis of pathogenic infectious bursal disease virus.

Susceptibility of DT40 cells to pathogenic field strains of infectious bursal disease virus (IBDV) including very virulent and classical virulent strains were studied. After the first and second passage of the virus in DT40 cells, IBDV-specific antigen was readily detected in DT40 cells inoculated with the pathogenic field strain infected bursal homogenates. Nucleotide sequence analysis in the VP2 hypervariable domain, which is critical for the virulence of IBDV, revealed no common amino acid substitutions among the pathogenic IBDVs in accordance with the propagation in DT40 cells. These results indicate that DT40 cells are a useful tool for rapid isolation of pathogenic field strains and successive in vitro analysis of IBDV.

Nucleotide sequence analysis of VP2 hypervariable domain of infectious bursal disease virus detected in Japan from 1993 to 2004.

Bursae of Fabricius were collected from 20 chickens diagnosed with infectious bursal disease virus (IBDV) infection from 15 prefectures in 1993 to 2004. Here we report the nucleotide sequence analysis of VP2 hypervariable domain of IBDV genome detected by reverse transcription-polymerase chain reaction from these samples. Ten sequences derived from 10 prefectures in 1996 to 2003 were of the classical type and other 10 sequences derived from 6 prefectures in 1993 to 2004 were of the highly virulent type. Of the classical type sequences, 9 sequences were closely related to the sequence of classical attenuated vaccines used in Japan. Furthermore, two were identical to the sequence of B-Chi5 which represents Vaccine B passaged 5 times in chickens and was reported to be reverted the virulence during the passages. The 10 highly virulent type sequences were classified into four sequences, none of which had been previously detected in Japan. However, the deduced amino acid sequences were identical to each other and to the sequences of highly virulent IBDVs previously detected in Japan. The most common nucleotide sequences, which accounted for 6 of the sequences, were identical to 34 highly virulent type sequences detected in various countries in BLAST search. This is the first report of detection of the sequence in Japan which is identical to highly virulent strains detected in other countries. These findings show the prevalence of classical IBDVs closely related to the attenuated vaccines and highly virulent IBDVs derived from other countries throughout Japan since 1993.

A practical tissue sampling method using ordinary paper for molecular detection of infectious bursal disease virus RNA by RT-PCR.

A practical sampling method for bursal tissue using ordinary paper for molecular diagnosis of infectious bursal disease (IBD) was established. IBD virus-infected bursa was directly smeared on chromatography paper, filter paper, or stationery copy paper and was then fixed with absolute ethanol, Tris-HCl-saturated phenol, or phenol:chloroform:isoamyl alcohol (25:24:1). Flinders Technology Associates (FTA) card, which is designed for the collection of biological samples for molecular detection, was also used. After storage at 37 C for up to 30 days, total RNA directly extracted from the tissue fixed on the papers and FTA card were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of IBD virus (IBDV) RNA. In addition, the ability of each chemical used in the fixation and the FTA card to inactivate IBDV was evaluated. Regardless of the paper quality, storage period, and fixation method, IBDV RNA was consistently detected in all of the samples. IBDV in the bursal tissue was inactivated with phenol but not with ethanol or the unknown chemicals in FTA card. These results show that ordinary papers sustain the viral RNA, as does FTA card, but phenol fixation is superior to FTA card in inactivating IBDV. The new sampling method using ordinary paper with phenol fixation is safe, inexpensive, simple, and easy, and is thus suitable for conducting a global survey of IBD even where laboratory resources are limited. This practical method should contribute to the control of IBD worldwide.