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Thanks for taking care of all the suggestions.
[# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #]
I am delighted with the explanation in the rebuttal letter and in the manuscript. After that clear explanation, the manuscript will benefit from the reference to the report.
Would you please add the WHO website that supports the statement that Thailand is H1N1-free in line 87 of the PDF:
Line 87: ...although one isolate of the H5N8 virus was found in chicken in 2008 (https://www.who.int/publications/m/item/influenza-a(h5n1)-highly-pathogenic-avian-influenza-timeline-of-major-events).
Please consider the minor comment by the reviewer. Also, please support, where possible, with WHO or other agencies the statement that Thailand is avian-flu free for the last 15 years. Please also check a recent note in Nature: https://www.nature.com/articles/d41586-022-03322-2
The authors have adequately answered all questions and suggestions raised. It was a great pleasure to read this manuscript. Great job!
Minor comment:
Conclusion, first sentence: Change “HA and NA genes are continue evolve” with “HA and NA gene continue to evolve”
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Extensive comments are made by the reviewers. A major concern that needs to be addressed before further consideration is that the current version of the manuscript excludes data from the past 12 years. Please provide an expanded analysis with the most recent genomic data from 2010-to date. Be advised that without further data inclusion the paper can not be considered for publication.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. #]
[# PeerJ Staff Note: The review process has identified that the English language must be improved. PeerJ can provide language editing services - please contact us at copyediting@peerj.com for pricing (be sure to provide your manuscript number and title) #]
Authors performed genetic analyses of influenza A H5N1 viruses isolated from Thailand between 2003 and 2010. A large number of outbreaks in domestic and wild birds and repeated spillovers into the human population make avian influenza A viruses a continuous threat to both animal and human health. Surveillance of genetic characteristics of avian influenza A viruses is essential for the detection of mutations that can change several important viral characteristics including virulence, transmissibility, host tropism, and antiviral drug susceptibility. There is a constant concern that their fast and unpredictable evolution may lead to the emergence of a strain with pandemic potential. Any investigation that gives us insight into the genetics of avian influenza A viruses is valuable and needs attention. Only continuous surveillance of influenza A viruses in general and particularly avian subtypes will help us in epidemic and pandemic preparedness. That’s why I think this research article, according to its topic, methodology and results deserves to be published.
However, before publishing the English language needs to be improved. Also, some parts of the manuscript have to be revised for a better understanding. You can find my suggestions in the section "Additional Comments".
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Please check carefully the English language. In my opinion, the following comments/suggestions could improve the quality of the manuscript:
Line 28: change “emergence of genetic related” with “the emergence of genetic-related”
Line 42: delete “Using”
Line 47: change “grouped to corresponding” with “grouped into corresponding”
Line 48: change “ranged of 92.4-96.8%” with “ranged from 92.4-96.8%”
Line 52: change “it was” with “they were”
Line 53: change “better” with “a better”
Line 54: change “on genetic” with “of genetic”
Line 62: “could” with “can”
Line 63: change “aquatic birds;” with “aquatic birds,”
Line 69: change “highest fatality of 53%” with “the highest fatality rate of 53%”
Line 70: change “evolutionary related” with “evolutionary-related”
Line 71: change “with combining different NA subtypes have been” with “in combination with different NA subtypes has been”
Line 75: change “resulting 18 cases with six deaths” with “and resulted in 18 cases and six deaths”
Line 76-77: change “but it is uncontrollable until present. Globally, since January 2003 to 02 June 2022, there have been 864 human cases with 456 deaths” with “and it is uncontrollable until the present. Globally, from January 2003 to June 2022, there have been 864 human cases including 456 fatalities…”
Line 78: change “from United State” with “from the United States”
Line 82: change “particular” with “particularly”
Line 83-84: change “Since 2014, 78 laboratory-confirmed H5N6 human cases with 32 deaths” with “A total of 78 laboratory-confirmed H5N6 human cases among which 32 with the fatal outcome…”
Line 85: change “in China during 2015-2022, and Lao People's Democratic Republic (Laos)” with “in China during 2015-2022 and in Laos in 2021. Use official or abbreviated names for both countries.
Line 86: change “; while seven positive cases” with “, while seven positive cases”
Line 88: change “human” with “humans”
Line 90: change “three more genomic” with “three new genomic”
Line 92 and 93: change “No H5Nx human infection was detected in Thailand, except only one isolate of H5N8 virus was found in chicken in 2008.” with “To date, no H5Nx human infections have been detected in Thailand, except one isolate of the H5N8 virus which was found in chicken in 2008.”
Line 94: change “Putative ancestor” with “A putative ancestor”
Line 96: change “from avian origin” with “of avian origin”
Line 100: please include in the text how many genotypes there are and how are they designated
Line 106: change “the new subclades…… for example, subclade” with “new subclades…… for example, subclades”
Line 113: change “Amonsin et al,. 2010” with “Amonsin et al., 2010”
Line 114: change “and H5N1 virus is no longer detect till present” with “and since then, it has not been detected” What about those 3 sequences reported in 2010?
Line 122: change “from Influenza Virus Resource” with “from the Influenza Virus Resource”
Line 130: change “with the references sequences” with “with the reference sequences”
Line 136: change “maximum likelihood” with “a maximum likelihood”
Line 137: change “boostrap” with “bootstrap”
Line 138: change “were the best-fit model” with “were the best-fit models”
Line 140: change “was showed” with “were shown”
Line 143: change “), H5N1 viruses reported” with “) and H5N1 viruses reported”
Line 145: change “time-scaled tree” with “The time-scaled tree”
149-150: change “3,000 generations were performed; with individually obtained effective sample sizes over 200 traced in Tracer v1.7.1 then were combined” with “3,000 generations were performed and individually obtained effective sample sizes over 200 traced in Tracer v1.7.1 were combined”
Line 160: change “Sequence Identity Matrix” with “the Sequence Identity Matrix”
Line 165-186: This paragraph needs to be revised. In the Results section, you must focus on the results of your study. Any information that does not present the result of your research should be left out of this section. There is no need to repeat sentences from the Introduction section, especially not in the Results section. Change the title “H5N1 viruses in Thailand between 2003 and 2010” into something like “Genetic characteristics of HA and NA…”
One question for authors: You have detected two more amino acids, HA 190E and 225G (Table 1), that confer avian receptor-binding preference but you do not mention them in the Results section. Why?
Line 190: change “references sequences” with “reference sequences”
Line 191: change “Phylogenetic” with “The phylogenetic”
Line 194: change “separated with showing” with “separated by”
Line 194-197: change “In addition, clade 1 was the major clade, contained viruses collected in 2003-2010, especially early introduction time period 2003-2005; whereas clade 2.3.4 was the minor clade, only 4 viruses collected in 2006-2007 from the eastern part of Thailand”. with “In addition, clade 1 was the major clade contained viruses collected in 2003-2010, mostly during the early introduction time period 2003-2005, whereas clade 2.3.4 was the minor clade with only 4 viruses collected in 2006-2007 from the eastern part of Thailand.”
Line 199: change “classified to two distinct major groups” with “classified into two distinct major groups”
Line 200: change “which genetically related to viruses” with “genetically related to viruses”
Line 201: change “was closely related to” with “which was closely related to”
Line 203: change “was ranged of 96.9-100%” with “ranged from 96.9-100%”
Line 210: change “descended from Gs/GD/1/96” with “descendant of Gs/GD/1/96,”
Line 214: change “Those viruses formed into the monophyletic clade” with “Those viruses formed the monophyletic clade”
Line 216-218: change “The introduction time of most circulating H5N1 viruses in Thailand during 2003-2010 were estimated as 2001.87 (95% HPD: 2001.34-2002.49); however, the PP support was poor possibility due to the low number of sampling viruses”
with “The introduction time of most H5N1 viruses circulating in Thailand during 2003-2010 was estimated as 2001.87 (95% HPD: 2001.34-2002.49). However, the PP support was poor possibly due to the low number of sampled viruses”
Line 220-221: change “The NA of Gs/GD/1/96 virus introduced to the adjacent region particularly Hunan, Yunnan, and Hong Kong” with “The NA of Gs/GD/1/96 virus was introduced into neighboring regions particularly Hunan, Yunnan, and Hong Kong”
Line 224: change “Open-billstork“ with “openbill stork”
Line 225: This part of the sentence needs to be revised: “and subsequently rapid spreading as investigated in short branch”.
Line 230-231: change the title, for example, “ML phylogenetic analysis of HA between Thai H5N1 clade 2.3.4 viruses and reference subtype H5Nx clade 2.3.4.4 viruses”
Line 232: change “four H5N1 clade 2.3.4” with “four H5N1 viruses belonging to clade 2.3.4”
Line 237-238: change “clearly separated to the clade 2.3.4.4” with “clearly separated from the clade 2.3.4.4”
Line 238: change “with ranged of 92.4-96.8%” with “ranging from 92.4-96.8%”
Line 239: change „human infection“ with „human infections“
Line 242-243: A sentence needs to be revised: “H5Nx viruses, which H5 HA in combination with various NA subtypes, caused global poultries outbreaks; particularly H5N1 subtype potentially caused pandemic in humans.”
Did you mean that: H5Nx viruses, which have H5 HA in combination with various NA subtypes, cause global poultry outbreaks. These viruses, particularly the H5N1 subtype, may become a pandemic threat to humans.
Line 248: change „Gs/GD/1/96 virus is a progenitor providing HA and NA genes“ with „Also, Gs/GD/1/96 virus was a progenitor which provided HA and NA genes“
Line 250: change „genetically evolved of descendant HA clade“ with „descendant HA clade“
Line 252: change “in both poultry and human” with “in both poultry and humans”
Line 254: change „2005); and several“ with „2005) and several“
Line 256 - change „Northeastern of the country“ with „in northeastern Thailand“
Line 257-258: change “lower north region” with “lower-north region”
Line 260: change “However, genotype V with clade” with “Genotype V within the clade”
Line 262-263: change „HA and NA genes, encoding major viral surface glycoproteins, are hypervariable with continuously evolved“ with „HA and NA genes, which encode major viral surface glycoproteins, are hypervariable and continuously evolve“
Line 268: change “in GenBank database” with “in the GenBank database”
Line 268-272: split and correct this sentence: „ML phylogenetic analysis demonstrated that two clades, clade 1 with predominantly detected and clade 2.3.4, were found in Thailand as corresponding to several previously reports (Uchida et al., 2008; Chaichoune et al., 2009; Amonsin et al., 2010), however clade 2.3.4 replaced clade 1 viruses were observed in Southeast Asian countries“ for example „ML phylogenetic analysis demonstrated that two clades, the predominant clade 1 and clade 2.3.4, were found in Thailand as corresponding to several previous reports (Uchida et al., 2008; Chaichoune et al., 2009; Amonsin et al., 2010). The replacement of clade 1 with clade 2.3.4 viruses was observed in Southeast Asian countries”
Line 275: change „they were avian receptor preference“ with „they showed an avian receptor preference“
Line 277: change “had 20-amino deletion” with “had a 20-amino deletion”
Line 278-279: change „indicating that the viruses contributed to the high pathogenicity and host adaptation“ with „which contributed to the high pathogenicity and host adaptation of virus“
Line 280: change “positon” with “position”
Line 283-284: change „; nevertheless, the earliest isolate found in China in 1996“ with „ , although the first isolate was reported in 1996 in China“
Line 285: change “, subsequently its re-emergence in 2003” with “and subsequently re-emerged in 2003”
Line 285-287: please revise this sentence to make it understandable “Furthermore, H5 HA clade 1 viruses prior arose in several cities in China and Hong Kong during 2003 by the emergence time was estimated around 1999, and then introduced to Thailand in 2001” Table 1.
Line 287: change “Notably, the virus strain A/Open-billedstork/Thailand/VSMU-12-BKK/2003” with “The virus strain A/openbilledstork/Thailand/VSMU-12-BKK/2003”
Line 291-292: change “The mean of rate nucleotide substitution” with “The mean rate of nucleotide substitution”
Line 294: change “in Asia particularly” with “in Asia, particularly”
Line 294: change “also” with “as well as”
Line 295-296: delete “subsequent”
Line 299: change “Northeastern” with “northeast”
Line 300: change “as genetically relationship” with “as genetically related”
Line 301: delete “Corresponding to HA,”
Line 303-306: please revise these two sentences to make them understandable, taking into account that “notably” means particularly/especially/specifically and that the term “divergence” means splitting/branching “The viruses emerged approximately in 1992, and then viruses diverged to new clade corresponding to H5 clade 1 in 1999 occurred in China and Hong Kong, and subsequently spread to Vietnam, Thailand, and Cambodia in 2001. Notably, the introduction to Thailand was possibly in 2002.”
Line 306: change “The mean of rate nucleotide substitution” with “The mean rate of nucleotide substitution”
Line 308-310: change “H5Nx viruses in which H5 HA belonged to genetic clade 2.3.4.4 were globally outbreak among wild birds and poultries, and the human infections with subtypes H5N6 and H5N8 have been recently reported” with “H5Nx viruses with H5 HA belonging to genetic clade 2.3.4.4, globally cause global outbreaks among wild birds and poultries. In addition, human infections with subtypes H5N6 and H5N8 have recently been reported.
Line 311-312: change “It is likely that H5Nx viruses become predominantly and replacing the H5N1 viruses.” with “H5Nx viruses will likely become predominant and replace H5N1 viruses in the future.”
Line 316-317: change “have been previously investigated” with “which has been previously investigated”
Line 317: change “with ranged of 92.4-96.8%” with “with a range of 92.4-96.8%”
Line 319-320: change “isolated in duck and chicken” with “isolated from ducks and chickens”
Line 320: change “of H5N8” with “of the H5N8”
Line 321: change “isolated in duck” with “isolated from a duck”
Line 329: change “2010); and” with “2010) and”
Line 330-331: change “Although, H5N1 virus is no longer detect till present,” with “Although, the H5N1 virus has not been detected in Thailand since 2010,”
Line 332: change “might be possibility” with “might be a possibility”
Line 338: change “might cause impact to Thailand in the future” with “might impact Thailand in the future”
Line 339: change “for better understanding on evolution, molecular epidemiology” with “for a better understanding of evolution and molecular epidemiology”
Line 340: change “continued” with “continuous”
Line 340: change “surveillance for” with “surveillance of”
Line 345: change “would like to thanks” with “would like to thank”
Table 1.
Please explain to me what is “HA1-17Y” and where can I find it in H5N1 Genetic Changes Inventory. You can include this explanation in the Results section..
change “ferret” with “ferrets”
The manuscript by Noisumdaeng et al is a retrospective analysis of H5N1 sequences from Thailand in 2003-2010. The authors discuss its genetic relationships with respect to other known H5 highly pathogenic avian influenza virus strains.
The writing is adequate and clear. The figures and tables are also relevant to the data being presented and discussed. Although there are some details that need to be revised. Please see Additional comments for specifics.
The phylogenetic analysis using BEAST and the reference sequences used are adequate and appropriate. However, the authors have only included sequences from 2003-2010. The inclusion of more contemporary strains from the past 15 years will potentially provide new insight into the ecology and epidemiology of H5 avian influenza viruses. Indeed, many developments have occurred since 2010 including the addition of further forks in the clade 2.3.4 such as 2.3.4.4A, 2.3.4.4B, 2.3.4.4C, and 2.3.4.4D.
While the methods used are appropriate, it is unclear whether new insight has been gained with the analysis. The finding that the H5N1 viruses from 2003-2010 does not come as a surprise and there have been publications indicating that H5N1 highly pathogenic avian influenza viruses from Southeast Asia are from the Goose-Guangdong lineage. The addition of more contemporary strains and/or sequencing performed by the authors will provide an analysis that would be worthy of publication.
Lines 68, 74, 76, 84, and others I may have missed: The percentage statistics should be referred to as “percent case fatality” instead of “fatality rate”. https://en.wikipedia.org/wiki/Case_fatality_rate
Line 112: Please check this sentence. Did you mean that the H5N1 virus has not been detected in the current situation?
Line 93: H5N1 subtype avian influenza viruses have been circulating for much longer than 2003. I believe the authors are referring to a particular lineage (sometimes referred to as clades) of H5N1 viruses called the A/goose/Guangdong/1/96 lineage (Gs/Gd) clade 2.3.4.4. The wording of this sentence makes it appear that all H5N1 viruses are Gs/Gd lineage. This is not the case, however. There are other H5N1 viruses that are not Gs/Gd. Here’s a better phrase for this sentence: The H5N1 subtype lineage called A/goose/Guangdong/1/96 clade 2.3.4.4 lineage likely emerged in 2003.
Lines 238: There has not been a pandemic of H5N1 in humans, although there have been human infections, these are relatively rare.
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